\r\n\tIn sum, the book presents a reflective analysis of the pedagogical hubs for a changing world, considering the most fundamental areas of the current contingencies in education.
",isbn:"978-1-83968-793-8",printIsbn:"978-1-83968-792-1",pdfIsbn:"978-1-83968-794-5",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"b01f9136149277b7e4cbc1e52bce78ec",bookSignature:"Dr. María Jose Hernandez-Serrano",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/10229.jpg",keywords:"Teacher Digital Competences, Flipped Learning, Online Resources Design, Neuroscientific Literacy (Myths), Emotions and Learning, Multisensory Stimulation, Citizen Skills, Violence Prevention, Moral Development, Universal Design for Learning, Sensitizing on Diversity, Supportive Strategies",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"September 14th 2020",dateEndSecondStepPublish:"October 12th 2020",dateEndThirdStepPublish:"December 11th 2020",dateEndFourthStepPublish:"March 1st 2021",dateEndFifthStepPublish:"April 30th 2021",remainingDaysToSecondStep:"3 months",secondStepPassed:!0,currentStepOfPublishingProcess:4,editedByType:null,kuFlag:!1,biosketch:"Dr. Phil. Maria Jose Hernandez Serrano is a tenured lecturer in the Department of Theory and History of Education at the University of Salamanca, where she currently teaches on Teacher Education. She graduated in Social Education (2000) and Psycho-Pedagogy (2003) at the University of Salamanca. Then, she obtained her European Ph.D. in Education and Training in Virtual Environments by research with the University of Manchester, UK (2009).",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"187893",title:"Dr.",name:"María Jose",middleName:null,surname:"Hernandez-Serrano",slug:"maria-jose-hernandez-serrano",fullName:"María Jose Hernandez-Serrano",profilePictureURL:"https://mts.intechopen.com/storage/users/187893/images/system/187893.jpg",biography:"DPhil Maria Jose Hernandez Serrano is a tenured Lecturer in the Department of Theory and History of Education at the University of Salamanca (Spain), where she currently teaches on Teacher Education. She graduated in Social Education (2000) and Psycho-Pedagogy (2003) at the University of Salamanca. Then, she obtained her European Ph.D. on Education and Training in Virtual Environments by research with the University of Manchester, UK (2009). She obtained a Visiting Scholar Postdoctoral Grant (of the British Academy, UK) at the Oxford Internet Institute of the University of Oxford (2011) and was granted with a postdoctoral research (in 2021) at London Birbeck University.\n \nShe is author of more than 20 research papers, and more than 35 book chapters (H Index 10). She is interested in the study of the educational process and the analysis of cognitive and affective processes in the context of neuroeducation and neurotechnologies, along with the study of social contingencies affecting the educational institutions and requiring new skills for educators.\n\nHer publications are mainly of the educational process mediated by technologies and digital competences. Currently, her new research interests are: the transdisciplinary application of the brain-based research to the educational context and virtual environments, and the neuropedagogical implications of the technologies on the development of the brain in younger students. Also, she is interested in the promotion of creative and critical uses of digital technologies, the emerging uses of social media and transmedia, and the informal learning through technologies.\n\nShe is a member of several research Networks and Scientific Committees in international journals on Educational Technologies and Educommunication, and collaborates as a reviewer in several prestigious journals (see public profile in Publons).\n\nUntil March 2010 she was in charge of the Adult University of Salamanca, by coordinating teaching activities of more than a thousand adult students. She currently is, since 2014, the Secretary of the Department of Theory and History of Education. Since 2015 she collaborates with the Council Educational Program by training teachers and families in the translation of advances from educational neuroscience.",institutionString:"University of Salamanca",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"University of Salamanca",institutionURL:null,country:{name:"Spain"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"23",title:"Social Sciences",slug:"social-sciences"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"301331",firstName:"Mia",lastName:"Vulovic",middleName:null,title:"Mrs.",imageUrl:"https://mts.intechopen.com/storage/users/301331/images/8498_n.jpg",email:"mia.v@intechopen.com",biography:"As an Author Service Manager, my responsibilities include monitoring and facilitating all publishing activities for authors and editors. From chapter submission and review to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. Whether that be identifying an exceptional author and proposing an editorship collaboration, or contacting researchers who would like the opportunity to work with IntechOpen, I establish and help manage author and editor acquisition and contact."}},relatedBooks:[{type:"book",id:"6942",title:"Global Social Work",subtitle:"Cutting Edge Issues and Critical Reflections",isOpenForSubmission:!1,hash:"222c8a66edfc7a4a6537af7565bcb3de",slug:"global-social-work-cutting-edge-issues-and-critical-reflections",bookSignature:"Bala Raju Nikku",coverURL:"https://cdn.intechopen.com/books/images_new/6942.jpg",editedByType:"Edited by",editors:[{id:"263576",title:"Dr.",name:"Bala",surname:"Nikku",slug:"bala-nikku",fullName:"Bala Nikku"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1591",title:"Infrared Spectroscopy",subtitle:"Materials Science, Engineering and Technology",isOpenForSubmission:!1,hash:"99b4b7b71a8caeb693ed762b40b017f4",slug:"infrared-spectroscopy-materials-science-engineering-and-technology",bookSignature:"Theophile Theophanides",coverURL:"https://cdn.intechopen.com/books/images_new/1591.jpg",editedByType:"Edited by",editors:[{id:"37194",title:"Dr.",name:"Theophanides",surname:"Theophile",slug:"theophanides-theophile",fullName:"Theophanides Theophile"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3092",title:"Anopheles mosquitoes",subtitle:"New insights into malaria vectors",isOpenForSubmission:!1,hash:"c9e622485316d5e296288bf24d2b0d64",slug:"anopheles-mosquitoes-new-insights-into-malaria-vectors",bookSignature:"Sylvie Manguin",coverURL:"https://cdn.intechopen.com/books/images_new/3092.jpg",editedByType:"Edited by",editors:[{id:"50017",title:"Prof.",name:"Sylvie",surname:"Manguin",slug:"sylvie-manguin",fullName:"Sylvie Manguin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3161",title:"Frontiers in Guided Wave Optics and Optoelectronics",subtitle:null,isOpenForSubmission:!1,hash:"deb44e9c99f82bbce1083abea743146c",slug:"frontiers-in-guided-wave-optics-and-optoelectronics",bookSignature:"Bishnu Pal",coverURL:"https://cdn.intechopen.com/books/images_new/3161.jpg",editedByType:"Edited by",editors:[{id:"4782",title:"Prof.",name:"Bishnu",surname:"Pal",slug:"bishnu-pal",fullName:"Bishnu Pal"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"72",title:"Ionic Liquids",subtitle:"Theory, Properties, New Approaches",isOpenForSubmission:!1,hash:"d94ffa3cfa10505e3b1d676d46fcd3f5",slug:"ionic-liquids-theory-properties-new-approaches",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/72.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1373",title:"Ionic Liquids",subtitle:"Applications and Perspectives",isOpenForSubmission:!1,hash:"5e9ae5ae9167cde4b344e499a792c41c",slug:"ionic-liquids-applications-and-perspectives",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/1373.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"57",title:"Physics and Applications of Graphene",subtitle:"Experiments",isOpenForSubmission:!1,hash:"0e6622a71cf4f02f45bfdd5691e1189a",slug:"physics-and-applications-of-graphene-experiments",bookSignature:"Sergey Mikhailov",coverURL:"https://cdn.intechopen.com/books/images_new/57.jpg",editedByType:"Edited by",editors:[{id:"16042",title:"Dr.",name:"Sergey",surname:"Mikhailov",slug:"sergey-mikhailov",fullName:"Sergey Mikhailov"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"371",title:"Abiotic Stress in Plants",subtitle:"Mechanisms and Adaptations",isOpenForSubmission:!1,hash:"588466f487e307619849d72389178a74",slug:"abiotic-stress-in-plants-mechanisms-and-adaptations",bookSignature:"Arun Shanker and B. 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1. Introduction
The importance of structural biology has been highlighted in the past few years not only as part of drug discovery programs in the pharmaceutical industry but also by structural genomics programs. Although the function of a protein can be studied by several biochemical and or biophysical techniques a molecular understanding of a protein can only be obtained by combining functional data with the three-dimensional structure. In principle three techniques exist to determine a protein structure, namely X-ray crystallography, nuclear magnetic resonance (NMR) and electron microscopy (EM). X-ray crystallography contributes over 90 % of all structures in the protein data bank (PDB) and emphasis the importance of this technique. Crystallization of a protein is a tedious route and although a lot of knowledge about crystallization has been gained in the last decades, one still cannot predict the outcome. The sometimes unexpected bottlenecks in protein purification and crystallization have recently been summarized and possible strategies to obtain a protein crystal were postulated [1]. This book chapter will tackle the next step: How to crystallize protein-ligand complexes or intermediate steps of the reaction cycle?
A single crystal structure of a protein however, is not enough to completely understand the molecular function. Conformational changes induced by for example ligand binding cannot be anticipated a priori. The determination of particular structures of one protein, for example with bound ligand(s) is required to visualize the different states within a reaction cycle. Ideally, one would trap an open conformation without any ligand, an open ligand-bound and a closed form with the bound molecule as well as the closed ligand-free protein to visualize the conformational changes occurring during catalysis in detail.
Within this chapter, the structural conformational changes induced by ligand binding with respect to the methods chosen for the crystallization are described. Here three distinct protein families are exemplarily described: first, where one substrate or ligand is bound, second, a protein with two or more bound substrates and finally, the structures of proteins, in which the product of the reaction cycle is present in the active site.
Specific methods or expressions written in bold italics are explained in the glossary box at the end of the chapter.
1.1. General approaches to obtain crystals with bound ligands and how to prepare the ground
Often the knowledge of the structure of a protein or enzyme without bound ligand(s) is not sufficiently significant since there is no or only little information provided about the catalytic mechanism. To gain further insights, it is important or at least helpful to obtain a binary or ternary structure of the protein of interest.
In theory there are different approaches to reach this goal even though it can be a difficult task in reality. All of them have in common that the naturally catalysed reaction must not occur. Apart from reporting all possible attempts we would like to give a general overview about several co-crystallization/soaking strategies first, followed by selected examples described within this bookchapter.
Possible co-crystallization or soaking trials:
(In order to keep it simple and coherent the expression „ligand“ in the following paragraph is used in terms of „substrate“, „cofactor“ or „binding partner“.)
first ligand without second ligand
second ligand without first ligand
first ligand with product of the second ligand
product of the first ligand with second ligand
substrate analogue/inhibitor or non-hydrolysable cofactor
application of substances that mimic transition state products (e. g. AlF3 which imitates a phosphate group)
usage of catalytically inactive mutants with bound ligand(s)
creation of an environment (i. g. buffer condition) which shifts the equilibrium constant so that the reaction cannot occur
The most important point concerning preparing co-crystallization trials is the knowledge of the corresponding kinetical parameters. Proteins bind their natural ligand(s) with high affinity, which means in the nM- up to low mM range. To successfully crystallize a protein with the ligand(s) bound, the affinity needs to be determined. There are numerous biophysical techniques to achieve this, for example Intrinsic Tryptophan Fluorescence, Isothermal Calorimetry, Surface Plasmon Resonance and many others. In principle the affinity is determined by the size of a ligand as well as the property of the binding site of the protein. As first approximation, one can state that affinity increases with a decrease in ligand size.
The application of a too low concentration of the ligand can lead to an inhomogeneous protein solution, which means that not all of the protein molecules are loaded with ligand (and this can prevent crystallization). It is also possible, that a low level of occupancy causes an undefined electron density so that the ligand cannot be placed or which even makes a structure solution impossible. As a rule of thumb the concentration of the ligand(s) should be applied to the crystallization trial about 5-fold of the corresponding KM value (the Michaelis constant KM is the substrate concentration at which the reaction rate is half of Vmax, which represents the maximum rate achieved by the system, at maximum (saturating) substrate concentrations).
Beyond that all requirements for the protein solution itself remain valid as described in [1] in more detail.
2. Binding protein with one ligand – How to crystallize and what can be deduced from the structure
A typical class of a protein binding one ligand are substrate-binding proteins (SBPs), and substrate-binding domains (SBDs) [2]. They form a class of proteins (or protein domains) that are often associated with membrane protein complexes for transport or signal transduction. SBPs were originally found to be associated with prokaryotic ATP binding cassette (ABC)-transporters, but have more recently been shown to be part of other membrane protein complexes as well such as prokaryotic tripartite ATP-independent periplasmic (TRAP)-transporters, prokaryotic two-component regulatory systems, eukaryotic guanylate cyclase-atrial natriuretic peptide receptors, G-protein coupled receptors (GPCRs) and ligand-gated ion channels [2].
Structural studies of a substantial number of SBPs revealed a common fold with a bilobal organization connected via a linker region [2]. In the ligand-free, open conformation, the two lobes or domains are separated from each other, thereby forming a deep, solvent exposed cleft, which harbors the substrate-binding site. Upon ligand binding, both domains of the SBP move towards each other through a hinge-bending motion or rotation, which results in the so-called liganded-closed conformation. As a consequence of this movement, residues originating from both domains generate the ligand-binding site and trap the ligand deeply within the SBP [3]. In the absence of a ligand, unliganded-open and unliganded-closed states of the SBP are in equilibrium, and the ligand solely shifts this equilibrium towards the liganded-closed state. This sequence of events has been coined the “Venus-fly trap mechanism” [4-6]; it is supported by a number of crystal structures in the absence and presence of a ligand [7, 8] and other biophysical techniques [3].
For the maltose binding protein (MBP) from Escherichia coli [9], it has been shown that both domains are dynamically fluctuating around an average orientation in the absence of the ligand [10]. NMR spectroscopy of MBP in solution revealed that the ligand-free form of MBP consists of a predominantly open species (95 %) and a minor species (5 %) that corresponds to a partially closed state; both forms co-exist in rapid equilibrium [11]. The open form of MBP observed by NMR is similar to the crystal structure of the unliganded-open conformation [12]. However, the partially closed species detected by NMR [11] does not correspond to the ligand-bound, fully closed form found in crystallographic studies. Instead, it represents an intermediate, partially closed conformation [13], suggesting that the substrate is required to reach the final, liganded-closed conformation.
Upon substrate binding, the closed conformation is stabilized, and the ligand is trapped within a cleft in between the two domains [14-16]. In principle one can divide the conformational changes in four (I-IV) states (highlighted in Figure 1). State I is the „open-unliganded“ where the protein adopts an open conformation and no substrate is bound to the protein. State II is the „closed-liganded“ conformation where the substrate is bound and induced a conformational change of both domains towards each other. This is likely the state within the cell before delivery of the substrate to its cognate transporter. Two other states are known to be present in solution although less frequent and the equilibrium is shifted towards the open-unliganded conformation. These forms are state III, the „closed-unliganded“ state and state IV, the „semi-closed-unliganded“ state. These are unfavorable conformations of the SBP, which occur due to the flexibility of the linker region in between both domains.
To fully understand the function as well as the structural changes happening upon ligand/substrate binding it would require structural information of at least states I and II, preferably also of states III and IV.
Figure 1.
Substrate binding proteins exist in four major conformations: I) unliganded-open II) liganded-closed III) unliganded-closed and IV) unliganded-semi-open. All states are in equilibrium with each other. In solution states I and II occur most frequently. To fully understand the opening and closing mechanism of the protein however snapshots of every state are needed to gain full knowledge.
2.1. Crystallization of the open-unliganded conformation (state I)
The crystallization of an open conformation of a rather flexible protein is not straight forward and most of the success came from „trial-and-error“ approaches. After purification of the protein, a reasonable concentration of the protein is taken to set up crystallization trials. Most commonly the vapor diffusion method with the hanging or sitting drop is used. SBPs mainly exist in the open-unliganded conformation in the absence of the substrate whereas only a small fraction is in a closed-unliganded conformation [5, 11, 17]. Thus, basically a standard crystallization approach is used to obtain crystals suitable for structure determination. This is reflected by the large number of structures solved in the unliganded-open conformation (see [2] for a recent summary of the available SBP structures). The open conformation basically gives an overall picture of the protein structures and in the case of SBPs the bilobal fold of the protein can be observed. In this conformation the binding site of the substrate is laid open and a detailed picture on how the substrate is bound cannot be deduced.
Most of the times the open conformation crystallizes differently from the ligand bound state. This is reflected in the different crystallization conditions as well as in changes of the crystal parameters (unit cell and/or spacegroup). One example is given below for the glycine betaine binding protein ProX.
2.2. Crystallization of the substrate bound closed conformation (state II)
The vast majority of substrate binding proteins have been crystallized in the closed-ligand bound conformation (for a detailed list see [2]). This is mainly due to the fact that the substrate bound protein adopts a stable conformation and possesses a drastically reduced intrinsic flexibility. In principle there are four methods to include the substrate into the crystallization trials: 1) co-crystallization 2) ligand soaking 3) micro or macro seeding 4) endogenously bound ligands.
The first method is co-crystallization. Here, normally the substrate is added prior to crystallization to the protein solution. As listed in Table 1 this is the method used the most in SBP crystallization trials. Knowledge about the affinity of the ligand is important, since the bilobal SPBs exist in equilibrium between the open and closed state in solution and the addition of substrate directs this equilibrium towards the latter. Exemplary, 11 SBPs are listed in Table 1 where the affinity of the corresponding ligand(s) as well as the concentration used in the crystallization trials is highlighted. In principle the concentrations used are 10-1000 times above the Kd.
Solved structures of selected SBPs. Listed are the proteins, the host organism, the substrate, whether the structure was solved in the unliganded-open and/or liganded-closed state, the highest resolution, the biochemically determined affinity, the used substrate concentration during crystallization and the method used: 1) co-crystallization 2) soaking 3) seeding 4) endogenously bound substrates.
2.2.1. Co-crystallization to obtain the ligand bound structure
The method of co-crystallization ensures the presence of only the substrate bound conformation of the SBP in solution. One major advantage of co-crystallization is the possibility to add different ligands into the crystallization trial. A prominent SBP member where several crystal structures were solved is the maltose binding protein (MBP). This protein binds a maltose molecule and delivers it to its cognate ABC transporter, which imports maltose into the cell for nutrient purposes. Substrate ranges from maltose, maltotriose, beta-cyclodextrin and many other sugar derivatives. All these structures were solved by using the addition of the substrates to the protein. Another example is the ectoine binding EhuB protein of S. meliloti [27]. Here, the structure was solved with both ligands, ectoine and hydroxyectoine, which yielded two high-resolution structures. The different binding modes of the substrates could be detected and the difference in affinity explained. The latter example was only crystallized in the closed-liganded state and no crystals could be obtained when the crystallization solution was depleted of substrate. This highlights the flexibility of the SBPs and the presence of multiple conformations of the SBPs in solution and in presence of the ligand. In many cases the ligand-closed conformation was crystallized under conditions, which differ greatly from the unliganded-open conformation also indicating the flexibility in the protein.
2.2.2. Ligand soaking to obtain the ligand bound state
The second method, which can be used to obtain a ligand bound protein structure, is ligand soaking. Soaking crystals with ligands is often the method of choice to obtain crystals of protein-ligand complexes owing to the ease of the method. However, there are several factors to consider. The crystals may be fragile and soaking in a stabilization buffer or cross-linking may be required. The soaking time and inhibitor concentration need to be optimized, as many protein crystals are sensitive to the solvents used to dissolve the ligands. Although for other proteins ligand soaking is successfully applied, for SBPs this method is not very commonly used as reflected by the low number of structures solved using this method. This is likely due to the fact that upon substrate binding the two domains undergo a relative large conformational change. Since crystal contacts are fragile and are disrupted easily, large conformational changes induced by soaking can damage crystal contacts resulting either in a massive drop in the resolution of the diffraction or the crystals crack/dissolve completely.
2.2.3. Seeding – A method to obtain the ligand bound state with unusual substrates
In some cases the ligand used for crystallization cannot be crystallized in a closed conformation. This occurs for example when the ligand is not stable during the time of crystallization. One such example is acetylcholine. During crystallization of the choline binding protein ChoX from S. meliloti, it became evident that besides the natural ligand choline also actylcholine is bound by this SBP [8]. To understand the binding properties of ChoX, a structure determination of ChoX in complex with acetylcholine was undertaken. For this purpose the protein was subjected to co-crystallization experiments. Acetylcholine presents a chemical compound, which is easily susceptible to hydrolysis especially at non-neutral pH values. Although the crystallization of ChoX was done at low pH values, a co-crystallization with intact acetylcholine was achieved. However, subsequent structural determination showed that the substrate was hydrolyzed to choline in the setup during the time of crystal growth. To overcome this limitation, a micro\n\t\t\t\t\t\tseeding strategy was devised. The application of micro seeding helped to crystallize ChoX complexed with acetylcholine within 24 hours. Structural analysis revealed that acetylcholine was not hydrolyzed in the drop during this short period of time required for crystal growth. Thereby, it was possible to solve the structure of ChoX in complex with acetylcholine. The quality of the crystals was good, resulting in diffraction up to 1.8 Å [28]. However, one drawback encountered, when crystals of ChoX were obtained by seeding, was that they all showed a high twinning fraction (up to 50 %). This effect is possibly due to the rapid growth process where crystals reach their final size within a day allowing the formation of merohedral twins, a phenomenon one has to take into account when using the streak seeding method.
2.2.4. Endogenously bound ligands
During purification of some proteins with high affinity for their substrate often the ligand is co-purified. Here, OppA from L. lactis is an excellent example. OppA belongs to peptide binding subgroup of the family of SBPs and is involved in nutrient uptake in prokaryotes and binds peptides of lengths from 4 to at least 35 residues and with no obvious specificity for a certain peptide sequence. These peptides bind so tightly that they remain associated with the protein throughout purification. The crystallization of the closed-ligand state therefor is relatively easy since the protein will stay only in the closed-liganded conformation. This results in a liganded bound structure. To obtain more different states of the protein one has to remove the ligand first, and afterwards add the wanted substrate. In the case of OppA the peptide was removed prior to crystallization and incubated either with a different ligand or no ligand to obtain a ligand free structure. In the case of OppA, the endogenous peptides can be removed from the protein only by partly unfolding using guanidium chloride, which generates ligand-free OppA. This removal of endogenous peptides was required to allow the binding of defined peptides which was used for crystallization. By this tour de force Bertnsson et al. were able to solve several structures with different ligands bound as well as a ligand free structure, explaining the substrate binding specificity of this protein in great detail [22].
2.3. Crystallization of the closed-unliganded state (state III)
The intermediate states of SBPs have been crystallized as well, although only a couple of structures have been reported. This energetically unfavorable state has been crystallized not on purpose in most cases. The choline binding protein ChoX from S. melioti has been crystallized in the absence of a ligand via micro seeding to gain structural insights into the open, ligand-free form of this binding protein. These attempts were not successful. Instead, the obtained crystals revealed a closed but ligand-free form of the ChoX protein. Nevertheless many structures are known of substrate binding proteins in either their unliganded-open or liganded-closed states [15].
2.4. Crystallization of a semi-open or semi-closed state (state IV)
During our efforts to solve the crystal structure of the choline-binding protein ChoX from S. meliloti we used the technique of micro seeding [15] to obtain ChoX crystals in the ligand-free form. To our surprise, a ligand-free structure, which was different from those that were expected for the ligand-free closed and/or open forms of SBPs described so far, was obtained. Here, ChoX was present in a ligand-free form whose overall fold was identical to the closed-unliganded structure. This structure however, represented a more open state of the substrate binding protein, which had not been observed before. From the crystal parameters such as the dimensions of the unit cell is was already obvious that the conformation of the protein had changed, since one axis of the unit cell appeared to be significantly larger (35 Å) when compared to the unliganded-closed crystal form of ChoX.
The structure revealed that the domain closure upon substrate binding does not occur in one step. Rather, a small subdomain in one of the two lobes is laid open and closes only after the substrate is bound. This observation was in line with data observed for the maltose importer system MalFEGK. Here it was observed that the ATPase activity of the ABC transporter was not stimulated by the maltose substrate binding protein when it was added in the unliganded-closed conformation. This is likely due to the fact that the subdomain is not fully closed and rotated outward, which does not activate the transporter. Thus, this biochemical phenomenon could only be explained by the semi-open/semi-closed structure of ChoX [14].
2.5. State I-IV: What do they tell about conformational changes
Substrate binding proteins are flexible proteins, which consist of two domains, which constantly fluctuate between several states of which the open and fully closed state are the most populated ones. Both domains together build up a deep cleft, which harbors the substrate-binding site. As described above the structural work on these proteins has been successful and in the next part a general outcome will be given of what these different states actually tell us about function and mode of action of this protein family.
The unliganded substrate binding proteins are thought to fluctuate between the open and closed state. The angle of opening varies between 26° up to 70° as observed in several open-unliganded structures, suggesting that the extent of opening is likely influenced by crystal packing. This has been observed very nicely for the ribose binding protein of which three different crystal structures have been described. Here the opening of the two domains varies between 43° and 63°. This suggests that the opening can be described as a pure hinge motion. The variation of the degree of opening has been elucidated by NMR in solution for the maltose binding protein MalF. Here 95 % of the protein adopts an open conformation fluctuating around one state with different degrees of opening.
2.5.1. Open and closed - An overall structure view
As an example for the closing movement observed when comparing the open-unliganded and closed-liganded structure the glycine betaine (GB) binding protein ProX from A. fulgidus is highlighted in more detail. ProX has been crystallized in different conformations: a liganded-closed conformation in complex either with GB or PB (proline betaine) as well as in an unliganded-open conformation [23]. From the crystallographic parameters it was already anticipated that crystals differ in the conformation of the protein. ProX crystals were grown using the vapor diffusion method. The authors attained four different crystal forms depending on the presence or absence of the ligand (hint 1). Liganded ProX crystallized in hanging drops using a reservoir solution containing 0.2 M ZnAc2, 0.1 M sodium cacodylate, pH 6.0-6.5, 10-12 % (w/v) PEG 4000 and they belonged to the space group P21 (crystal form I). In a different setup, liganded ProX crystallized in sitting drops equilibrated against a reservoir containing 30 % (w/v) PEG 1500 and belong to the space group P43212 (crystal form II). Unliganded ProX crystallized in hanging drops against a reservoir solution containing 0.3 M MgCl2, 0.1 M Tris, pH 7.0-9.0, 35 % (w/v) PEG 4000. The first crystals appeared after 2-3 months, and belong to space group C2 (crystal form III). Again using a different setup, unliganded ProX crystallized in hanging drops equilibrated against a reservoir containing 0.1 M ZnAc2, 0.1 M MES, pH 6.5, 25-30 % (v/v) ethylene glycol. These crystals grew within 4 weeks, reached a final size of 200 × 150 × 20 μm3, and belong to space group P212121 (crystal form IV) [23]. Thus, the different crystallization conditions as well as space group already suggested that several different conformations had been crystallized. Initial phases were obtained by two-wavelength anomalous dispersion of ProX-PB crystals of form IV. All other structures were determined by molecular replacement.
In Figure 2 the opening and closing of the glycine betaine binding protein ProX from A. fulgidus is highlighted. Here domain II was taken as an anchor point.
Figure 2.
Equilibrium between the open and closed states of substrate binding proteins (ProX from A. fulgidus). The unliganded structure (highlighted in green) of an SBP is fluctuating between the open and closed state (highlighted in orange). In the absence of substrate this equilibrium is pointing towards the open conformation. In the presence of the substrate this equilibrium is changed towards the closed conformation. Here the two domains are close together and side chains of both domains bury the substrate in a deep cleft in between them. (PDB entries: 1SW2, 1SW5). All Figures containing structures were prepared with pymol (“www.pymol.org”).
Figure 2 highlights the open conformation (green), which is in equilibrium with the closed state although only a small percentage will be present in the closed unliganded state. Upon the addition of glycine betaine a stable closed conformation is reached and the equilibrium is shifted towards this state. Besides the crystal structure of the substrate bound state with glycine betaine, proline betaine and betaine as a substrate also the open conformation was crystallized. This allowed a detailed analysis of the closing and opening motion mediated by the hinge region between both domains. The comparison of the ligand-free and liganded conformation of other binding proteins showed an approximate rigid body motion of the two domains highlighting a total rotation of domain II by ~ 58° with respect to domain I (Figure 2). The total rotation has two components: 1) the hinge angle between the two domains of ~ 40° with its axis going through the above-mentioned hinges in the polypeptide and 2) a rotation perpendicular to the hinge axis of ~ 42°. Although the domains behave more or less as rigid bodies, there are a few changes of the binary complex in two regions of ProX. If one succeeds in crystallizing several conformation of a protein one can search for and visualize small distinct changes in the overall structure. This has been also observed in ProX, the α-helical conformation (in the open form) of residues 144–148 (domain I) change either to an isolated-β-bridge or to a turn conformation (in the closed form). This conformational change may be caused by the proximity to Arg149, which plays an important role in ligand binding as discussed below. Furthermore, residues 222–225 (domain II), which are in turn and 310-helix conformation (in the open form), become rearranged to a short α-helix in the closed form. These structural changes highlight an important point in the function of such a protein (more detail below).
2.5.2. Open and closed - An active site view
A closer look at the binding site or the amino acids involved in substrate binding shows that small but distinct conformational changes of the amino acids involved in ligand binding occur upon substrate binding. Again as an example the glycine-betaine binding protein ProX from A. fulgidus is used.
The binding site is located in the cleft between domains I and II and can be subdivided into two parts, one binding the quaternary ammonium head group and the other binding the carboxylic tail of these compounds. The quaternary ammonium head group is captured in a box formed by Asp109 and the four tyrosine residues Tyr63, Tyr111, Tyr190, and Tyr214 being oriented almost perpendicular to each other. The tyrosine side chains provide a negative surface potential that is complementary to the cationic quaternary ammonium head group of GB. The carboxylic tail of GB is pointing outward of this partially negatively charged environment forming interactions with Lys13 (domain I), Arg149 (domain II), and Thr66 (domain I), respectively. Furthermore the structure was solved at a resolution sufficient to locate water molecules. An important water molecule was observed, which was held in place by residues Tyr111 and Glu145, and stabilizes domain closure. Here it is important to mention that this water molecule was not observed in the open unliganded structure and its importance would therefore be easily overlooked when no comparison between the two states were possible.
The superposition of the open-unliganded form and the closed-liganded form of ProX allowed an unambiguous identification of residues of domain II that are involved in ligand binding. They show virtually the same orientation in the open and closed forms (see Figure 3). Residues Tyr63, Tyr214, Lys13, and Thr66 superimpose very well. Only the main chain carbonyl of Asp109 from domain I is slightly out of place compared to the closed form because of the enormous main chain rearrangement between Asp110 and Tyr111 upon domain closure. The residues contributed by domain II behave quite differently. Tyr111 and Tyr190 are not only moved as parts of domain B but they undergo a major conformational change to adopt the conformation of the closed-liganded binding site. The side chain conformation of Arg149 shows only small changes between the open and closed conformations although it undergoes a large movement as part of domain II.
Figure 3.
The binding site of ProX is highlighted in the open (depicted in ball and stick in green-left picture) and the substrate bound closed conformation (depicted in ball and stick in orange middle picture). As observed some of the ligand binding amino acid change their conformation. The right picture shows an overlay of both structures to visualize these conformation changes (PDB entries: 1SW2, 1SW5 and 3MAM).
Recently, another structure of ProX was solved in the liganded but open conformation [29]. This conformation represents a state of which only very few structures are known. In other words, the protein has a ligand bound and is on its way to close up the binding site. This structure provided an even more detailed picture on the function of ProX and finally highlighted the crucial role of Arg149. In addition to the direct interaction with GB and residues that are part of the substrate-binding pocket (Tyr111, Thr66), Arg149 is a major determinant in domain-domain interactions in the closed structure. As such, Arg149 interacts with Val70 (domain I) and Asp151 (domain II), thereby acting as a linking element between the two domains enforcing stable domain closure. These interactions complement those mediated by Pro172 of domain II, where Pro172 interacts via its Cα-atom and a water molecule with Glu155 of domain II. Together, this provides a further explanation for the crucial role of Arg149 for the stability of the liganded-closed state, which has been observed in mutagenis studies. Here, the binding affinity of GB was dramatically lowered when Arg149 was mutated to alanine, a phenomenon that could not be explained since the aromatic cage which dominates the binding affinity was still present to bind glycine betaine. This suggested that Arg149 is the final amino acid to interact with the substrate and, thereby, terminate the molecular motions that result in the high affinity closed state of ProX. Besides this crucial role of switching from a low affinity to a high affinity state via the interaction of Arg149 the open liganded structure also shed light on the movement that the amino acids undergo during closure of the protein. In the open-liganded structure the presence of glycine betaine is communicated to Arg149 through interactions of the side chains of Tyr190, Tyr111, and Phe146 via a side-chain network [29]. Interestingly when comparing the open and closed structures of other SBPs, the maltose binding protein (MBP) [9] and the ribose binding protein (RBP) from E. coli and the N-Acetyl-5-neuraminic acid binding protein (SIAP) from H. influenza [25] a similar network can be identified in these proteins, something which had not been identified before due to the lack of an open-liganded structure.
In summary, the “Venus fly trap” model describes the opening and closing of SBPs. Here the equilibrium between these two conformations is shifted towards the closed state upon substrate binding. Many crystal structures of SBPs have been solved in the unliganded-open, liganded-closed, and, more rarely, in the liganded-open or unliganded-closed state [3, 14, 15, 23]. The crystal structure of one of these states will give information on the overall structure of the protein as well as the ligand binding site. Several SBPs have been crystallized in two or more states and quite clearly the increasing amount of states will shed a more detailed look on how domain closure is occurring. Thus, although crystallization is trial and error and sometimes tedious, it is worth to search for crystals in the liganded-closed conformation as well as crystals of another state since every stage will visualize the cleverness of nature to use conformational changes for the formation of ligand binding sites.
3. Protein with multiple ligands – How to crystallize the different ligand bound intermediate states
Besides proteins that bind one substrate, a large number of enzymes are binding two or more substrates and convert these into a product. Here, the crystallization of the apo-enzyme (protein without any ligand bound) often reveals the binding site of these ligands. However, the exact influence of the binding of these ligands can only be deduced from several structures, where different ligands are bound or one structure with all ligands bound. The different states are called apo-enzyme, when the enzyme is depleted of all ligands, the binary complex when the first substrate is bound, the ternary complex when the second ligand is bound as well. A quaternary complex would describe the protein with three ligands bound.
Figure 4.
Overview of the conformations a protein can adopt with multiple ligands. A) The apo-enzyme B) binary complex where the first ligand is bound. This ligand with the highest affinity induces a stable conformation of the enzyme which allows the binding of the second ligand (ternary complex CI or CII). D) Enzyme complex where all ligands are bound.
Most of these proteins are enzymes. In reactions mediated by enzymes, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates sufficient for life. Since enzymes are selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes synthesized in a cell determines which metabolic pathways are utilized. Obtaining a snapshot of the substrate bound enzyme is difficult, because the enzymatic reaction will proceed immediately after substrate binding. One “trick” mostly used to solve this problem is to inhibit the reaction by either the reaction condition, meaning by varying pH of the buffer to a value where the reaction is not occurring. Another approach often appied in crystallography is to use a mutant, which cannot catalyze the reaction anymore; however it is still capable of binding the substrate. This has been proven to be successful in many cases. For example the catalytic cycle of nucleotide binding domains has been unraveled by such a mutation. In the latter case the ATP hydrolysis, in the wild type the measure for activity, has been abolished by mutation of a crucial amino acid, which still allowed binding of ATP but prevented hydrolysis. Thereby the dimeric state of the protein was stabilized and the active form of the NBD (nucleotide binding domain) could be crystallized in the presence of ATP [30-32].
Below the structural studies of the octopine dehydrogenases (OcDH) from P. maximus will be described in more detail. This enzyme catalyses the reductive condensation of L-arginine with pyruvate forming octopine under the simultaneous oxidation of NADH (reduced form of nicotinamide adenine dinucleotide). This oxidation of NADH is the terminal step in the anaerobiosis, meaning the generation of ATP when organisms are suffering from low oxygen levels. A prominent member of these terminal pyruvate oxidoreductases is the lactate dehydrogenase, which catalyzes the transfer of a hydride ion from NADH to pyruvate, with produces NAD+ (nicotinamide adenine dinucleotide) and lactate. Thereby the redox state in vertebrates is maintained during functional anaerobiosis. OcDH fulfills the same function in the invertebrate P. maximus.
This enzyme has been chosen due to the fact that three substrates need to be bound simultaneously for the reaction, in contrast to the lactate dehydrogenase, which has only two subtrates, NADH and pyruvate. Furthermore this enzyme was crystallized as wildtype protein and in all substrate bound states (binary and ternary complex CI and CII) and the corresponding structures were elucidated. The state where all substrates were present did not yield a structure due to the immediate conversion to the product. However, the other structure allowed a detailed view on how the latter state might look like.
In 2007 Mueller and co-workers achieved cloning and heterologously expression of this enzyme using E. coli as expression system [33]. After the purification the enzyme was characterized and the authors proposed a sequential binding mode of the substrates. Here, NADH was bound first followed by either L-arginine or pyruvate. The order of the last two was not revealed by the enzymatic analysis. Furthermore, a catalytic triad was proposed consisting of three highly conserved amino acid, building up a protein rely-system for the reduction of NADH. This triad has been observed in the sequence and structure of the lactate dehydrogenase as well. Sequence analysis of different proteins from this family revealed that the protein contained two distinct domains where domain I contained the characteristic Rossmann-fold, a domain responsible for the binding of NADH. Domain II was assigned as octopine dehydrogenase domain, which is specific for this protein family and was suggested to contain the binding site for both L-arginine and pyruvate. Both domains are connected via a linker region of 5-8 amino acids suggesting that these domains might undergo large conformational changes.
3.1. The crystallization of apo-enzyme and the binary complex
Parallel to the biochemical characterization, the crystallization of the enzyme was started. Due to the two-domain structure OcDH can adopt multiple conformations in solution, which prevents crystal formation. However, purified OcDH-His5 yielded small crystals that appeared to be multiple on optical examinations (Figure 5 A). They diffracted to a resolution of 2.6 Å. However the diffraction showed multiple lattices in one diffraction image and could not be used for structure determination (Figure 5 A) [34]. All attempts to improve these crystals using for example seeding, temperature ramping or various crystallization conditions failed. Finally, the primary ligand, NADH, was added prior to crystallization. This produced crystals under conditions similar to those in the absence of NADH. Here, the incubation temperature appeared to be critical and needs to be kept at 285 K. The crystals obtained were single and diffracted to 2.1 Å resolution, which allowed processing of the data and subsequent structure determination (Figure 5 B). The structure of OcDH was solved as binary complex with NADH [34, 35].
Cofactors like NADH are often observed to be co-purified. This was assumed to be the case for OcDH as well, however, no activity was ever observed without NADH, but in the presence of the other two substrates. This implies that OcDH is not homogenous and multiple conformations exist as observed in the multiple crystal lattices of the diffraction image. This is in line with the only other available three-dimensional structure of an enzyme of the OcDH superfamily, the apo-form of N-(1-D-carboxylethyl)-L-norvaline dehydrogenase (CENDH) from Arthrobacter sp. strain 1C [36]. CENDH catalyzes the NADH-dependent reductive condensation of hydrophobic L-amino acids such as L-methionine, L-isoleucine, L-valine, L-phenylalanine or L-leucine with α-keto acids such as pyruvate, glyoxylate, α-ketobutyrate or oxaloacetate with (D, L) specificity [37]. The structure of the binary complex of CENDH with NAD+ was determined to a resolution of 2.6 Å. Although NAD+ was added in the crystallization trials the cofactor could not be observed unambiguously in the electron density. This was likely due to the concentration of NAD+, which was below the Kd. As a result not all proteins had the substrate bound, which led to a not very well defnied electron density. Only the nicotinamide ribose moiety was of moderate quality and the density of the nicotinamide ring was very weak. This has been attributed to low NAD+\n\t\t\t\t\toccupancy in this crystal, hence the co-factor has been omitted from the high resolution refinement [36].
This highlights the importance to verify the affinity of substrate prior to crystallization. Since NAD+ is the product of the reaction and to ensure the release of the product, the affinity of NAD+ must be lower than the affinity of NADH. In a recent study on the OcDH the affinities have been determined to be 18 µM for NADH and 200 µM for NAD+ [38]. As described above the addition of substrate in crystallization trials need to be at least a 10-fold above the Kd. For OcDH 0.8 mM NADH was used for the crystallization of the binary complex, which represents a 40-fold excess.
The structure of the OcDH-NADH binary complex revealed why the initial crystallization attempt of the apo-enzyme failed. NADH is bound by the Rossmann-fold located in domain I as well as by an arginine residue in domain II. Thereby the OcDH captured in a state which enables the binding of the other substrates, pyruvate and L-arginine (see below) [34, 35].
Figure 5.
Crystallization of OcDH in the absence and presence of NADH. A) Absence of NADH. The crystals obtained are multiple (upper panel) and the diffraction pattern yielded showed several lattices (middle panel). The structure of OcDH shows two distinct domains connected by a flexible linker, which can rotate freely in the absence of NADH (lower panel). B) Crystals obtained in the presence of NADH (upper panel). The diffraction showed a single lattice diffracting up to 2.1 Å (middle panel). The structure revealed the binding site of NADH and an interaction of an arginine residue from domain II with NADH, which locks OcDH in one stable conformation (lower panel) (PDB entries: 3C7A and 3C7D).
In summary, the apo-state of multiple ligand binding enzymes is difficult to crystallize when the enzyme undergoes large conformational changes. In the case of the OcDH only the binary complex in the presence of NADH could be crystallized. Here crystals were of sufficient quality to determine the structure.
3.2. The crystallization of the ternary complexes CI and CII
OcDH catalyzes the condensation of L-arginine with pyruvate to form octopine under the oxidation of NADH. Biochemical analysis as well as the crystal structure revealed that NADH is the first substrate to bind to OcDH. The structure of this binary complex exhibited a stable conformation of the protein in solution with an Arg-sensor, which binds NADH, and thereby stabilizes the protein in one conformation (see above).
So the next step was to determine the structure of the OcDH in the presence of the second and third substrate, L-arginine and pyruvate, respectively. Initially, the protein and the substrate were mixed and an extensive search for suitable crystallization conditions was started. However, no crystals were obtained for OcDH in the presence of L-arginine and/or pyruvate. Instead only needles were grown which were multiple and very fragile similar to the crystals obtained for the apo-enzyme. This is in line with the biochemical data, which highlights the order of substate binding which show that NADH has to be bound prior to binding of L-arginine as well as pyruvate [38, 39]. Here the authors used two other techniques, NMR and ITC (isothermal titration calorimetry) repectively, to show that L-arginine only binds after saturation of the apo-enzyme with NADH. Pyruvate was shown to be bound only after L-arginine binding to the enzyme. This suggests that OcDH undergoes a conformational change when NADH is bound and thereby the binding site of L-arginine is formed. Furthermore the binding site for pyruvate is only created when L-arginine is bound.
Since crystallization was not successful the next step was to use co-crystallization with the OcDH protein and L-arginine and/or pyruvate to obtain structural information of the ternary complex (CI and CII). This yielded crystals of OcDH only in the presence of NADH and no additional density was observed for neither L-arginine nor pyruvate. So, soaking the ligand into preformed OcDH-NADH crystals was the last method chosen. Crystallization trials were carried out using the hanging-drop vapor diffusion method and crystals of OcDH were grown in the presence of 0.8 mM NADH. L-arginine-bound crystals were obtained by soaking NADH-bound OcDH crystals in 100 mM MES pH 7.0, 1.15 M Na-citrate, 0.8 mM NADH containing 10 mM L-arginine for at least 24 hours. Pyruvate-bound crystals were obtained also by soaking the crystals in 100 mM MES pH 7.0, 1.15 M Na-citrate, 0.8 mM NADH and 10 mM pyruvate for at least 8 hours. Both concentrations were chosen relatively high but they resemble the in vivo concentration as well as were backed up by the affinity observed for both substrates in biochemical and biophysical studies, being 5.5 mM L-arginine and 3.5 mM pyruvate, respectively. During soaking a cracking of the crystals was observed after the first minutes. However, the crystals recovered completely from this cracking within the following hours and showed no fissures or other damages after that soaking procedure. Desprite this, the diffraction analysis revealed a loss in diffraction. Initally the crystals diffracted to 2.1 Å. After soaking in L-arginine or pyruvate the diffraction potential was reduced to 3.0 Å and 2.6 Å, respectively. The phenomenon of crystal cracking and decline of the diffraction already was a good indication that the substrates diffused into the crystal. A dataset was collected from crystals where either one of the ligands was soaked in and besides the decrease in diffraction potential also the unit cell parameters changed (see Table 2).
\n\t\t
\n\t\t
\n\t\t
\n\t\t\t
\n\t\t\t\tCrystal Complex\n\t\t\t
\n\t\t\t
\n\t\t\t\tUnit cell parameters ( a,b,c in Å)\n\t\t\t
\n\t\t
\n\t\t
\n\t\t\t
OcDH-NADH
\n\t\t\t
99.8, 99.8, 126.5
\n\t\t
\n\t\t
\n\t\t\t
OcDH-NADH/L-arginine
\n\t\t\t
95.9, 95.9, 117.9
\n\t\t
\n\t\t
\n\t\t\t
OcDH-NADH/pyruvate
\n\t\t\t
95.0, 95.0, 120.2
\n\t\t
\n\t
Table 2.
Crystallographic parameters of the unit cell of the binary OcDH-NADH complex and after soaking of the ternary complex CI: OcDH-NADH/L-arginine and CII: OcDH- NADH/pyruvate
The change in unit cell parameters suggested that a conformational change occurred during the soaking with the ligand. This was further observed after the structure was resolved and electron density was clearly defined for L-arginine in the one and for pyruvate in the other dataset. The structure of OcDH-NADH/L-arginine showed a rotational movement of domain II towards the NADH binding domain I, and a stronger interaction of the Arginine residue with NADH. A domain closure was also observed in the pyruvate bound structure. So stable binding of NADH to the Rossman fold of domain I, the first step in the reaction sequence of OcDH, occurs without participation of domain II. A comparison of the OcDH-NADH (colored light-purple in Figure 6) and the OcDH-NADH/L-arginine complexes revealed a 42° rotation of domain II towards the NADH binding domain (domain I) in the latter complex. This domain closure is triggered by the interaction of Arg324 (domain II) with the pyrophosphate moiety of NADH bound to the Rossman fold in domain I.
A comparison of the two ternary complexes suggests that both, pyruvate and L-arginine, are capable to trigger domain closure to a similar extent. However, in the OcDH-NADH/pyruvate complex, pyruvate partially blocks the entrance for L-arginine, while in the OcDH-NADH/L-arginine complex, the accessibility of the pyruvate binding site is not restricted by L-arginine [34, 35]. From these structures it could be deduced that L-arginine binds to the OcDH-NADH complex in a consecutive step and induces a rotational movement of domain II towards domain I. This semi-closed active center, which is further stabilized using the pyrophosphate moiety of the bound NADH and by interactions of L-arginine with residues from both domains is then poised to accept pyruvate and consequently the product octopine can be formed. With regard to the structures it was proposed that instead of a random binding process, an ordered sequence of substrate binding in the line of NADH, L-arginine and pyruvate will occur.
This ordered sequence of substrate binding was then biochemically proven by ITC studies where the binding affinities of the substrates were measured. Here, the binding of L-arginine was only observed when NADH was bound primarily and the binding of pyruvate only when the complex was preloaded with L-arginine [38, 39]. Furthermore this ordered binding mechanism explains why no lactate is found in side P. maximus which is normally formed when NADH and pyruvate is bound by lactate dehydrogenases. Here, it is worth mentioning that the conformational changes induced by ligand soaking into the crystal were also observed in NMR studies that were perfomed in solution. So the apparent conformational changes in the crystal resemble the changes the protein undergoes in solution.
Figure 6.
Overlay of the OcDH-NADH binary complex with the OcDH-NADH/L-arginine ternary complex CI. As seen in the superposition the binding of L-arginine induces a conformational change. Domain II is rotated towards domain I which is thereby creating the pyruvate binding site. In the overlay the pyruvate structure is not shown due to clarity (PDB entries: 3C7A and 3C7D).
The crystal structures of the different states of OcDH, delivered snapshots elucidating for the first time the precise and very distinct binding order [35]. Unfortunally the crystals with the endproduct octopine did not diffract X-ray with a resolution and quality high enough for structure determination. The same hold true for a complex with all three substrates present at once. This is likely due to the fact that the immediate condensation occured and the product was formed.
To show how proteins can be crystallized with their enzymatic endproducts we chose another enzyme family as example and will describe the different procedures during the next paragraphs.
4. Enzymatic products in protein structures – How to crystallize this rather unfavored states
The state found to be important within an enzyme reaction cycle is supposedly the product bound state. After the reaction occurs the product is still sitting within the protein and will be released. Often these product have a low(er) affinity to the protein than the substrates and are therefor less often found to be successfully crystallized.
Examples of prosperous structure determination however are the shikimate dehydrogenase (SDH or AroE) of Thermus thermophilus (TthSDH), Aquifex aeolicus (AaeSDH) and the recently deposited structures of the SDH of Helicobacter pylori (HpySDH) as well as the bifunctional dehydroquinase-shikimate dehydrogenase (AthDHQ-SDH) from Arabidopis thaliana which were crystallized with its reaction product shikimic acid ([40-43]. Similar to that the closely related quinate dehydrogenase (QDH) of Corynebacterium glutamicum (CglQDH) was structurally characterized in four different states: as apo-enzyme and at atomic resolution with bound cofactor NAD+ as well as in complex with quinic acid (QA) and the reduced cofactor or shikimic acid (SA) and NADH [44].
Figure 7.
Schematic diagram of the conformational changes within a protein (blue ellipses) during the catalyzed reaction. 1.) Before a substrate (red trapezium) is bound the proteins exhibits an open conformation. 2.) – 4.) Binding of the substrate induces a slight domain closure before the cofactor (green hexagon) is bound. 5.) + 6.) In order to facilitate the conversion from substrate to the product (orange rhombus) both protein domains need to be in close contact. 7.) -9.) A stepwise domain opening allows the changed cofactor (light green pentagon) to leave the protein domain, followed by the product. The protein itself is not modified at all during the whole reaction and mostly all steps are reversible.
Shikimate-/quinate dehydrogenases belong to the superfamily of NAD(P)-dependent (nicotinamide adenine dinucleotide phosphate) oxidoreductases whereas SDHs catalyse the reversible reduction of 3-dehydroshikimate to shikimate under oxidation of NAD(P)H (reduced form of nicotinamide adenine dinucleotide phosphate) and QDHs the oxidation of quinate to 5-dehydroquinate with reduction of NAD(P), respectively. The overall fold consists of a N-terminal or substrate binding domain and a C-terminal or cofactor-binding domain and is highly conserved within that subfamily (schematically shown in Figure 7). Compared to other proteins, like the above-mentioned SBPs, the structural changes occurring during catalysis are less prominent and comprise a movement of the two domains against each other in a range of several Ångstrom.
4.1. Shikimate dehydrogenase from Aquifex aeolicus
Crystals of the native (apo-) AaeSDH were obtained with non-His-tagged protein, whereas the ternary complex crystals were obtained with His-tagged SDH. To get these complexes the protein solution was mixed with substrate and cofactor (i. e. with both natural products) to final concentrations of 5.0 mM shikimic acid and 5.0 mM NADP+ before crystallization. The hanging-drop vapor diffusion method was used for crystallization trials. The drops were prepared by mixing 3 µl of the protein-ligand solution with 1 µl of well solution [41].
KM values were determined to be 42.4 µM for both ligands, which means that there was a 100-fold excess in the crystallization drop. The bound products SA and NADP+ in the protein could be explained by the low activity of the enzyme and the equilibrium constant favoring the formation of SA and NADP+, both of which are caused by the low pH. The equilibrium constant ([SA][NADP+]/[DHSA][NADPH]) was determined by Yaniv and Gilvarg (1955) to be 27.7 at pH 7 and 5.7 at pH 7.8 [45]. As of any dehydrogenase reaction, the equilibrium position of the AaeSDH-catalysed reaction depends on the hydrogen ion concentration of the environment. The pH of the well solution (0.2 M ammonium acetate, 30 % w/v PEG 4000, 0.1 M sodium acetate) was 4.6 and therefore the drop became more acidic during crystallization. They estimated the equilibrium constant at pH 5 to be around 3000 in favor of the formation of SA and NADP+. The geometry of NADP+ is not distinguishable from that of the NADPH at this resolution (2.2 Å) but the geometry of SA containing a tetrahedral (sp3) C3 atom is distinct from that of DHSA, in which the geometry of C3 is planar (sp2) [41].
There were eight (apo) and four (ternary complex) crystallographically independent AaeSDH molecules in the asymmetric unit of apo-AaeSDH or AaeSDH-NADP+-SA, respectively. According to the structure of the apo-protein and the ternary complex a fully open (molecule F in apo-AaeSDH) and a closed conformation with bound ligands (molecule D in AaeSDH-NADP+-SA; Figure 8) were observed as well as several intermediate states. From the fully open to the closed form there is a movement of three loops in the catalytic domain towards the NADP-binding domain by around 5 Å sealing the active site of the enzyme. SA and NADP+ are brought in close contact in that cavity: the C3-O3 bond of SA is parallel to the C4-C5 bond of the nicotine amide ring of NADP+, and the distance between the two bonds is 3.5 Å. This represents a typical distance for a hydride transfer.
The open conformation therefor represents the protein structure in state 9.) (or 1.), respectively) in Figure 7, the closed conformation correlates to state 6.) in that scheme.
Figure 8.
Shikimate dehydrogenase from Aquifex aeolicus. The cartoon depicted in cyan represents the open (apo) conformation of the enzyme (PDB entry: 2HK8), the structure coloured in black illustrates the closed conformation (PDB entry: 2hk9) with the bound ligands shikimic acid and NADP+, shown as sticks.
4.2. Shikimate dehydrogenase from Thermus thermophiles
In case of TthSDH, crystals of the native protein were grown in microbatch plates. Co-crystallization trials were only successful with added NADP+ but failed with shikimate. To obtain complexes with bound shikimate crystals of the apo-protein or the SDH-NADP+ complex were soaked for several seconds in cryosolution supplemented with shikimate. The final concentration of all added ligands was 5 mM. Although the kinetical parameters were not determined prior to crystallization, all KM values of closely related SDHs are in a µM range so that there was at least a 20-fold excess of substrate and cofactor [40].
Evaluation of the complex structures revealed an open and a closed conformation of the two domains but neither the binding of shikimate nor NADP+ seem to induce that conformational change. Shikimate could bind to the closed as well as to the open form, whereas NADP+ was found only in closed conformation. As described for AaeSDH, the crystallization condition was in an acidic range of about pH 4.6, which explains that the reaction did not occur. An alignment of the three structures (apo-SDH, SDH-SA, SDH-SA-NADP+) of T. thermophilus illustrates the domain closure while/after SA and/or NADP+ binding (Figure 9). Surprisingly there seems to be no further movement of the substrate binding domain against the NADP(H) binding domain when the cofactor is bound. Thus, the apo-structure represents state 9.) (or 1.) in Figure 7 and both the binary and the ternary complex may match a state between 6.) and 7.) of that scheme.
Figure 9.
Shikimate dehydrogenase from T. thermophilus. The cartoons depicted in green (left and right side) represent the open (apo) conformation of the enzyme (PDB entry: 1WXD), the structure coloured in black illustrates the closed form with bound shikimic acid (PDB entry: 2D5C), whereas the red one corresponds to the ternary complex (PDB entry: 2EV9) with shikimic acid and NADP+, shown as sticks.
4.3. Bifunctional dehydroquinase-shikimate dehydrogenase (AthDHQ-SDH) from Arabidopis thaliana
Remarkable are the co-crystallization trials of Singh and Christendat with the bifunctional enzyme dehydroquinase-shikimate dehydrogenase from Arabidopsis thaliana (AthDHQ-SDH). First crystals were obtained with the product shikimate at the SDH site and tartrate as a substrate analogue at the DHQ site. Later they could crystallize AthDHQ-SDH with its natural products shikimate and NADP+.
For the shikimate-tartrate complex crystals they used the vapor diffusion\n\t\t\t\t\thanging-drop technique. Protein solution with a final concentration of 1 mM of shikimate was mixed with the reservoir solution containing 0.4 M potassium sodium tartrate tetrahydrate [42]. To obtain ligand bound crystals of the three different protein conditions were tested: protein only, protein with 1 mM shikimate or protein with 1 mM NADP+. The protein-shikimate approach was the only one that yielded crystals (under the same conditions as mentioned above). To gain crystals of the ternary complex a further treatment was necessary: The above-mentioned crystals were soaked with a NADP+ solution (final concentration 10 mM) for about 8 hours at pH 5.8. The KM values were determined to be 0.6 mM for shikimic acid and 0.13 mM for NADP+, respectively [42].
Not only that the closed conformation of the enzyme after binding of both products could be demonstrated (Figure 10) but also the activity of that ternary complex was proven as the oxidation of shikimate was evidenced by the generation of dehydroshikimate, – the product of the DHQ moiety – found in the DHQ site [43].
Figure 10.
Bifunctional dehydroquinase-shikimate dehydrogenase (AthDHQ-SDH) from Arabidopis thaliana. The cartoon coloured in grey reveals the binary complex (PDB entry: 2GPT) with the bound product shikimate (grey lines), the structure depicted in red shows the protein with bound substrate dehydroshikimate (red lines; PDB entry: 2O7Q), whereas the cartoon in green represents the ternary complex (PDB entry: 2O7S) with bound dehydroshikimate and the cofactor NADP(H).
The structures of the AthDHQ-SDH binary complexes with bound product shikimate or substrate dehydroshikimate illustrate therefore the states 8.) or 2.), while the ternary complex corresponds to the transition state 5.) in Figure 7.
4.4. Shikimate dehydrogenase from Helicobacter pylori
Recently three different catalytic states of the HpySDH were deposited in the PDB. Unfortunately the results are not published so that detailed information about the crystallization trials are lacking. Apparently they obtained all crystals by means of the hanging-drop vapor diffusion method.
However, the structure is ideally suited to visualize structural changes during cofactor binding Figure 11.
Figure 11.
Binary (left; PDB entry: 3PHI) and ternary structure (right; PDB entry: 3PHH) of the shikimate dehydrogenase from Helicobacter pylori. The substrate dehydroshikimate and the cofactor NADP(H) are presented as sticks. The red circle indicates the loop region in the N-terminal domain which acts as a lid during cofactor binding.
In the binary structure of the HpySDH with bound shikimate there is a large loop in the C-terminal domain that obstructs the entrance to the cofactor-binding cleft and virtually acts as a lid. For cofactor binding this loop has to move away from the cleft in order to create space for NADP(H). Comparing these two structures with the overall conformation of the apo-protein, these two conformational stages represent stages 2.)/3.) or 6./7.), in the catalytic cycle shown in Figure 7.
4.5. Quinate dehydrogenase from Corynebacterium glutamicum
Last but not least the bacterial quinate dehydrogenase of C. glutamicum could be structurally solved in four different catalytic states: apo-enzyme, with bound cofactor NAD+ and in complex with quinate (QA) and the reduced cofactor or shikimate (SA) and NADH, i. e. with the natural substrate and the natural cofactor as product of the reaction.
For growing the crystals of the apo-form the protein solution was mixed with the reservoir solution and a NADH solution (2 µg/ml) in a drop ratio 1:1:1. The reduced cofactor could not be detected in the electron density due to the very low concentration [46].
For the co-crystallization trials (with the cofactor NAD+, the substrate quinate (QA) and the reduced cofactor or shikimate (SA) and NADH) the kinetical parameters were determined first in order to get an idea of the concentrations necessary for successful ligand binding. The KM values for NAD+, QA and SA are 0.28 mM, 2.37 mM and 53,88 mM, respectively (Hoeppner et al.; publication in progress).
To obtain the binary and both of the ternary complexes the protein solution was mixed with either NAD+ or QA plus NADH or SA plus NADH to a final concentrations of 1 mM for NAD+ or NADH and 35 mM for QA or SA. These mixtures were incubated on ice for about 1 hour prior to crystallization. All substrates and the cofactor were bound during co-crystallization experiments by means of the sitting drop method with drop size of 2-4 µl in 1:1 ratio of protein and reservoir solution.
Figure 12.
Comparison of the crystal shapes of the four different catalytical states of the CglQDH and the corresponding crystallization conditions.
Crystals of the binary and ternary complexes were different in shape compared to the crystals of the apo-enzyme and grew under diffenrent conditions (Figure 12), which was a hint to (structural) changes within the protein molecules.
The crystals of all three CglQDH complexes diffracted to atomic resolution and allowed us to assign the position of all ligand atoms unambiguously within the electron density (Figure 13).
Figure 13.
Representative sections of electron density maps of the CglQDH complexes at 1.0 Å (CglQDH-NAD+) or 1.16 Å (CglQDH-QA-NADH and CglQDH-SA-NADH) resolution. A) electron density defining protein side chains, B) density around the nicotinamide ring of the cofactor NAD(H), C) bound substrate quinate, D) bound substrate shikimate (electron density maps in A)-C) contoured at 1 σ and in D) 0.7 σ).
By comparing the overall structures of all these states an open, a semi-open and a closed conformation of the enzyme (Figure 14) was observed. Surprisingly, the apo-structure of the CglQDH exhibits the closed form although one would intuitively expect the open conformation. But it is possible that these findings were a crystallization artifact since the reservoir solution was quite acidic (pH 4.6) compared to the CglQDH pH optimum, which is 9.0-9.5 for quinate and 10.0-10.5 for shikimate (Hoeppner et al.; publication in progress).
Within the cofactor binding domain of CglQDH the glycine rich loop, which is highly conserved within SDH proteins and represents a classical Rossmann fold, is flapped down towards the cofactor binding cleft in the apo-structure, but moved away when the cofactor is bound. With regard to the overall arrangement of the apo-state compared to the NAD+-bound state there is a clearly visible opening of the two domains. After forming the ternary complex the two domains are brought closer to each other, if only more slightly compared to the apo-conformation and thus adopt a semi-closed conformation (Hoeppner et al.; publication in progress).
Figure 14.
Structural alignments of the binary structure of CglQDH with bound NAD+ (black; PDB entry: 3JYO) and A) the apo-protein (green; PDB entry: 2NLO) or B) the ternary structure with bound quinic acid and NADH (red; PDB entry: 3JYP). The substrate and the cofactor NAD(H) are presented as sticks. The red arrow indicates the conformational changes within the glycine rich loop.
4.6. Insights into the structural changes during catalysis and elucidation of substrate and cofactor specificity, using the example of CglQDH
4.6.1. Structure overview of C. glutamicum QDH
All CglQDH structures presented here are determined from crystals that were nearly isomorphous and belong to the same space group C2. The unit-cell parameters are very similar with one monomer per asymmetric unit.
The 282 residues in the QDH molecule form two structural domains (Figure 15): the N-terminal or catalytic domain (residues 1 to 113 and 256 to 283), which binds the substrate molecule, and the C-terminal or nucleotide binding domain (residues 114 to 255). The catalytic domain forms an open α/β sandwich, which is characteristic for enzymes of the S/QDH family but different from all other known proteins. The domain consists of a six-stranded, mainly parallel β sheet (strand order β2, β1, β3, β5, β6 and β4, where β5 is antiparallel. This β sheet is flanked by helices α1 and α11 at one side and α2, one 310-helix and α4 at the other. The C-terminal domain contains a six-stranded parallel β sheet (strand order β9, β8, β7, β10, β11 and β12) sandwiched by three helices (α7, α6, α5) on one face and by helices α8, α10 and a 310-helix on the other. The nucleotide binding domain exhibits a glycine rich loop with the sequence motive GXGGXG. The overall fold of this functional domain is very similar to that observed for other SDH proteins [47, 48] and represents the classical Rossmann fold. Both domains are linked together by helices α5 and α11. The arrangement of these two domains creates a deep active site groove in which cofactor and substrate are located.
Figure 15.
Schematical overview of the CglQDH fold
4.6.2. Description and analysis of QDH active site
Cofactor Binding Site: The electron densities for NAD(H) were of high quality and allowed us to assign the position of these ligand unambiguously at 1.0 Å. CglQDH crystallizes in the presence of NAD+ in the same space group with similar unit cell dimensions, but under different crystallization conditions compared to the apo-enzyme. With regard to the overall structure we found that the catalytic domain moves away from the nucleotide-binding domain after cofactor binding making the interdomain cavity larger. Concerning the steric configuration of the residues there are only little but fundamental variances, especially in the glycine rich loop. In comparison to the QDH apo-enzyme (PDB entry 2NLO) the residues of the loop (Gly136-Val138) move out of the cavity after cofactor binding and therefore clear space for the NAD(H) molecule (Figure 14). Cofactor binding occurs in an extended groove between the N-terminal and C-terminal domain, whereas most of the molecular interactions result from the C-terminal domain. The adenine part of the adenosine moiety form hydrogen bonds only to some water molecules, while the ribose is bound by the side chains of Asp158 and Arg163. The phosphate moiety contacts the glycine rich loop and forms hydrogen bonds to Arg163 and the backbone nitrogen atom of Val138. The following ribose moiety again interacts only with water molecules, whereas the nicotinamide moiety is cramped by the backbone nitrogen of Ala255 and backbone oxygens of Val228 and Gly251, respectively. Gly251 and Ala255 are the only residues of the N-terminal domain involved in cofactor binding (Figure 16). The nucleotide-binding motive GXGGXG comprises the residues Gly134-Ala135-Gly136-Gly137-Val138-Gly139 (Hoeppner et al.; publication in progress).
Figure 16.
Interactions between the cofactor NAD(H) and CglQDH. Residues involved in hydrogen bonds (dotted lines) and bound ligand are shown as sticks, water molecules are depicted as red stars.
The strict specificity for NAD(H) is determined by the negatively charged aspartate residue 158, the neutral Leu159 and the bulk side chain of Arg163, which would result in steric hindrance with the additional phosphate group in the NADP(H) molecule.
Substrate Binding Site: We examined the substrate binding site of CglQDH by analysis of the two different ternary complexes QDH-QA-NADH and QDH-SA-NADH. The substrate binding site is located in the N-terminal domain, close to the nicotinamide ring of the cofactor, and is characterized by a number of highly conserved residues.
After quinate binding a slight closure of the N- and C-terminal domain of CglQDH so that the crevice becomes closer by about 0.5 Å was observed. The substrate quinate is anchored by numerous key interactions with these residues: the carbonyl group of quinate is bound by the hydroxyl groups of Ser17 and Thr19; the hydroxyl groups of the C1 and C3 atom of the substrate form hydrogen bonds to side chain of Thr69, whereas the nitrogen atom of Lys73 binds to the hydroxyl groups of C3 and C4, the latter furthermore interacts with the side chains of Asn94 and Asp110; the fourth hydroxyl group at C5 forms hydrogen bonds to the amide group of Asp110 and the oxygen atom of the Gln258 side chain, respectively. A total of eleven hydrogen bonds cause a forcipate anchorage of the substrate molecule (Figure 17 B).
Figure 17.
Active site residues of CglQDH. A) Apo-CglQDH (PDB entry: 2NLO) with bound glycerol (purple), B) ternary complex (PDB entry: 3JYP) with bound quinate (orange), C) ternary complex (PDB entry: 3JYQ) with shikimate (wheat). Residues involved in hydrogen bonds (dotted lines) and bound ligand are shown as sticks, water molecules are depicted as red stars.
In comparison to the apo-enzyme QDH (Figure 17 A) it is noteworthy that the side chain of Lys73 exhibits a sprawled conformation after quinate binding, which is required for interaction with the C3 and C4 hydroxyl groups of the substrate. For the hydride ion transfer from C3 of quinate to C4 of NAD+ a particular distance between these atoms is very important. In the crystal structure the nicotinamide ring is located in a suitable orientation for the H- transfer. After quinate binding and resulting closure of the domains the cofactor approaches to the substrate-binding site, whereby the distance of interest amounts to 4.27 Å.
In the case of shikimate binding a somewhat different situation was observed. In principle the above mentioned residues except Thr19 are involved in shikimate binding (Figure 17 C), but only eight polar interactions are achieved (compared to eleven when QA is bound), from which some are furthermore weaker pronounced: Thr19 is not involved in polar contacts to SA, Thr69 has contact only to the hydrogen group of C3, Asn94 is about 0.2 Å farer apart from the hydrogen atom of C4 and has no contact to the OH-group of C5. Remarkable is the appearance of an alternative side chain conformation of Lys73, as evidenced by the excellent electron density in this region. The first conformation of the Lys73 side chain in the crystal exhibits the sprawled conformation as found for the quinate binding; the second conformation reveals an angled rotamer as it occurs in apo-CglQDH. The latter conformation makes hydrophobic interactions with the shikimate molecule impossible (Hoeppner et al.; publication in progress). Furthermore the shikimate molecule exists in a half-chair conformation, whereas the quinate molecule adopts a chair conformation. Hence the distance of the C4 atom of the cofactor and the C3 atom of the substrate increases to 4.67 Å. All residues involved in cofactor and substrate binding identified here are consistent with these of further reported structures (i. e. TthSDH, AaeSDH, AthDHQ-SDH).
4.6.3. Substrate and cofactor specificity and discrimination
All results of the structural analysis are also in excellent agreement with the findings of the kinetical assays. The higher affinity of CglQDH to the substrate quinate (as obvious by means of the clearly unequal KM values) arises from the major quantity of hydrogen bonds between protein and the substrate quinate. Considering the known structures of shikimate dehydrogenases as SDH from T. thermophilus [40] SDH from A. aeolicus [41] or the SDH domain of A. thaliana [42, 43] they all possess at least eleven hydrogen bonds to the substrate molecule shikimate, comparable to the quinate binding in CglQDH. In contrast there are only eight polar interactions present between the enzyme and the shikimate molecule, because shikimate offers a somewhat different conformation (half-chair instead of chair) and exhibits no hydroxyl group at the C1 atom. Furthermore the formed hydrogen bonds between shikimate and the enzyme are accomplished weaker. The higher catalytic efficiency of CglQDH regarding to quinate (as obvious on the basis of significantly higher kcat/KM values) possibly results from the slightly lower distance between the C4 atom of the cofactor NAD(H) and the C3 atom of the quinate molecule (4.27 Å versus 4.67 Å) and the improved orientation of the substrate quinate.
A further occasion for the lower affinity and catalytic efficiency regarding shikimate results from the appearance of an alternative conformation of the Lys73 side chain (Figure 17 C), which leads to a loss of an important hydrogen bond. At last we compared the substrate binding residues of CglQDH with those of AaeQDH, AthDHQ-SDH and TthSDH, which convert shikimate. We detected two differences possibly jointly responsible for quinate binding: in all the above-mentioned structures a tyrosine residue is involved in shikimate binding but not in CglQDH (Tyr230). Furthermore the second serine, which forms a hydrogen bond to the carbonyl group of shikimate, is replaced by a threonine residue in CglQDH (Thr19). Since the carbonyl group of shikimate bound in CglQDH is twisted about 90° compared to the situation of the aforesaid enzymes, Thr19 cannot take part in polar interaction with the substrate shikimate. Concerning the usage of the cofactors NAD(H) and NADP(H) the classical dinucleotid fold were identified in the past. Characteristic for all nucleotide binding proteins is the glycine rich loop with the common sequence GXGXXG, in which the number of glycine residues changes [49]. Enzymes using NAD or FAD possess a well conserved negatively charged amino acid at the C-terminus of the second β-strand of the nucleotide binding βαβ unit, mostly aspartate or glutamate. This residue interacts with the 2´-hydroxyl group of the ribose. In the majority of the NADP binding proteins this negatively charged residue is absent since the additional 2´-phosphate group is located at this position. Moreover, various NADP dependent proteins exhibit a charged amino acid (like arginine) in the position of the 2´-phosphate group which stabilizes the cofactor molecule. The CglQDH described here offers a negative charged residue (Asp158) forming a hydrogen bond to 2´-hydroxyl group of the ribose, followed by a neutral amino acid (Leu159) which is unable to interact with an additional phosphate group as present in NADP. Furthermore the bulk side chain of Arg163 constrict the cofactor binding site, which would result in steric hindrance with the additional phosphate group in a NADP(H) molecule. Due to all results of the kinetical and structural analysis we conclude that CglQDH is strictly NAD(H) specific and not able to bound NADP(H).
5. Conclusions
Crystal structures of proteins and enzymes are important to fully understand the mechanism and mode of action. Although the crystallization of a large number of proteins was successful and delivered valuable information the goal must be to fully understand the function. When crystallizing a protein a snapshot of the protein in a certain conformation is observed in the electron density. It is known that proteins are flexible and can obtain several states in solution.
Within that book chapter we explained the importance to acquire structural information of different catalytical states of proteins or enzymes, to fully understand how the protein behaves during catalysis or how the substrate bound state differs from the apo-enzyme.
The open and closed structures of the substrate binding protein ProX as apo-protein or with different substrates bound revealed enormous conformational changes during ligand binding and clearly visualzes how flexible a protein can be and elucidates the side chain movements within the substrate site upon ligand binding.
All described crystallization trials of the different transition states of the OcDH showed impressively that protein crystallization is a trial and error approach and that knowledge of the protein (especially the kinetical parameters beside others) is the essential thing to be successful. At best and as recompenses for ones effort one will achieve important insights that clearly explains the catalytic mechanism.
Last but not least the different structural information of the enzymes of the shikimate dehydrogenase family could bring to light how substrate and cofactor specificity and discrimination can be achieved throught detailed analysis of apo-, binary and ternary structure information about involved amino acids in substrate and cofactor binding.
So with these three examples the difficulties in crystallization on one hand and on the other hand the beauty of looking at proteins at work is shown.
PDB entries used
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\n\t\t\t\tProtein\n\t\t\t
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\n\t\t\t\tPDB Code\n\t\t\t
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\n\t\t\t\tTitle\n\t\t\t
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ChoX
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2RF1
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Crystal structure of ChoX in an unliganded closed conformation
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3HCQ
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Structural analysis of the choline binding protein ChoX in a semi-closed and ligand-free conformation
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2REJ
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ABC-transporter choline binding protein in unliganded semi-closed conformation
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2RIN
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ABC-transporter choline binding protein in complex with acetylcholine
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2REG
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ABC-transporter choline binding protein in complex with choline
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ProX
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1SW1
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Crystal structure of ProX from Archeoglobus fulgidus in complex with proline betaine
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1SW4
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Crystal structure of ProX from Archeoglobus fulgidus in complex with trimethyl ammonium
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1SW2
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Crystal structure of ProX from Archeoglobus fulgidus in complex with glycine betaine
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3MAM
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A molecular switch changes the low to the high affinity state in the substrate binding protein AfProX
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1SW5
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Crystal structure of ProX from Archeoglobus fulgidus in the ligand free form
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CenDH
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1BG6
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Crystal structure of the N-(1-D-carboxylethyl)-L-norvaline dehydrogenase from Arthrobacter sp. strain 1C
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OcDH
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3C7C
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A structural basis for substrate and stereo selectivity in octopine dehydrogenase (OcDH-NADH-L-arginine)
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3C7D
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A structural basis for substrate and stereo selectivity in octopine dehydrogenase (OcDH-NADH-pyruvate)
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3C7A
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A structural basis for substrate and stereo selectivity in octopine dehydrogenase (OcDH-NADH)
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AroE
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2HK8
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Crystal structure of shikimate dehydrogenase from Aquifex aeolicus\n\t\t\t
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2HK9
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Crystal structure of shikimate dehydrogenase from Aquifex aeolicus in complex with shikimate and NADP+\n\t\t\t
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1WXD
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Crystal structure of shikimate 5-dehydrogenase (AroE) from Thermus thermophilus HB8
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2D5C
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Crystal structure of shikimate 5-dehydrogenase (AroE) from Thermus thermophilus HB8 in complex with shikimate
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2EV9
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Crystal structure of shikimate 5-dehydrogenase (AroE) from Thermus thermophilus HB8 in complex with NADP(H) and shikimate
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3PHG
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Crystal structure of the shikimate 5-dehydrogenase (AroE) from Helicobacter pylori\n\t\t\t
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3PHH
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Crystal structure of the shikimate 5-dehydrogenase (AroE) from Helicobacter pylori in complex with shikimate and NADP(H)
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3PHI
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Crystal structure of thesShikimate 5-dehydrogenase (AroE) from Helicobacter pylori in complex with dehydroshikimate
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DHQ-SDH
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2GPT
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Crystal structure of Arabidopsis dehydroquinate dehydratase-shikimate dehydrogenase in complex with tartrate and shikimate
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2O7Q
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Crystal structure of the A. thaliana DHQ-dehydroshikimate-SDH in complex with dehydroshikimate
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2O7S
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Crystal structure of the A. thaliana DHQ-dehydroshikimate-SDH in complex with dehydroshikimate, NADP(H) and tartrate
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QDH
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3JYO
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Quinate dehydrogenase from Corynebacterium glutamicum in complex with NAD
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3JYP
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Quinate dehydrogenase from Corynebacterium glutamicum in complex with quinate and NADH
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3JYQ
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Quinate dehydrogenase from Corynebacterium glutamicum in complex with shikimate and NADH
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2NLO
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Crystal structure of the quinate dehydrogenase from Corynebacterium glutamicum\n\t\t\t
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Glossary
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\n\t\t\t\tApo-protein/apo-enzyme\n\t\t\t
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Enzymes that require a cofactor but do not have one bound are called apo-enzymes or apo-proteins. An apo-enzyme together with its cofactor(s) is called a holoenzyme.\n\t\t\t
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\n\t\t\t\tAffinity\n\t\t\t
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The dissociation constant is commonly used to describe the affinity between a ligand and a protein, i.e. how tightly a ligand binds to a particular protein. Ligand-protein affinities are influenced by non-covalent intermolecular interactions between the two molecules such as hydrogen bonding, electrostatic interactions, hydrophobic and Van der Waals forces. They can also be affected by high concentrations of other macromolecules, which causes macromolecular crowding. The smaller the dissociation constant K\n\t\t\t\td, the more tightly bound the ligand is, or the higher the affinity between ligand and protein.
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\n\t\t\t\tBinary complex\n\t\t\t
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A binary complex refers to a protein complex containing two different molecules which are bound together. In structural biology, the term binary complex can be used to describe a crystal containing a protein with one small molecule bound, for example the cofactor or the substrate; or a complex formed between two proteins.
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\n\t\t\t\tCo-crystallization\n\t\t\t
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Co-crystallization means that the protein solution is mixed with one or more ligand prior to the crystallization. Often the protein-ligand mixture is preincubated before setting up the crystallization drops.
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\n\t\t\t\tCofactor\n\t\t\t
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A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein\'s biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations. Cofactors are either organic or inorganic. They can also be classified depending on how tightly they bind to an enzyme, with loosely bound cofactors termed coenzymes and tightly-bound cofactors termed prosthetic groups. Examples of widespread cofactors are ATP, coenzyme A, FAD, and NAD+, vitamins or metal ions.
In chemistry, biochemistry, and pharmacology, a dissociation constant K\n\t\t\t\td is a specific type of equilibrium constant that measures the propensity of a larger object to separate (dissociate) reversibly into smaller components, as when a complex falls apart into its component molecules, or when a salt splits up into its component ions.
In biochemistry, Michaelis-Menten kinetics is one of the simplest and best-known models of enzyme kinetics. The Michaelis constant K\n\t\t\t\tM is the substrate concentration at which the reaction rate is half of V\n\t\t\t\tmax (which represents the maximum rate achieved by the system, at maximum (saturating) substrate concentrations).
Binding of ligands to proteins frequently causes changes to their three-dimensional structure. Exampes of this include the binding of substrates, inhibitors, cofactors or allosteric modulators to enzymes or of hormons to receptors. If this structural change has an effect on the environment of an intrinsic or extrinsic fluorophore in the protein, this can result in measurable changes in the fluorescence spectrum. Provided that the fluorophore has a unique location in the protein, such changes of fluorescence at a particular wavelength can be used to determine the dissociation constant (K\n\t\t\t\td) of the protein for the ligand where K\n\t\t\t\td is a measure of the affinity of the protein for the ligand [50].
This technique is useful for protein concentrations in the range of mg/ml. A typical experiment involves measurement of heat change as a function of addition of small quantities of a reagent to the calorimeter cell containing other components of the system under investigation. For example, this reagent could be a protein ligand or substrate/ inhibitor of an enzyme. At the beginning of the experiment, there is a large excess of protein compared to ligand. This means that Δ H values associated with each aliquot can be individually measured. Initially, these values are large but, as aliquots are progressively added, eventually decrease to values similar to the Δ H of dilution of ligand into the solution in the calorimeter cell. The Δ H measured is the total enthalpy change which includes heat associated with processes such as formation of noncovalent bonds between interacting molecules and with other equilibria in the system such as conformational changes, ionization of polar groups (e.g. deprotonation) and changes due to interactions with solvent. ITC provides a useful method for studying binding processes such as those involving a protein and a ligand. It allows estimation of both the binding constant (K\n\t\t\t\tb) and of the dissociation constant (K\n\t\t\t\td) [50].
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\n\t\t\t\tLigand\n\t\t\t
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In biochemistry a ligand is a substance (usually a small molecule), that forms a complex with a biomolecule to serve a biological purpose. In the context of this chapter ligand is used as a more general expression for substrate, product or cofactor.
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\n\t\t\t\tLigand soaking\n\t\t\t
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Ligand soaking means the addition of ligands into the mother liquid with preformed crystals. The idea is that the ligand diffuses into the crystals and binds at the active site. This technique was initially used for the incorporation of heavy atoms into protein crystals for phasing purposes.
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\n\t\t\t\tMacro and Micro Seeding\n\t\t\t
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During Macro Seeding the protein crystal is replaced into a freshly made mother liquid which allows the further enlargement of the crystals size. In Micro Seeding a suspension of microcrystals is prepared by either resuspending or crushing a protein crystal cluster or single crystals. These seeds are then used (often streaked through a new droplet of precipitant and fresh protein) to serve a crystallization starting point.
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\n\t\t\t\tMicrobatch\n\t\t\t
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Microbatch is a method in which the molecule to be crystallized is mixed with the crystallizing agents at the start of the experiment. The concentration of the ingredients is such that supersaturation is achieved immediately upon mixing, thus the composition and the volume of a trial remain constant and crystals will only form if the precise conditions have been correctly chosen.
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\n\t\t\t\tOccupancy\n\t\t\t
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Occupancy means the degree of protein molecules in solution or in a crystal with bound ligand. If every second protein has attached a ligand the occupancy is 50 %.
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\n\t\t\t\tSubstrate\n\t\t\t
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In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate(s). In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or more products, which are then released from the active site. The active site is now free to accept another substrate molecule. In the case of more than one substrate, these may bind in a particular order to the active site, before reacting together to produce products.
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\n\t\t\t\tSurface Plasmon Resonance (SPR)\n\t\t\t
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SPR is an optical technique which depends on changes in refractive index or mass changes near metal surfaces. When two surfaces, one a metal and the other a dielectric material are exposed to a beam of plane-polarized light of wavelength, λ, a longitudinal charge density wave (a surface plasmon) is propagated along the interface between them. This only happens when one of the surfaces is a metal and works best with silver, gold, copper and aluminium. This is because metals contain free oscillating electrons called plasmons. When light traveling through an optically dense medium such as glass arrives at an interface with a lower optical density (e.g. liquid), it is reflected back into the more optically dense medium, a phenomenon called total internal reflectance. Any process altering n\n\t\t\t\ts (the refractive index of the dielectric medium) can be sensitively detected by SPR so the technique has found applications in the study of kinetics and thermodynamics of binding processes (e.g. protein-ligand, protein-protein) [50].
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\n\t\t\t\tTernary complex\n\t\t\t
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A ternary complex refers to a protein complex containing three different molecules which are bound together. In structural biology ternary complex can be used to describe a crystal containing a protein with two small molecules bound, for example cofactor and substrate; or a complex formed between two proteins and a single substrate.
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\n\t\t\t\tVapor diffusion Hanging or Sitting drop\n\t\t\t
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Two of the most commonly used methods for protein crystallization fall under the category of vapor diffusion. These are known as the hanging drop and sitting drop methods. Both entail a droplet containing purified protein, buffer, and precipitant being allowed to equilibrate with a larger reservoir containing similar buffers and precipitants in higher concentrations. Initially, the droplet of protein solution contains an insufficient concentration of precipitant for crystallization, but as water vaporizes from the drop and transfers to the reservoir, the precipitant concentration increases to a level optimal for crystallization. Since the system is in equilibrium, these optimum conditions are maintained until the crystallization is complete [51].
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\n',keywords:null,chapterPDFUrl:"https://cdn.intechopen.com/pdfs/43121.pdf",chapterXML:"https://mts.intechopen.com/source/xml/43121.xml",downloadPdfUrl:"/chapter/pdf-download/43121",previewPdfUrl:"/chapter/pdf-preview/43121",totalDownloads:2712,totalViews:858,totalCrossrefCites:1,totalDimensionsCites:5,hasAltmetrics:0,dateSubmitted:"May 8th 2012",dateReviewed:"October 4th 2012",datePrePublished:null,datePublished:"February 20th 2013",dateFinished:null,readingETA:"0",abstract:null,reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/43121",risUrl:"/chapter/ris/43121",book:{slug:"advanced-topics-on-crystal-growth"},signatures:"Astrid Hoeppner, Lutz Schmitt and Sander H.J. Smits",authors:[{id:"83819",title:"Dr.",name:"Sander H.J.",middleName:null,surname:"Smits",fullName:"Sander H.J. Smits",slug:"sander-h.j.-smits",email:"sander.smits@hhu.de",position:null,institution:{name:"Heinrich Heine University Düsseldorf",institutionURL:null,country:{name:"Germany"}}},{id:"159750",title:"Dr.",name:"Astrid",middleName:null,surname:"Hoeppner",fullName:"Astrid Hoeppner",slug:"astrid-hoeppner",email:"astrid.hoeppner@hhu.de",position:null,institution:null},{id:"159751",title:"Prof.",name:"Lutz",middleName:null,surname:"Schmitt",fullName:"Lutz Schmitt",slug:"lutz-schmitt",email:"lutz.schmitt@hhu.de",position:null,institution:null}],sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_1_2",title:"1.1. General approaches to obtain crystals with bound ligands and how to prepare the ground ",level:"2"},{id:"sec_3",title:"2. Binding protein with one ligand – How to crystallize and what can be deduced from the structure",level:"1"},{id:"sec_3_2",title:"2.1. Crystallization of the open-unliganded conformation (state I)",level:"2"},{id:"sec_4_2",title:"2.2. Crystallization of the substrate bound closed conformation (state II)",level:"2"},{id:"sec_4_3",title:"2.2.1. Co-crystallization to obtain the ligand bound structure",level:"3"},{id:"sec_5_3",title:"2.2.2. Ligand soaking to obtain the ligand bound state",level:"3"},{id:"sec_6_3",title:"2.2.3. Seeding – A method to obtain the ligand bound state with unusual substrates",level:"3"},{id:"sec_7_3",title:"2.2.4. Endogenously bound ligands",level:"3"},{id:"sec_9_2",title:"2.3. Crystallization of the closed-unliganded state (state III)",level:"2"},{id:"sec_10_2",title:"2.4. Crystallization of a semi-open or semi-closed state (state IV)",level:"2"},{id:"sec_11_2",title:"2.5. State I-IV: What do they tell about conformational changes",level:"2"},{id:"sec_11_3",title:"2.5.1. Open and closed - An overall structure view",level:"3"},{id:"sec_12_3",title:"2.5.2. Open and closed - An active site view",level:"3"},{id:"sec_15",title:"3. Protein with multiple ligands – How to crystallize the different ligand bound intermediate states",level:"1"},{id:"sec_15_2",title:"3.1. The crystallization of apo-enzyme and the binary complex",level:"2"},{id:"sec_16_2",title:"3.2. The crystallization of the ternary complexes CI and CII",level:"2"},{id:"sec_18",title:"4. Enzymatic products in protein structures – How to crystallize this rather unfavored states",level:"1"},{id:"sec_18_2",title:"4.1. Shikimate dehydrogenase from Aquifex aeolicus",level:"2"},{id:"sec_19_2",title:"4.2. Shikimate dehydrogenase from Thermus thermophiles",level:"2"},{id:"sec_20_2",title:"4.3. Bifunctional dehydroquinase-shikimate dehydrogenase (AthDHQ-SDH) from Arabidopis thaliana",level:"2"},{id:"sec_21_2",title:"4.4. Shikimate dehydrogenase from Helicobacter pylori",level:"2"},{id:"sec_22_2",title:"4.5. Quinate dehydrogenase from Corynebacterium glutamicum",level:"2"},{id:"sec_23_2",title:"4.6. Insights into the structural changes during catalysis and elucidation of substrate and cofactor specificity, using the example of CglQDH",level:"2"},{id:"sec_23_3",title:"4.6.1. Structure overview of C. glutamicum QDH",level:"3"},{id:"sec_24_3",title:"4.6.2. Description and analysis of QDH active site",level:"3"},{id:"sec_25_3",title:"4.6.3. Substrate and cofactor specificity and discrimination ",level:"3"},{id:"sec_28",title:"5. Conclusions",level:"1"},{id:"sec_29",title:"PDB entries used",level:"1"},{id:"sec_30",title:"Glossary",level:"1"}],chapterReferences:[{id:"B1",body:'Abts A, Schwarz CK, Tschapek B, Smits SH, Schmitt L. Rational and Irrational Approaches to Convince a Protein to Crystallize, Modern Aspects of Bulk Crystal and Thin Film Preparation, 2012 Nikolai Kolesnikov and Elena Borisenko (Ed.), ISBN: 978-953-307-610-2, InTech'},{id:"B2",body:'Berntsson RP, Smits SH, Schmitt L, Slotboom DJ, Poolman B. A structural classification of substrate-binding proteins. FEBS Lett. 2010 Jun 18;584(12):2606-17.'},{id:"B3",body:'Wilkinson J, Verschueren KHG. Crystal structures of periplasmic solute-binding proteins in ABC transport complexes illuminate their function. In: Holland IB, Cole SPC, Kuchler K, Higgins CF, editors. ABC proteins: from bacteria to man. London: Academic Press (Elsevier Science); 2003. p. 187-208.'},{id:"B4",body:'Quiocho FA, Ledvina PS. Atomic structure and specificity of bacterial periplasmic receptors for active transport and chemotaxis: variation of common themes. Mol Microbiol. 1996 Apr;20(1):17-25.'},{id:"B5",body:'Mao B, Pear MR, McCammon JA, Quiocho FA. Hinge-bending in L-arabinose-binding protein. The "Venus\'s-flytrap" model. J Biol Chem. 1982 Feb 10;257(3):1131-3.'},{id:"B6",body:'Sack JS, Saper MA, Quiocho FA. Periplasmic binding protein structure and function. J Mol Biol. 1989;206:171-91.'},{id:"B7",body:'Oh BH, Pandit J, Kang CH, Nikaido K, Gokcen S, Ames GF, et al. Three-dimensional structures of the periplasmic lysine/arginine/ornithine-binding protein with and without a ligand. J Biol Chem. 1993 May 25;268(15):11348-55.'},{id:"B8",body:'Loh AP, Pawley N, Nicholson LK, Oswald RE. An increase in side chain entropy facilitates effector binding: NMR characterization of the side chain methyl group dynamics in Cdc42Hs. Biochemistry. 2001;40(15):4590-600.'},{id:"B9",body:'Davidson AL, Dassa E, Orelle C, Chen J. Structure, function, and evolution of bacterial ATP-binding cassette systems. Microbiol Mol Biol Rev. 2008 Jun;72(2):317-64, table of contents.'},{id:"B10",body:'Shilton BH. The dynamics of the MBP-MalFGK(2) interaction: a prototype for binding protein dependent ABC-transporter systems. Biochim Biophys Acta. 2008 Sep;1778(9):1772-80.'},{id:"B11",body:'Tang C, Schwieters CD, Clore GM. Open-to-closed transition in apo maltose-binding protein observed by paramagnetic NMR. Nature. 2007 Oct 25;449(7165):1078-82.'},{id:"B12",body:'Sharff AJ, Rodseth LE, Quiocho FA. Refined 1.8-A structure reveals the mode of binding of beta-cyclodextrin to the maltodextrin binding protein. Biochemistry. 1993 Oct 12;32(40):10553-9.'},{id:"B13",body:'Spurlino JC, Lu GY, Quiocho FA. The 2.3-A resolution structure of the maltose- or maltodextrin-binding protein, a primary receptor of bacterial active transport and chemotaxis. J Biol Chem. 1991 Mar 15;266(8):5202-19.'},{id:"B14",body:'Oswald C, Smits SH, Hoing M, Bremer E, Schmitt L. Structural analysis of the choline-binding protein ChoX in a semi-closed and ligand-free conformation. Biol Chem. 2009 Nov;390(11):1163-70.'},{id:"B15",body:'Oswald C, Smits SH, Hoing M, Sohn-Bosser L, Dupont L, Le Rudulier D, et al. Crystal structures of the choline/acetylcholine substrate-binding protein ChoX from Sinorhizobium meliloti in the liganded and unliganded-closed states. J Biol Chem. 2008 Nov 21;283(47):32848-59.'},{id:"B16",body:'Pittelkow M, Tschapek B, Smits SH, Schmitt L, Bremer E. The Crystal Structure of the Substrate-Binding Protein OpuBC from Bacillus subtilis in Complex with Choline. J Mol Biol. 2011 Aug 5;411(1):53-67.'},{id:"B17",body:'Bermejo GA, Strub MP, Ho C, Tjandra N. Ligand-free open-closed transitions of periplasmic binding proteins: the case of glutamine-binding protein. Biochemistry. 2010 Mar 9;49(9):1893-902.'},{id:"B18",body:'Borths EL, Locher KP, Lee AT, Rees DC. The structure of Escherichia coli BtuF and binding to its cognate ATP binding cassette transporter. Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16642-7.'},{id:"B19",body:'Linke C, Caradoc-Davies TT, Young PG, Proft T, Baker EN. The laminin-binding protein Lbp from Streptococcus pyogenes is a zinc receptor. J Bacteriol. 2009 Sep;191(18):5814-23.'},{id:"B20",body:'Zou JY, Flocco MM, Mowbray SL. The 1.7 A refined X-ray structure of the periplasmic glucose/galactose receptor from Salmonella typhimurium. J Mol Biol. 1993 Oct 20;233(4):739-52.'},{id:"B21",body:'Quiocho FA, Spurlino JC, Rodseth LE. Extensive features of tight oligosaccharide binding revealed in high-resolution structures of the maltodextrin transport/chemosensory receptor. Structure. 1997 Aug 15;5(8):997-1015.'},{id:"B22",body:'Berntsson RP, Doeven MK, Fusetti F, Duurkens RH, Sengupta D, Marrink SJ, et al. The structural basis for peptide selection by the transport receptor OppA. EMBO J. 2009 May 6;28(9):1332-40.'},{id:"B23",body:'Schiefner A, Holtmann G, Diederichs K, Welte W, Bremer E. Structural basis for the binding of compatible solutes by ProX from the hyperthermophilic archaeon Archaeoglobus fulgidus. J Biol Chem. 2004 Nov 12;279(46):48270-81.'},{id:"B24",body:'Machius M, Brautigam CA, Tomchick DR, Ward P, Otwinowski Z, Blevins JS, et al. Structural and biochemical basis for polyamine binding to the Tp0655 lipoprotein of Treponema pallidum: putative role for Tp0655 (TpPotD) as a polyamine receptor. J Mol Biol. 2007 Oct 26;373(3):681-94.'},{id:"B25",body:'Muller A, Severi E, Mulligan C, Watts AG, Kelly DJ, Wilson KS, et al. Conservation of structure and mechanism in primary and secondary transporters exemplified by SiaP, a sialic acid binding virulence factor from Haemophilus influenzae. J Biol Chem. 2006 Aug 4;281(31):22212-22.'},{id:"B26",body:'Lecher J, Pittelkow M, Zobel S, Bursy J, Bonig T, Smits SH, et al. The crystal structure of UehA in complex with ectoine-A comparison with other TRAP-T binding proteins. J Mol Biol. 2009 May 29;389(1):58-73.'},{id:"B27",body:'Hanekop N, Hoing M, Sohn-Bosser L, Jebbar M, Schmitt L, Bremer E. Crystal structure of the ligand-binding protein EhuB from Sinorhizobium meliloti reveals substrate recognition of the compatible solutes ectoine and hydroxyectoine. J Mol Biol. 2007 Dec 14;374(5):1237-50.'},{id:"B28",body:'Oswald C, Smits SH, Bremer E, Schmitt L. Microseeding - a powerful tool for crystallizing proteins complexed with hydrolyzable substrates. Int J Mol Sci. 2008 Jun;9(7):1131-41.'},{id:"B29",body:'Tschapek B, Pittelkow M, Sohn-Bosser L, Holtmann G, Smits SH, Gohlke H, et al. Arg149 Is Involved in Switching the Low Affinity, Open State of the Binding Protein AfProX into Its High Affinity, Closed State. J Mol Biol. 2011 Aug 5;411(1):36-52.'},{id:"B30",body:'Zaitseva J, Oswald C, Jumpertz T, Jenewein S, Wiedenmann A, Holland IB, et al. A structural analysis of asymmetry required for catalytic activity of an ABC-ATPase domain dimer. EMBO J. 2006 Jul 26;25(14):3432-43.'},{id:"B31",body:'Karpowich NK, Huang HH, Smith PC, Hunt JF. Crystal structures of the BtuF periplasmic-binding protein for vitamin B12 suggest a functionally important reduction in protein mobility upon ligand binding. J Biol Chem. 2003 Mar 7;278(10):8429-34.'},{id:"B32",body:'Chen J, Lu G, Lin J, Davidson AL, Quiocho FA. A tweezers-like motion of the ATP-binding cassette dimer in an ABC transport cycle. Mol Cell. 2003 Sep;12(3):651-61.'},{id:"B33",body:'Muller A, Janssen F, Grieshaber MK. Putative reaction mechanism of heterologously expressed octopine dehydrogenase from the great scallop, Pecten maximus (L). Febs J. 2007 Dec 7;274(24):6329-39.'},{id:"B34",body:'Smits SH, Mueller A, Grieshaber MK, Schmitt L. Coenzyme- and His-tag-induced crystallization of octopine dehydrogenase. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Sep 1;64(Pt 9):836-9.'},{id:"B35",body:'Smits SH, Mueller A, Schmitt L, Grieshaber MK. A structural basis for substrate selectivity and stereoselectivity in octopine dehydrogenase from Pecten maximus. J Mol Biol. 2008 Aug 1;381(1):200-11.'},{id:"B36",body:'Britton KL, Asano Y, Rice DW. Crystal structure and active site location of N-(1-D-carboxylethyl)-L-norvaline dehydrogenase. Nat Struct Biol. 1998 Jul;5(7):593-601.'},{id:"B37",body:'Asano Y, Yamaguchi K, Kondo K. A new NAD+-dependent opine dehydrogenase from Arthrobacter sp. strain 1C. J Bacteriol. 1989 Aug;171(8):4466-71.'},{id:"B38",body:'van Os N, Smits SH, Schmitt L, Grieshaber MK. Control of D-octopine formation in scallop adductor muscle as revealed through thermodynamic studies of octopine dehydrogenase. J Exp Biol. 2012 May 1;215(Pt 9):1515-22.'},{id:"B39",body:'Smits SH, Meyer T, Mueller A, van Os N, Stoldt M, Willbold D, et al. Insights into the mechanism of ligand binding to octopine dehydrogenase from Pecten maximus by NMR and crystallography. PLoS One. 2010;5(8):e12312.'},{id:"B40",body:'Bagautdinov B, Kunishima N. Crystal structures of shikimate dehydrogenase AroE from Thermus thermophilus HB8 and its cofactor and substrate complexes: insights into the enzymatic mechanism. J Mol Biol. 2007 Oct 19;373(2):424-38.'},{id:"B41",body:'Gan J, Wu Y, Prabakaran P, Gu Y, Li Y, Andrykovitch M, et al. Structural and biochemical analyses of shikimate dehydrogenase AroE from Aquifex aeolicus: implications for the catalytic mechanism. Biochemistry. 2007 Aug 21;46(33):9513-22.'},{id:"B42",body:'Singh SA, Christendat D. Structure of Arabidopsis dehydroquinate dehydratase-shikimate dehydrogenase and implications for metabolic channeling in the shikimate pathway. Biochemistry. 2006 Jun 27;45(25):7787-96.'},{id:"B43",body:'Singh SA, Christendat D. The DHQ-dehydroshikimate-SDH-shikimate-NADP(H) Complex: Insights into Metabolite Transfer in the Shikimate Pathway. Cryst Growth Des. 2007;7(11):2153-60.'},{id:"B44",body:'Schoepe J, Niefind K, Chatterjee S, Schomburg D. Cloning, expression, purification and preliminary crystallographic characterization of a shikimate dehydrogenase from Corynebacterium glutamicum. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Jul 1;62(Pt 7):635-7.'},{id:"B45",body:'Yaniv H, Gilvarg C. Aromatic biosynthesis. XIV. 5-Dehydroshikimic reductase. J Biol Chem. 1955 Apr;213(2):787-95.'},{id:"B46",body:'Schoepe J, Niefind K, Schomburg D. 1.6 angstroms structure of an NAD+-dependent quinate dehydrogenase from Corynebacterium glutamicum. Acta Crystallogr D Biol Crystallogr. 2008 Jul;D64(Pt 7):803-9.'},{id:"B47",body:'Singh S, Korolev S, Koroleva O, Zarembinski T, Collart F, Joachimiak A, et al. Crystal structure of a novel shikimate dehydrogenase from Haemophilus influenzae. J Biol Chem. 2005 Apr 29;280(17):17101-8.'},{id:"B48",body:'Michel G, Roszak AW, Sauve V, Maclean J, Matte A, Coggins JR, et al. Structures of shikimate dehydrogenase AroE and its Paralog YdiB. A common structural framework for different activities. J Biol Chem. 2003 May 23;278(21):19463-72.'},{id:"B49",body:'Wierenga RK, De Maeyer MCH, Hol WGJ. Interaction of Pyrophosphate Moieties with a-Helixes in Dinucleotide Binding Proteins. Biochemistry. 1985;24:1346-57.'},{id:"B50",body:'Sheenan D. Physical Biochemistry. Principles and Applications: Wiley; 2009.'},{id:"B51",body:'McRee DE. Practical Protein Crystallography. San Diego: Academic Press; 1993.'}],footnotes:[],contributors:[{corresp:null,contributorFullName:"Astrid Hoeppner",address:null,affiliation:'
X-Ray Facility and Crystal Farm, Heinrich Heine University, Duesseldorf, Germany
Institute of Biochemistry, Heinrich Heine University, Duesseldorf, Germany
'}],corrections:null},book:{id:"3349",title:"Advanced Topics on Crystal Growth",subtitle:null,fullTitle:"Advanced Topics on Crystal Growth",slug:"advanced-topics-on-crystal-growth",publishedDate:"February 20th 2013",bookSignature:"Sukarno Olavo Ferreira",coverURL:"https://cdn.intechopen.com/books/images_new/3349.jpg",licenceType:"CC BY 3.0",editedByType:"Edited by",editors:[{id:"150520",title:"Dr.",name:"Sukarno",middleName:"Olavo",surname:"Ferreira",slug:"sukarno-ferreira",fullName:"Sukarno Ferreira"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},chapters:[{id:"43121",title:"Proteins and Their Ligands: Their Importance and How to Crystallize Them",slug:"proteins-and-their-ligands-their-importance-and-how-to-crystallize-them",totalDownloads:2712,totalCrossrefCites:1,signatures:"Astrid Hoeppner, Lutz Schmitt and Sander H.J. 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1. Introduction
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Systemic sclerosis is a chronic rheumatic autoimmune disease characterized by abnormal fibrotic processes, microvascular damage, and excessive deposition of collagen into connective tissues. The vascular alterations and immunological activation lead to progressive fibrosis of multiple organ systems including the skin, the gastrointestinal tract, kidneys, and the lungs. Disease-associated changes can have a negative impact on sexual functioning [1, 2]. Generally, sexuality in systemic sclerosis has been a neglected area so far, especially female sexual dysfunction. Impaired sexual functioning in women was probably less studied due to the complexity and multifactorial nature of female sexual response. A little bit more attention was paid to erectile dysfunction, where etiology is more pronounced even though women are affected by this disease more often [3, 4, 5]. The etiology of sexual dysfunctions in systemic sclerosis is not well known; the causes are multifactorial and are related to both the disease symptoms and the therapy. The most common physical problems associated with female sexual dysfunction include vaginal dryness, dyspareunia, vaginal tightness, Raynaud’s phenomenon, fatigue, generalized pain, muscle weakness, joint contractures, heartburn, and dyspnea. Presence of depression, fear, changes in face and body appearance, and lack of self-esteem are the psychological aspects, which can play a key role in the pathogenesis of sexual dysfunction in systemic sclerosis patients [6]. The etiology of erectile dysfunction is a little bit more understood. It is considered to be a result of microangiopathic changes. Due to corporal fibrosis and myointimal proliferation, the blood flow in the penile arteries is reduced.
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Several studies have suggested that sexual dysfunction is a widespread problem in both men and women with SSc. It is more prevalent than in general population and other chronic diseases [7]. The most common symptoms of female sexual dysfunction are vaginal tightness, dryness, and dyspareunia [7, 8]. More severe sexual dysfunction is usually associated with depression symptoms, aging, and functional impairment [2, 9, 10]. The prevalence of erectile dysfunction is about 80% [11, 12, 13]. In women, more than half of the SSc patients experience some sexual problems [7, 8]. The management of erectile dysfunction has been more studied compared to female sexual dysfunction treatment. However, the number of publications regarding the efficacy of erectile dysfunction treatment in SSc patients is still very limited and further research is needed. The treatment of female sexual dysfunction in SSc women has not been paid much attention so far. There are only general recommendations available.
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2. Definition and classification of sexual dysfunction
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In order to better understand why and how systemic sclerosis may affect sexual functioning, there is an overview of sexual response models, developed over the past few years, which led to the current diagnostic and classification criteria for sexual dysfunction. The first model of female sexual response was described by Masters and Johnson in 1966. They published that a normal female sexual response consists of four consecutive phases including desire, plateau phase, orgasm, and resolution. It was supposed that in both women and men, the sexual response is commenced by desire which is influenced by the activity of two brain centers—dopamine sensitive excitatory center and serotonin sensitive inhibitory center. These centers send a signal going through the descendent nervous system into the spinal cord from where the genital sexual reaction is triggered. The arousal phase is mediated by the parasympathetic nervous system, which leads to vascular and genital changes such as enlargement of the clitoris, dilatation of perivaginal arterioles, and lubrication and expansion of two-thirds of the vagina. The following level of excitement is referred to the plateau phase that lasts until the orgasm. The orgasm phase is accompanied by contractions of pelvic floor muscles, increased heart rate, respiratory rate, and blood pressure. After reaching orgasm, the body usually calms down and this phase is called the refractory or resolution phase (Figure 1) [14].
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Figure 1.
The four-phase model of female sexual response cycle; BP = blood pressure, HR = heart rate, and RR = respiratory rate. Modified according to [14].
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In 1979, this model was modified by Kaplan into a three-phase model, which consists of desire, arousal, and orgasm [15]. Based on this linear model, the diagnostic and classification system was developed. The World Health Organization International Classifications of Diseases-10 (ICD-10) and Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) by the American Psychiatric Association were established. The ICD-10 focused on how physical factors influenced sexual response, whereas DSM-IV classification emphasized more emotional and psychological aspects of female sexual dysfunction. Because both approaches followed the linear model of sexual response, which was later criticized for not taking into consideration the complexity of female sexual response, the new classification was needed [16].
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In 1998, the Sexual Function Health Council of the American Foundation for Urologic Disease (AFUD) convened an interdisciplinary congress, which was attended by 19 experts on female sexual dysfunction selected from five countries. The aim was to develop a consensual definition and classification based on the ICD-10 and DSM-IV. The new developed classification has been extended to include psychogenic and organic causes of desire, arousal, orgasm, and sexual pain disorders. An essential point of this classification is the personal distress criterion considering sexual complaint as a disorder only if it causes a subjective feeling of distress [17].
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Further research has shown that the model of female sexual response is still incomplete and that many aspects affecting women’s sexual function have been omitted. Studies reported that women often describe overlapping phases of sexual response in variable sequences. For instance, the unfounded assumption that desire always precedes arousal has been mistaken, and based on the women self-report and research data, it was proven that arousal and desire co-occur and reinforce each other. It was also found that motivation for sexual activity is much more complex than the mere presence of sexual desire defined as thinking or fantasizing about sex. Women in different surveys cited that increased desire for sexual activity may be caused by the emotional closeness of a partner or intimacy that increases female well-being and self-image, which may include the sense of feeling attractive, appreciated, loved, or desired. If enough appropriate sexual stimulation is provided, enough time and intimacy are available, the woman’s enjoyment and excitement can be intensified. The type of stimulation, time needed, and interpersonal context are highly individual. Moreover, spontaneous desire can be affected by the menstrual cycle, which usually decreases with age and grows with a new relationship. These new findings have surpassed the original hypothesis that women’s sexual response must always begin with sexual desire (thoughts and fantasies) and its absence is the result of a disorder. In addition, it was confirmed that, unlike men, there is no correlation between female subjective excitement and genital congestion. Subjective excitement could be influenced by interpersonal relationships, contextual factors, privacy, appropriateness, general emotional status, emotional relationships, biological factors, presence of depression, the influence of hormones (dopamine, testosterone), and others. In 2003, therefore, a revision of the current definition was done. The International Definitions Committee consisting of 13 experts from seven countries convened and proposed new definitions, which take into account new findings in the field of female sexual response [18, 19, 20].
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Current definition was again revised in 2010 by the International Consensus of Sexual Medicine, where the movement away from the nonoverlapping linear model toward a more circular model depicting the variety of triggers of women’s desire was accepted. It was emphasized that innate sexual fantasies and thoughts are not necessary for healthy sexual functioning and that desire is the result of sexual incentive that may be physically or subjectively perceived. Based on the previous findings, the arousal disorder was reclassified into subjective arousal disorder, genital arousal disorder, combined genital and subjective arousal disorder, and persistent genital arousal disorder. In May 2013, DSM-V was released, which also takes the focus away from the four-phase model, removed the sexual aversion disorder and merged vaginismus and dyspareunia into a new genito-pelvic pain/penetration disorder. It was finally noted that sexual dysfunction is a result of both psychological and biological and many other contributing factors [15, 21]. In 2015, the Fourth International Consultation on Sexual Medicine presented the new set of definitions of all forms of sexual dysfunction in women and men, which was based on ICD-10 and DSM-V (Figure 2) [22].
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Figure 2.
Nonlinear model of female sexual response cycle. The initial stage of female sexual response is sexual neutrality, but with positive motivation (left). The reasons why a woman is willing to initiate or agree to sexual activity can be that she wants to feel loved, share physical pleasure or be emotionally closer to her partner, please her partner, or she wants to increase her own satisfaction. Stimuli for sexual activity are being processed in the woman’s mind, influenced by biological and psychological factors, and result in subjective sexual arousal. If sexual stimuli last sufficiently long, sexual arousal and enjoyment will intensify, and it can trigger a desire for further sexual activity. It is important to note that desire appears at this point, not in the initial phase. When the stimulation continues and no negatives outcomes are involved, the process results in sexual satisfaction (with or without orgasm). Modified according to [23].
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The newest changes in nomenclature of female sexual dysfunction came in May 2018, when the World Health Organization developed the eleventh revision of the International Classification of Diseases and Related Health Problems (ICD-11). The ICD-10 classification of sexual dysfunctions was separated to two main groups: “organic” and “nonorganic” conditions. The nonorganic sexual diseases were classified as mental and behavioral disorders and organic belonged to diseases of the genitourinary system chapter. However, since ICD-10 definition, lots of evidences have been accumulated regarding the causes of sexual dysfunction, which often involve a combination of physical and psychological factors. The ICD-10 classification was therefore not consistent with clinical approaches in sexual health. ICD-11 diagnostic guidelines organize sexual dysfunctions into four main groups:
sexual desire and arousal dysfunctions;
orgasmic dysfunctions;
ejaculatory dysfunctions; and
other specified sexual dysfunctions.
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Moreover, a separate grouping of sexual pain disorders has been established. Where possible, categories in this new classification of sexual dysfunctions apply to both men and women even though the differences in sexual response are known. On the other hand, the neural pathways and neurotransmitters mediating sexual response are the same for both men and women. Separate sexual dysfunction categories are provided where clinical manifestations differ [24].
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The overview of current diagnostic criteria of sexual dysfunctions is presented below in Table 1. The present definition according the WHO ICD-11 is: “Sexual Dysfunctions are syndromes that comprise the various ways in which adult people may have difficulty experiencing personally satisfying, noncoercive sexual activities. Sexual response is a complex interaction of psychological, interpersonal, social, cultural and physiological processes and one or more of these factors may affect any stage of the sexual response. In order to be considered a sexual dysfunction, the dysfunction must: (1) occur frequently, although it may be absent on some occasions; (2) have been present for at least several months; and (3) be associated with clinically significant distress” [25].
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ICD-11 (2018)
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DSM-5 (2013)
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Chapter: conditions related to sexual health
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Grouping: sexual dysfunctions
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Grouping: sexual dysfunctions
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Category: hypoactive sexual desire dysfunction
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Category: female sexual interest/arousal disorder; male hypoactive sexual desire disorder
The eleventh revision of the International Classification of Diseases and Related Health Problems (ICD-11) and Diagnostic and Statistical Manual of Mental Disorders (DSM-5). Adapted from [25, 26].
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3. Sexual functioning in women with systemic sclerosis
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Persons with systemic sclerosis can experience a variety of symptoms that may affect all aspects of life, including sexual functions. The exact etiopathogenesis of sexual dysfunctions in systemic sclerosis is not well known; the causes are multifactorial and are related to both the disease symptoms and the therapy. Medical, pathophysiological, psychological, and social components may be involved in sexual dysfunction. Both physical and psychological problems arising from disease-related condition contribute to partnership difficulties, less active and less enjoyable sexual life [3, 27, 28].
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The most common physical symptom is skin tightness. Due to skin tightness, the fingers becomes fixed in bent position, which could interfere with sexual foreplay, touch, and masturbation. If skin tightening causes the mouth to shrink, kissing or oral sex could become difficult. Sometimes the skin become stiffer around the vaginal introitus, which often leads to painful penetration, and changes in the vaginal mucosae causing lubrication disorder contribute to this [27, 29, 30]. Specifically, 56% of SSc female patients reported painful penetration during the intercourse [31]. The sexual difficulties such as vaginal tightness, dryness, and dyspareunia were reported by more than half of systemic sclerosis female patients [7, 8]. It was also published that vaginal tightness (71%), dyspareunia (56%), and ulceration (23%) were the most common symptoms of sexual dysfunction observed in 60 women with systemic sclerosis [31]. These genital tract abnormalities could be associated with a decrease in number and intensity of orgasm, which are also often observed in SSc individuals [32].
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A majority of the systemic sclerosis patients experience Raynaud’s phenomenon, which can affect not only fingers and toes but also tongue and nipples. This is another reason, why the cuddling, foreplay, and oral sex could become uncomfortable and unpleasant [27]. In addition, a lot of patients suffer from secondary Sjögren’s syndrome characterized by drying of oral, nasal, ocular, and vaginal mucosae. The prevalence of Sjögren’s syndrome in systemic sclerosis ranged in different studies from 20 to 69% depending on the criteria used and sample size. In Saad’s study, 37% from 83 systemic sclerosis female patients reported Sjögren’s syndrome, 56% of them had impaired sexual function, and vaginal dryness was the most presented symptom [29].
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Another disease-related problem that impedes sexual activity is the affection of musculoskeletal system. The presence of joint contractures, stiffness, or pain leads to limited range of motion and it could restrict the ability to engage in sexual activities. Other aspects that reduced exercise capacity are muscle weakness and fatigue. It can be difficult to become sexually aroused when extremely tired. The consequence of skin thickening and other physical changes is the impaired body image, self-esteem, and sexuality. However, it has been also reported that body image dissatisfaction does not correlate with reduced sexual function [30]. In a different study, the major reasons for decreased sexual activity in married women with SSc were fatigue, altered body image, and pain [2]. Regarding psychological factors, depression is another often presented symptom that has been significantly associated with a sexual function disorder [8].
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There are a few more causes that could lead to less active sexual life in SSc patients. For instance, in rare cases, the fibrotic process of visceral vessels leads to renal impairment that may have an impact on sexual desire and orgasm. It is usually the medication used to treat kidney problems rather than the problems themselves. Also, gastrointestinal problems such as heartburn or chronic diarrhea may disrupt sexual activities [7].
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To sum up, existing studies of sexual function among women with SSc have concluded that sexual dysfunction is common in comparison to the general population. It was even showed a significantly greater decrease in orgasm and its intensity in SSc female patients compared to other systemic rheumatic diseases—rheumatoid arthritis and systemic lupus erythematosus [31]. What is more, Knafo et al. [4] published that the prevalence of sexual dysfunction is higher in SSc than in other chronic diseases such as breast cancer, gynecological cancer, or HIV positivity. On the other hand, Impens et al. [33] maintain that SSc women remain sexually active despite the psychological and physical difficulties caused by the disease. In this study, only 17% of women suffered from female sexual dysfunction primarily caused by systemic scleroderma. Other reasons for sexual inactivity were the absence of a partner (37%), personal choice (32%), and the health status of respondents’ partners (20%).
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4. Diagnostic approach of female sexual dysfunction
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The first step in the diagnosis of female sexual dysfunction is a detailed personal, sexual, pharmacological, and psychosocial history. Where necessary, further examination by a psychiatrist, gynecologist, or physiotherapist is indicated. There are several objective methods assessing female sexual function, such as laboratory tests including hormonal profile or Doppler ultrasonography. However, laboratory tests in clinical practice are used only as auxiliary diagnostic methods. With duplex Doppler ultrasonography, it is possible to display a blood flow in cavernous tissue vessels, which is performed either under basal conditions or during sexual stimulations (vibrational or audiovisual) and objectified sexual arousal reactions. The most commonly used physiological method for evaluating sexual response is vaginal photoplethysmography, which investigates vascular reactivity during sexual arousal. It is based on the assessment of congestion of the vaginal mucosae. Other methods include electromyography, measurement of changes in vaginal pressure, and measurement of pH [34, 35].
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Another widespread screening method is the questionnaire survey. Self-report questionnaires have a long history of use in psychological and sociological studies of sexual behavior. For example, Derogatis Sexual Function Inventory is a 245-item, multidimensional scale that evaluates a wide range of sexual behavior in 10 separate domains. Despite the very strong psychometric properties, this scale is not widely used in clinical trials due to its excessive length and complexity. Instead, several short evaluation scales have been developed [36, 37]. Currently, the female sexual function index (FSFI) is the “gold standard” in assessment of female sexual functioning [38]. FSFI is a widely used tool that has been validated for use across multiple populations including women of various age groups, in various health problems and sexual dysfunctions. It has been developed as a simple multidimensional self-assessment tool for assessing the key domains of female sexual function. The questionnaire consists of 19 items that evaluate sexual function over the last 4 weeks in six domains: sexual desire, sexual arousal, lubrication, orgasm, satisfaction, and pain [39]. Since the first validation study, it has been translated into more than 20 languages and validated in over 30 countries [40, 41].
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It has to be noted that both objective methods and questionnaire screenings have their limitations. The main disadvantage of objective methods is the absence of an intimate condition that can play a key role in the female subjective arousal. Basson et al. published that intimacy and desire are essential to women’s sexual activity. Raina et al. reported that intimacy leads to emotional arousal of a woman, followed by sexual desire and physical arousal that result in sexual satisfaction. These findings suggest that the presence of intimacy can be crucial in examining sexual arousal, and its absence can lead to a lack of emotional excitement and consequently to incomplete sexual responses [35, 42, 43]. On the contrary, the questionnaires are filled in private and evaluate women’s real sexual experiences. Moreover, this method is not time-consuming and costly. However, as with any brief self-report scale of a complex psychophysiological construct, the questionnaire assessment has notable practical and theoretical limitations. Self-reported screening does not provide objective information but only patient’s subjective perception.
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In terms of FSFI, its drawback is the assessment of sexual activity in the past 4 weeks. Of course, there are number of reasons why women may be sexually inactive during a 4-week period and it does not necessarily imply significant sexual dysfunction. For example, the absence of sexual partner is a very common reason, why patients are not sexually active. Specifically, the FSFI questionnaire contains 15 questions, which could be answered “No sexual activity” or “Did not attempt intercourse.” Both possible responses are scored as zero that could become problematic when lower scores indicate severer sexual dysfunction. In that case, the FSFI could produce biased results. Other inaccurate data may be the result of relatively vague terminology. A problematic issue seems to be to define and measure sexual desire and subjective sexual arousal. Studies suggest that women often have difficulty distinguishing desire and arousal in their sexual experience. Another drawback of the FSFI questionnaire is that questions in orgasm subscale are basically focused on the orgasmic function associated with penile-vaginal contact. In fact, the vagina is primarily a reproductive organ with little sensitivity, and clitoral stimulation is more important for female orgasm. Therefore, achieving orgasm should be judged more in conjunction with masturbation or oral sex than sexual intercourse [38, 44].
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5. The use of the FSFI questionnaire in systemic sclerosis patients
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The FSFI is also the most common questionnaire evaluating sexual function in women with systemic sclerosis. It has been used by an Italian team to evaluate the prevalence of sexual dysfunction in 46 women with SSc. The second purpose of this trial was to investigate the association with sociodemographic, physical, psychological, and disease-related variables. Compared to healthy controls, only the FSFI desire subscale score was significantly lower. The overall score did not substantially differ from healthy controls. The association with health status, functional ability, mouth affection, hand disability, and presence of depression was reported [10]. A very similar study has been conducted in the Netherlands. The FSFI was used to assess the sexual function of 69 SSc women aged 18–60 years. It was that the FSFI total score and the subscale scores for lubrication, orgasm, arousal, and pain were significantly lower in comparison with healthy population in the same age. Impaired sexual functioning and sexual distress were associated with marital distress and depressive symptoms [9]. Levis et al. first detected women with SSc who had engaged in sexual activities with their partner in the past 4 weeks, and then only sexually active patients completed a 9-item version of the FSFI. The aim of this Canadian cross-sectional multicenter study was to evaluate sociodemographic and clinical variables that distinguish sexually active from inactive patients and identify the source of pain during and after sexual activity in sexually active patients. The results showed that in total only 17% of 547 women were sexually active without sexual disorder [45]. The same group of scientists in different project used a shortened version of FSFI to compare sexual activity and impairment rates of women with systemic sclerosis to general population data. Among women with SSc, 296 of 730 (41%) were sexually active and 181 (61%) of sexually active patients reported sexual dysfunction. It means that only 115 of 730 (16%) patients engaged in sexual activities without impairment. It was also confirmed that SSc patients are significantly less likely to be sexually active and more likely to be sexually impaired than the general population of women [46]. Severe sexual dysfunction was also observed in married women with systemic sclerosis. About 8 out of 10 women achieved low scores in the FSFI questionnaire assessed, and all the subscales were affected in this study. The reasons why patients reported decrease in the frequency of intercourse since the onset of their disease and a diminished desire for a sexual relationship were fatigue, altered body image, and pain [2].
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6. Treatment and recommendation for female sexual dysfunction
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The management of female sexual dysfunction in the general population is based on the understanding of the basic physiology of female sexual response. Currently, several approaches of female sexual function treatment are available. It is known that some antidepressants can cause sexual dysfunction as a side effect. Antidepressants, such as SSRIs, are commonly associated with hypoactive sexual desire disorder (HSDD) and they have to be eliminated or dosed at lower levels. In women with major and disabling mood disorders, the adjustment of antidepressants requires a continuous collaboration with the prescribing psychiatrist, because dosage adjustments of antidepressants must be done very gradually [47]. Bupropion, buspirone, mirtazapine, vortioxetine, and vilazodone have been found to have lower rates of antidepressant-induced sexual dysfunction than other antidepressants, and they can be a suitable medication options for the treatment of depression [48, 49, 50].
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Another option, primarily used in menopausal women, is hormonal therapy. Estrogen therapy may be used to increase clitoral sensitivity and libido and to reduce pain during and after sexual intercourse. In women with menopause-related sexual dysfunction that have estrogen treatment experience improved sexual desire, vaginal atrophy and vaginal dryness [49]. Transdermal estradiol has been found to be a preferred therapy for depleted estrogen. It is considered as the most effective therapy available for reducing vasomotor symptoms and associated menopausal symptoms with minimal adverse effects. Intravaginal estrogens combined with mechanical dilatations are also highly effective for treatment of vaginal atrophy [47]. Testosterone replacement therapy may also be considered to increase sexual desire and libido. Several high-quality sources documenting that transdermal testosterone is effective in restoring sexual desire are available [47]. Intramuscular testosterone combined with estradiol in postmenopausal women had a positive impact on sexual desire, arousal, and frequency of sexual fantasies compared with women without testosterone treatment [51].
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Flibanserin is the first nonhormonal treatment for female sexual dysfunction to be approved by the Food and Drug Administration (FDA). The approval, in August 2015, occurred fully 18 years after the approval of sildenafil, the first treatment for erectile dysfunction. Flibanserin is a 5-HT 1A agonist and 5-HT 2A antagonist and is indicated for acquired hypo-active sexual desire disorder in premenopausal women. It is strongly recommended to avoid consumption of alcohol while using flibanserin treatment, because several serious adverse events were found: dizziness, loss of consciousness, hypotension, and circulatory collapse [49]. Another drug approved by the FDA is ospemifene, which is indicated in treatment of dyspareunia in postmenopausal women. It is a selective estrogen receptor modulator that can be prescribed when hormone replacement therapies fail. The daily dose of 60 mg of ospemifene has been found to be effective and tolerable for postmenopausal women with vaginal dryness and atrophy [52, 53]. To improve sexual functioning in domains of arousal, orgasm, and satisfaction, bupropion can be used, whose efficacy is based on the influence of dopamine and norepinephrine reuptake [54]. Although phosphodiesterase type 5 (PDE-5) inhibitors are mostly used in erectile dysfunction treatment, they can be used in women as well. Sildenafil has been found to significantly improve arousal, orgasm, and enjoyment in women without sexual dysfunction. In women with antidepressant-associated sexual dysfunction, sildenafil (in dose ranges of 50–100 mg) has also shown good efficacy [49].
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Rehabilitation treatment can be useful in patients suffering from vaginism, dyspareunia, and anorgasmia due to pelvic muscle spasms. Adequate exercises can lead to normalization of muscle tension and relaxation [55]. Psychotherapy is recommended where the pathogenesis of sexual dysfunction has a psychological nature. Because recent studies have suggested that more severe sexual dysfunctions in patients with SSc are significantly associated with the presence of depression [2, 9, 10], the psychotherapy should be an integral part of the treatment of sexual dysfunction in SSc patients. In terms of sexual pain disorder, psychotherapy seems to be immediately helpful although objective research on the long-term efficacy of psychotherapy for sexual pain disorder is limited and difficult to evaluate [47].
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There are some general recommendations for women with systemic sclerosis that may help to continue enjoying an active, fulfilling sexual life. If sexual activity is reduced due to pain, it can be alleviated by use of pain medication. The sexual activity can be scheduled for a time the pain will be at a minimum. A warm bath or shower before sexual activity often eases arthritic stiffness. The range of motion exercises before sex may help to reduce the stiffness. If the range of motion is limited and do not allow comfortable position, then it seems to be a good solution to experiment with sexual positions and try to find those that are the most suitable [3, 56]. If sclerosis has caused the mouth to shrink, physical therapist or occupational therapist can teach patients how to do the exercises to stretch the mouth. The regular stretching can improve the range of motion of the mouth and make kissing and oral sex more enjoyable. When fingers become fixed in a bent position, its possible to integrate stretching exercises as well and use the other part of hands (thumbs, wrists, or backs of the hands) to touch yourself or partner. An auxiliary material such as vibrators, creams, and lotions can also be used to enhance sexual activity. In order to avoid fatigue, patients with SSc may schedule their sexual activity for that part of the day when they still have enough energy, because becoming sexually aroused, when tired, is difficult. To prevent a Raynaud episode, it is necessary to keep entire body warm. It is possible to turn up the thermostat, leave some clothes on, or use extra blankets. When vaginal dryness and dyspareunia occur, the use of vaginal moisturizers on a regular basis along with lubricants as needed for sexual activity is the initial step in managing these symptoms. Women can choose from a number of commercially available lubricants that are either water based, mineral or plant oil based, or silicone based [47, 55]. Dilator therapy is an another option that can be useful in treatment of vaginal tightness. This method offers a nonsurgical approach to restore vaginal capacity and elasticity and alleviate sexual discomfort. If the penetration is still painful, there are alternative sexual activities like clitoral stimulation that can be sometimes more enjoyable than intercourse. If there is no interest in sex, still it is possible to stay physically close by holding or caressing one another [3, 56].
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Several options for the treatment of female sexual dysfunction in normal population are available. The management of impaired sexuality in women with systemic sclerosis was less studied and the further research is strongly needed. What is essential and beneficial for the patients is the team-based model of care for management of sexual dysfunction including a medical provider, physical therapist, occupational therapist, psychotherapist, and sex therapist [57].
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7. Sexual functioning in male with systemic sclerosis
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Systemic sclerosis is an autoimmune connective tissue disorder characterized by following typical findings: endothelial changes, microangiopathic damages, and progressive fibrosis. These pathological processes may affect various organs including penile arteries leading to erectile dysfunction (ED). Male erectile dysfunction is defined as the consistent inability to reach and maintain an erection sufficient to permit satisfactory sexual performance. It is a widespread issue in men with systemic sclerosis. Sexual dysfunction in men has been given more attention than female sexual dysfunction, and the etiology is more obvious compared to women. Erectile dysfunction is a result of microangiopathic changes, when the blood flow is reduced in the small penile arteries due to corporal fibrosis and myointimal proliferation [5]. It was proven that damage of the penile cavernous arteries occurs in almost all SSc patients regardless of clinical symptoms. They are characterized by the presence of hyperechoic spots, suggesting fibrotic changes and low peak systolic velocities that are signs for vascular alterations [58].
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The prevalence of erectile dysfunction in SSc patients ranged from 12 to 81% in different studies [28]. However, most studies agree that about 80% of SSc men are affected [11, 12, 13]. It was also found that ED is more prevalent in systemic sclerosis than other inflammatory rheumatic diseases. In a majority of men with systemic sclerosis, ED started to manifest after the onset of the disease. The mean duration from the onset of the first SSc symptom to erectile dysfunction was around 3 years [12]. Risk factors of erectile dysfunction such as smoking, hypertension, diabetes, and steroid use have been investigated. It was found that only self-reported history of nerve damage and diabetes are significant for predicting the likelihood of ED in systemic sclerosis. There are also risk factors that are presented in non-SSc men as well, like older age or alcohol consumption. The ED association with more severe diseases in terms of worse skin involvement, elevated pulmonary arterial pressures, presence of restrictive lung disease, and muscular and renal involvement in SSc patients was also confirmed [5, 12, 13, 59].
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The most likely hypothesis of ED in SSc is a combination of vascular and fibrotic abnormalities. In men with SSc, decreased penile blood pressure, impaired peak systolic and diastolic blood flow in the penile arteries, and the presence of veno-occlusive dysfunction were found. Also, a decreased penile temperature and a slow recovery after cold exposure were reported. A duplex sonography was conducted to reveal the thickening of tunica albuginea and diffuse hyperechogenic spots within the corpora cavernosa. All these findings point to the microangiopathic cause of ED in male SSc patients. This is confirmed by the fact that no carotid artery thickening has been found in SSc, which would predict atherosclerotic macroangiopathy. From a histological point of view, the ED cause in SSc men is the presence of severe corporal fibrosis, increased collagen production by penile smooth muscle cells, and increased accumulation of extracellular matrix. Due to these changes, penile hypoxia arises, which can lead to overexpression of platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β1 and TGF-β1 receptors in the corpora cavernosa, which are important profibrotic regulators of collagen synthesis and production of extracellular matrix. In addition, endothelin (ET-1) is also released by penile smooth muscle cells. Thus, penile hypoxia stimulates penile fibrosis. Therefore, it can be assumed that once ED in patients with SSc is manifested (due to disease), its next mechanisms are similar to those of non-SSc population. Other causes such as hormonal abnormalities or neurological causes have not been confirmed. No disturbances have been found in follicle-stimulating hormone, luteinizing hormone, serum testosterone, prolactin, estradiol, and thyroid hormones in SSc patients [5, 11, 59, 60].
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8. Diagnostic approach of erectile dysfunction
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The diagnosis of erectile dysfunction in SSc patients is not always easy. We are following the common steps starting with taking a detailed history. Then assessment with appropriate questionnaires and dynamic penile Duplex ultrasound is required. The ambiguity lies in the fact that penile vascular damage occurs in almost all SSc patients, regardless of clinical symptoms and the questionnaire results that often do not match with vascular findings. Thus, it is always better to carry out both investigations: the duplex ultrasound to document the degree of vascular involvement and self-administered questionnaire. The International Index of Erectile Function is the standardized and most widely used tool for evaluating erectile dysfunction. As mentioned above, penile temperature in SSc patients is lower than in healthy individuals. Since cutaneous temperature depends on cutaneous blood flow and thermal exchanges with deeper tissues, these findings could suggest the presence of functional alterations of both tissue properties and blood flow. Therefore, assessing changes in thermal properties and temperature control processes of the penis in SSc patients could provide a potential clue in diagnosis of erectile dysfunction. It has to be noted that with the progression of micro/macrovascular damage in the natural course of the disease, a concomitant penile fibrosis and veno-occlusive dysfunction occur and usually lead to difficult-to-treat ED. We should pay attention in cases where the reduced blood flow is observed, for example, on the hands (Raynaud’s phenomenon), because it can suggest that the penile arterial flow will be also altered, and it may be a sign of initial stage of ED in SSc patients [61, 62].
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9. Treatment and recommendation for erectile dysfunction
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It is not a mistake to initiate ED treatment by eliminating general cardiovascular risk factors including lifestyle, psychological, or drug-related factors, but such treatment is often unsatisfactory. This step usually has beneficial effect on erectile function in the general population [63]. Phosphodiesterase-5 (PDE-5) inhibitors are recommended as a first-line option for pharmacotherapy. In non-SSc men, this group of drugs causes the relaxation of smooth muscle cells and temporarily increases arterial blood flow in the penis. However, to achieve an erection, sexual stimulation is required, because this class of drugs is not considered as an initiator of erection. Several types of PDE-5 inhibitors are currently available. The most commonly used are sildenafil (Viagra), tadalafil (Cialis), and vardenafil (Levitra). Except these, newer molecules of PDE-5 inhibitors can be used, for example, mirodenafil, udenafil, and avanafil. All types of PDE-5 inhibitors are effective and safe regarding the treatment of ED in the normal population. The differences are only in their half-lives, when, for instance, tadalafil is effective for up to 36 hours whereas the effectiveness of sildenafil is only 12 hours. The choice of a PDE5-I depends on the frequency of intercourse and the patient’s personal experience [64, 65]. However, research on the efficacy of PDE-5 inhibitors is very limited in SSc patients [5]. Proietti et al. reported that once-daily tadalafil improved both erectile function and vascular measures of cavernous arteries in men with SSc-related erectile dysfunction. Also, an increase in frequency of morning erections and decrease in plasma ET1 levels were found. They suggest that daily tadalafil dose could play a potential role in preventing progression of penile fibrosis and erectile dysfunction in male SSc patients [58]. Furthermore, long-acting PDE-5 may lead to a decrease in the frequency and severity of Raynaud’s phenomenon and the promotion of digital ulceration [66].
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Patients who do not respond to PDE-5 inhibitors may be offered to try vacuum constriction devices. It was reported that patients who used the vacuum therapy system for a month to increase blood oxygenation in the corpora cavernosa and then employed the vacuum constriction device to maintain penile erection for sexual intercourse significantly improved their erectile function and sexual satisfaction [67]. However, there are no reports about the use of this system in patients with SSc.
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Another option for the treatment of erectile dysfunction is prostaglandin analogues, which can be administered via intracavernous injections or intraurethral application. Alprostadil is a stable form of prostaglandin E1 that increases the concentration of cyclic adenosine monophosphate and decreases the intracellular calcium concentration, resulting in the relaxation of smooth muscle cells. Several studies reported the efficiency of alprostadil in the general population, but in terms of SSc patients, it was reported that a substantial percentage of SSc patients did not respond adequately to intracavernous prostaglandin E1 injections [68].
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When pharmacotherapy fails and the patient wants a permanent solution, the surgical implantation of a penile prosthesis may be considered as the third-line option. Penile prosthesis improved erectile dysfunction in over 70% of men in the general population. Available prostheses are either malleable (semirigid) or inflatable (two or three pieces), but it should be considered that there are two main complications of penile prosthesis implantation—the mechanical failure and infection [5, 64, 65].
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Most of the treatment options described above have not been verified in patients with systemic sclerosis yet. In spite of the fact that erectile dysfunction is common in men with systemic sclerosis, demographics, risk factors, and ED treatments have not been sufficiently investigated. Only a small case series has described unsatisfactory results with on-demand sildenafil (25–50 mg). The higher dose of sildenafil has not been investigated. Tadalafil has been slightly better evaluated in the treatment of SSc-related erectile dysfunction. The efficiency of 20 mg tadalafil on demand and 20 mg tadalafil in a fixed alternate day regimen has been compared. The results showed that flow-mediated dilatation and peak systolic velocities of cavernous arteries at penile duplex ultrasound improved significantly with the alternate day treatment; but no significant changes were observed after the on-demand tadalafil dosing. In addition, the alternate day regimen also reduced the plasma levels of ET-1 and vascular cell adhesion molecule as markers of endothelial function [68]. Therefore, long-term administration of tadalafil and its constant plasma level seems to have a positive effect on the treatment of ED in male SSc patients.
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10. Conclusion
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Sexual dysfunction is a common problem in both men and women with systemic sclerosis. Erectile dysfunction is the dominant issue in males, which seems to be tightly linked to vascular dysfunction. Sexual dysfunction in the female patient is not less prevalent, but it is considerably more complex and it has been less studied. Several diagnostic approaches have been established to assess sexual dysfunction. Also, there are some treatment options available, but most of them have not been sufficiently verified in patients with systemic sclerosis. Further research regarding sexual dysfunction in patients with systemic sclerosis is strongly needed.
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Acknowledgments
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This work was supported by grants AZV NV18-01-00161A, 16-33542A, 16-33574A, and institutional support of the Ministry of Health of the Czech Republic for the Institute of Rheumatology number: 023728 and GA UK 1578119.
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\n',keywords:"systemic sclerosis, sexual functioning, female sexual dysfunction, erectile dysfunction",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/66966.pdf",chapterXML:"https://mts.intechopen.com/source/xml/66966.xml",downloadPdfUrl:"/chapter/pdf-download/66966",previewPdfUrl:"/chapter/pdf-preview/66966",totalDownloads:306,totalViews:0,totalCrossrefCites:1,dateSubmitted:"July 16th 2018",dateReviewed:"April 5th 2019",datePrePublished:"May 3rd 2019",datePublished:null,dateFinished:null,readingETA:"0",abstract:"Systemic sclerosis (SSc) may affect all aspects of life including sexual function. Although sexual dysfunction is a neglected area of quality of life in patients with SSc, it turns out that it is an important issue for both men and women characterized by high prevalence. The etiology of sexual dysfunction in systemic sclerosis is multifactorial and includes factors associated with both physical and emotional (psychological) status. The most common physical problems in women are vaginal dryness, dyspareunia, and vaginal tightness. Erectile dysfunction is not only a frequent but also often underestimated clinical symptom in men with SSc. The incidence of erectile dysfunction in patients with SSc ranged from 12 to 81% in different studies. The main psychological factors that may affect sexuality are depression, fear, and changes in the appearance of the face and body that usually leads to impaired self-esteem. This chapter is a review about the impact of systemic sclerosis on sexual functioning.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/66966",risUrl:"/chapter/ris/66966",signatures:"Barbora Heřmánková",book:{id:"8269",title:"New Insights into Systemic Sclerosis",subtitle:null,fullTitle:"New Insights into Systemic Sclerosis",slug:"new-insights-into-systemic-sclerosis",publishedDate:"September 18th 2019",bookSignature:"Michal Tomcik",coverURL:"https://cdn.intechopen.com/books/images_new/8269.jpg",licenceType:"CC BY 3.0",editedByType:"Edited by",editors:[{id:"193284",title:"Dr.",name:"Michal",middleName:null,surname:"Tomcik",slug:"michal-tomcik",fullName:"Michal Tomcik"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:null,sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Definition and classification of sexual dysfunction",level:"1"},{id:"sec_3",title:"3. Sexual functioning in women with systemic sclerosis",level:"1"},{id:"sec_4",title:"4. Diagnostic approach of female sexual dysfunction",level:"1"},{id:"sec_5",title:"5. The use of the FSFI questionnaire in systemic sclerosis patients",level:"1"},{id:"sec_6",title:"6. Treatment and recommendation for female sexual dysfunction",level:"1"},{id:"sec_7",title:"7. Sexual functioning in male with systemic sclerosis",level:"1"},{id:"sec_8",title:"8. Diagnostic approach of erectile dysfunction",level:"1"},{id:"sec_9",title:"9. Treatment and recommendation for erectile dysfunction",level:"1"},{id:"sec_10",title:"10. Conclusion",level:"1"},{id:"sec_11",title:"Acknowledgments",level:"1"}],chapterReferences:[{id:"B1",body:'Heřmánková B. Sexual dysfunction in patients with systemic sclerosis. London: IntechOpen; 2019. DOI: 10.5772/intechopen.86219\n'},{id:"B2",body:'Frikha F, Masmoudi J, Saidi N, Bahloul Z. Sexual dysfunction in married women with systemic sclerosis. 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World Psychiatry. 2016;15(3):205-221\n'},{id:"B25",body:'WHO. World Health Organization [Online]. 2018. Available from: https://icd.who.int/browse11/l-/en#/http://id.who.int/icd/entity/160690465 [Accessed: March 31, 2019]\n'},{id:"B26",body:'Association AP. Diagnostic and Statistical Manual of Mental Disorders (DSM-5®). American Psychiatric Pub; 2013\n'},{id:"B27",body:'Tristano AG. The impact of rheumatic diseases on sexual function. Rheumatology International. 2009;29(8):853-860\n'},{id:"B28",body:'Impens AJ, Seibold JR. Vascular alterations and sexual function in systemic sclerosis. International Journal of Rheumatology. 2010;2010:139020\n'},{id:"B29",body:'Saad SC, Pietrzykowski J, Lewis SS, Stepien AM, Latham VA, Messick S, et al. Vaginal lubrication in women with scleroderma and Sjogren\'s syndrome. Sexuality and Disability. 1999;17(2):103-113\n'},{id:"B30",body:'Bruni C, Raja J, Denton CP, Matucci-Cerinic M. The clinical relevance of sexual dysfunction in systemic sclerosis. Autoimmunity Reviews. 2015;14(12):1111-1115\n'},{id:"B31",body:'Bhadauria S, Moser DK, Clements PJ, Singh RR, Lachenbruch PA, Pitkin RM, et al. Genital tract abnormalities and female sexual function impairment in systemic sclerosis. American Journal of Obstetrics and Gynecology. 1995;172(2 Pt 1):580-587\n'},{id:"B32",body:'Sampaio-Barros PD, Samara AM, Marques Neto JF. Gynaecologic history in systemic sclerosis. Clinical Rheumatology. 2000;19(3):184-187\n'},{id:"B33",body:'Impens A, Rothman J, Schiopu E, Cole J, Dang J, Gendrano N, et al. Sexual activity and functioning in female scleroderma patients. Clinical and Experimental Rheumatology. 2009;27(3):S38\n'},{id:"B34",body:'Wylie K, Daines B, Jannini EA, Hallam-Jones R, Boul L, Wilson L, et al. Loss of sexual desire in the postmenopausal woman. The Journal of Sexual Medicine. 2007;4(2):395-405\n'},{id:"B35",body:'Basson R. Women’s sexual desire—Disordered or misunderstood? Journal of Sex and Marital Therapy. 2002;28(1):17-28\n'},{id:"B36",body:'Rosen RC. Assessment of female sexual dysfunction: Review of validated methods. Fertility and Sterility. 2002;77(Suppl 4):S89-S93\n'},{id:"B37",body:'Brotto L, Atallah S, Johnson-Agbakwu C, Rosenbaum T, Abdo C, Byers ES, et al. Psychological and interpersonal dimensions of sexual function and dysfunction. The Journal of Sexual Medicine. 2016;13(4):538-571\n'},{id:"B38",body:'Stephenson KR, Toorabally N, Lyons L, Meston CM. Further validation of the female sexual function index: Specificity and associations with clinical interview data. Journal of Sex and Marital Therapy. 2016;42(5):448-461\n'},{id:"B39",body:'Wiegel M, Meston C, Rosen R. The female sexual function index (FSFI): Cross-validation and development of clinical cutoff scores. Journal of Sex and Marital Therapy. 2005;31(1):1-20\n'},{id:"B40",body:'Rosen RC, Revicki DA, Sand M. Commentary on “critical flaws in the FSFI and IIEF”. The Journal of Sex Research. 2014;51(5):492-497\n'},{id:"B41",body:'Nowosielski K, Wróbel B, Sioma-Markowska U, Poręba R. Development and validation of the polish version of the female sexual function index in the polish population of females. The Journal of Sexual Medicine. 2013;10(2):386-395\n'},{id:"B42",body:'Raina R, Pahlajani G, Khan S, Gupta S, Agarwal A, Zippe CD. Female sexual dysfunction: Classification, pathophysiology, and management. Fertility and Sterility. 2007;88(5):1273-1284\n'},{id:"B43",body:'Basson R. Rethinking low sexual desire in women. BJOG: An International Journal of Obstetrics and Gynaecology. 2002;109(4):357-363\n'},{id:"B44",body:'Puppo V. Female sexual function index (FSFI) does not assess female sexual function. Acta Obstetricia et Gynecologica Scandinavica. 2012;91(6):759\n'},{id:"B45",body:'Levis B, Hudson M, Knafo R, Baron M, Nielson WR, Hill M, et al. Rates and correlates of sexual activity and impairment among women with systemic sclerosis. Arthritis Care and Research. 2012;64(3):340-350\n'},{id:"B46",body:'Levis B, Burri A, Hudson M, Baron M, Thombs BD, Canadian Scleroderma Research G. Sexual activity and impairment in women with systemic sclerosis compared to women from a general population sample. PLoS One. 2012;7(12):e52129\n'},{id:"B47",body:'Buster JE. Managing female sexual dysfunction. Fertility and Sterility. 2013;100(4):905-915\n'},{id:"B48",body:'Jacobsen PL, Mahableshwarkar AR, Palo WA, Chen Y, Dragheim M, Clayton AH. Treatment-emergent sexual dysfunction in randomized trials of vortioxetine for major depressive disorder or generalized anxiety disorder: A pooled analysis. CNS Spectrums. 2016;21(5):367-378\n'},{id:"B49",body:'Harsh V, Clayton AH. Sex differences in the treatment of sexual dysfunction. Current Psychiatry Reports. 2018;20(3):18\n'},{id:"B50",body:'Clayton AH, Pradko JF, Croft HA, Montano CB, Leadbetter RA, Bolden-Watson C, et al. Prevalence of sexual dysfunction among newer antidepressants. The Journal of Clinical Psychiatry. 2002;63(4):357-366\n'},{id:"B51",body:'Sherwin BB, Gelfand MM, Brender W. Androgen enhances sexual motivation in females: A prospective, crossover study of sex steroid administration in the surgical menopause. Psychosomatic Medicine. 1985;47(4):339-351\n'},{id:"B52",body:'Portman DJ, Bachmann GA, Simon JA, Ospemifene Study G. Ospemifene, a novel selective estrogen receptor modulator for treating dyspareunia associated with postmenopausal vulvar and vaginal atrophy. Menopause. 2013;20(6):623-630\n'},{id:"B53",body:'Portman D, Palacios S, Nappi RE, Mueck AO. Ospemifene, a non-oestrogen selective oestrogen receptor modulator for the treatment of vaginal dryness associated with postmenopausal vulvar and vaginal atrophy: A randomised, placebo-controlled, phase III trial. Maturitas. 2014;78(2):91-98\n'},{id:"B54",body:'Segraves RT, Clayton A, Croft H, Wolf A, Warnock J. Bupropion sustained release for the treatment of hypoactive sexual desire disorder in premenopausal women. Journal of Clinical Psychopharmacology. 2004;24(3):339-342\n'},{id:"B55",body:'Vegunta S, Kling JM, Faubion SS. Sexual health matters: Management of female sexual dysfunction. Journal of Women’s Health. 2016;25(9):952-954\n'},{id:"B56",body:'Kocher A, Adler S, Spichiger E. Skin and mucosa care in systemic sclerosis–patients’ and family caregivers’ experiences and expectations of a specific education programme: A qualitative study. Musculoskeletal Care. 2013;11(3):168-178\n'},{id:"B57",body:'Rullo J, Faubion SS, Hartzell R, Goldstein S, Cohen D, Frohmader K, et al. Biopsychosocial management of female sexual dysfunction: A pilot study of patient perceptions from 2 multi-disciplinary clinics. Sexual Medicine. 2018;6(3):217-223\n'},{id:"B58",body:'Proietti M, Aversa A, Letizia C, Rossi C, Menghi G, Bruzziches R, et al. Erectile dysfunction in systemic sclerosis: Effects of longterm inhibition of phosphodiesterase type-5 on erectile function and plasma endothelin-1 levels. The Journal of Rheumatology. 2007;34(8):1712-1717\n'},{id:"B59",body:'Aversa A, Proietti M, Bruzziches R, Salsano F, Spera G. The penile vasculature in systemic sclerosis: A duplex ultrasound study. The Journal of Sexual Medicine. 2006;3(3):554-558\n'},{id:"B60",body:'Lally EV, Jimenez SA. Erectile failure in systemic sclerosis. The New England Journal of Medicine. 1990;322(19):1398-1399\n'},{id:"B61",body:'Merla A, Romani G, Tangherlini A, Di Romualdo S, Proiettii M, Rosato E, et al. Penile cutaneous temperature in systemic sclerosis: A thermal imaging study. International Journal of Immunopathology and Pharmacology. 2007;20(1):139-144\n'},{id:"B62",body:'Aversa A, Bruzziches R, Francomano D, Rosato E, Salsano F, Spera G. Penile involvement in systemic sclerosis: New diagnostic and therapeutic aspects. International Journal of Rheumatology. 2010;2010:706087\n'},{id:"B63",body:'Gupta BP, Murad MH, Clifton MM, Prokop L, Nehra A, Kopecky SL. The effect of lifestyle modification and cardiovascular risk factor reduction on erectile dysfunction: A systematic review and meta-analysis. Archives of Internal Medicine. 2011;171(20):1797-1803\n'},{id:"B64",body:'Wespes E, Eardley I, Giuliano F, Hatzichristou D, Hatzimouraditis K. Guidelines on male sexual dysfunction: Erectile dysfunction and premature ejaculation. European Association of Urology. 2013;2013:1-54\n'},{id:"B65",body:'Hatzimouratidis K, Amar E, Eardley I, Giuliano F, Hatzichristou D, Montorsi F, et al. Guidelines on male sexual dysfunction: Erectile dysfunction and premature ejaculation. European Urology. 2010;57(5):804-814\n'},{id:"B66",body:'Shenoy PD, Kumar S, Jha LK, Choudhary SK, Singh U, Misra R, et al. Efficacy of tadalafil in secondary Raynaud’s phenomenon resistant to vasodilator therapy: A double-blind randomized cross-over trial. Rheumatology. 2010;49(12):2420-2428\n'},{id:"B67",body:'Li P, Shen YJ, Liu TQ , Ma M, Zhang SJ, Wang YX, et al. Vacuum therapy for erectile dysfunction that fails to respond to PDE-5i: Report of 70 cases. Zhonghua nan ke xue = National Journal of Andrology. 2013;19(3):236-240\n'},{id:"B68",body:'Walker UA, Tyndall A, Ruszat R. Erectile dysfunction in systemic sclerosis. Annals of the Rheumatic Diseases. 2009;68(7):1083-1085\n'}],footnotes:[],contributors:[{corresp:"yes",contributorFullName:"Barbora Heřmánková",address:"herman.barbora@gmail.com",affiliation:'
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