Two-component systems (TCS) are ubiquitous among bacteria. They play essential roles in signaling events in bacteria, such as cell-cell communication, adaptation to environments, and pathogenesis in the case of pathogens. Due to their absence in humans and other mammals, TCS proteins are considered potential targets for developing new antibiotics. Most bacterial TCSs consist of two proteins, a sensor histidine kinase (HK) and a response regulator (RR). The HK senses specific signals, and that leads to activation of the kinase activity and autophosphorylation of a conserved histidine residue. The phosphoryl group is subsequently transferred to a cognate response regulator to activate its activities. Most RRs are transcription regulators and turn on or off gene transcriptions in response to the signals received by sensor HKs. Recent years have seen a rapid expansion of structural data of bacterial TCS proteins. In this chapter, I will review structures of HKs and RRs and discuss their structure-function relationship and their signaling mechanisms. The chapter contains three main sections: structures of histidine kinases, structures of response regulators, and structures of complexes between a histidine kinase and a response regulator. I will conclude the chapter with the implications of these structural and functional data on developments of new therapeutics against bacterial pathogens. Analysis of structures and reaction mechanisms will focus on the extracytosolic sensor HKs and OmpR/PhoB subfamily transcription regulator RRs.
2. Structures of histidine kinases
The prototypical histidine kinase is a homodimeric integral membrane protein (Figure 1). Each protomer has two transmembrane (TM) helices with the N-terminus in the cytosol. An extracytosolic sensor domain lies between the two TM helices. After the second TM helix is a HAMP domain, which is commonly found in Histidine kinases, Adenylyl cyclases, Methyl-accepting chemotaxis proteins, and Phosphatases , hence the name. The HAMP domain connects TM2 to the dimerization and histidine phosphorylation domain (often abbreviated as DHp). A catalytic and ATP-binding (CA) domain lies at the carboxyl terminus. The combination of DHp and CA is sometimes referred to as the kinase domain. The TM helices, HAMP domains, and DHp domains are all involved in homodimerization. The sensor domain is likely to dimerize in the context of the entire protein. Sensor domains share little sequence identity, as is expected from their diverse functions of sensing various signals. However, available structures of sensor domains fall into a few common structural folds, suggesting conserved signal sensing mechanisms. The HAMP, DHp, and CA domains are common modules of HKs and have well conserved structures and sequences, especially DHp and CA, whose sequences contain several conserved motifs. There is an absolutely conserved histidine residue that is phosphorylated, and the phosphoryl group is then donated to an RR, in response to signals sensed by the sensor domain. How the sensor domain regulates the kinase activities is still unknown, due to the lack of full-length structures of the transmembrane sensor HKs. However, a large accumulation of structures of isolated domains in recent years has started to shed light on possible molecular mechanisms of the signal transduction. In this section, I will summarize these structures and discuss their conformational changes that transmit signals from the sensor domain to the kinase domain.
2.1. Structures of sensor domains
Sensor domains of histidine kinases are located in cytosol, in membrane, or outside of cell membrane (extracytosolic). Currently, there is little structural information of the membrane-embedded sensor domains. The prototypical HK has an extracytosolic sensor domain that senses extracellular signals or conditions in the cell envelope. These sensor domains have highly diverse sequences. However, most of the known structures of extracytosolic sensor domains fall into three distinct structural folds, mixed αβ, all-helical, and β-sandwich. Unlike extracytosolic sensor domains, many cytosolic sensor domains can be annotated on the sequence level as PAS or GAF domains, which have related structural folds and are named from their occurrence in Period circadian, Aryl hydrocarbon receptor nuclear translocator, and Single-minded proteins (PAS) , or in cGMP-regulated cyclic nucleotide phosphodiesterases, Adenylate cyclases, and the bacterial transcriptional regulator FhlA (GAF) .
DosS and DosT from
The most common structural fold of the extracytosolic sensor domains is the mixed αβ fold, which has an identical topology to that of PAS domains . The structures consist of a central 5-stranded antiparallel β-sheet, flanked by α-helices on both sides (Figure 2, top row). These extracytosolic sensor domains are slightly different in structure from the PAS and GAF domains. Their structures fall into a group by themselves and are named as PDC domains, referring to first three structures of this group, i.e. sensor domains of PhoQ, DcuS, and CitA . Among the PDC domains, the structures are similar to each other, especially the N-terminal helix and the position of the C-terminus. The PDC domains have a long N-terminal helix, possibly continuous from the TM1 helix. The N- and C-termini of the domain are next to each other, and both are facing the same direction toward the membrane for connecting to the transmembrane helices. In some structures, there is a short helix at the C-terminus, possibly continuous to the TM2 helix.
Some HK sensor domains are all-helical, represented by those of NarX  and TorS  (Figure 2). The NarX sensor domain is an antiparallel four-helix bundle. Most of the helices have kinks or bends, suggesting mobility. TorS has two antiparallel four-helix bundles stacked along their bundle axes, with the second one inserted between the last two helices of the first bundle. Their mechanisms for signal sensing are different. NarX binds directly to nitrite and nitrate; while TorS interacts with a periplasmic binding protein TorT to detect trimethylamine-N-oxide.
The crystal structure of the sensor domain of RetS from
2.2. Structure and function of HAMP domains
HAMP domains are widely occurring domains of prokaryotic transmembrane receptors. They follow the last TM helix and thus connect the extracytosolic sensory domain to the cytosolic signaling domains. The sequences of HAMP domains have heptad repeats, in which hydrophobic residues occupy positions a and d (Figure 3). The structures of HAMP domains are dimers with each protomer of ~50 residues forming two helices (referred to as Hα1 and Hα2) connected by a linker of ~14 residue. The four helices from two protomers associate to form a parallel four-helix bundle. The linker residues have an extended structure that spans the length of the four-helix bundle [13, 14]. Other than the hydrophobic side chains of the heptad repeats and a conserved glycine residue at the beginning of the linker, HAMP domains do not have strong sequence conservation. Nevertheless, the domains can be exchanged, yielding hybrid proteins that are still functional [15, 16], which suggests that HAMP domains share a conserved mechanism for propagating signals.
The HAMP domain of Af1503, a hypothetical receptor from the archaeon
The crystal structure of a tri-HAMP unit from the
2.3. The dimerization and phosphorylation domain
The dimerization and phosphorylation domain is a conserved domain of histidine kinases. This domain is often abbreviated as DHp for dimerization and histidine phosphotransfer. It is also referred to as dimerization domain or histidine kinase domain A. The sequence of DHp contains the H box that is part of the signature motifs defining histidine kinases (Figure 4). The histidine residue in the H box motif is absolutely conserved and is the site for autophosphorylation and subsequent transfer of the phosphoryl group to downstream proteins. This domain participates in dimerization of the histidine kinase. DHp is located in the cytosol and directly follows the HAMP domain, and it is immediately followed by the CA domain in the prototypical histidine kinases (Figure 1).
The structure of the DHp domain consists of two α-helices (referred to as Dα1 and Dα2), that form an antiparallel coiled-coil (also referred to as helical hairpin), and two protomers associate to form a four-helix bundle (Figure 5). The conserved histidine residue is on helix Dα1. Similar to HAMP domains, sequences of the DHp domains have heptad repeats. Several structures of DHp have been determined. The NMR structure of the
helix Dα2, especially around the conserved H-box residues. The intra-subunit interface is mostly hydrophobic; the dimer interface, however, contains two acidic clusters. A related phosphotransferase Spo0B from
The structure of the entire cytoplasmic portion of HK853, a putative sensor HK from
2.4. ATP-binding domain
In prototypical sensor histidine kinases, the ATP-binding domain is located at the carboxyl terminus of the polypeptide (Figure 1). The ATP-binding domain is often abbreviated as CA for catalytic and ATP-binding. This domain binds ATP, which then donates its γ-phosphate group to the conserved histidine residue on the DHp domain. Sequences of the CA domains have well conserved N, G1, F, G2, and G3 box sequence motifs (Figure 4). Together with the H box at the DHp domain, they are the markers to annotate histidine kinases from DNA sequences.
The structures of the CA domains are well conserved, as expected from their highly conserved sequences. The structures have an αβ sandwich fold containing two layers, a layer of mixed 5-stranded β-sheet and a layer of three α-helices (Figure 6). The CA domain of DesK from
The ATP-binding site is at one end of the domain (Figure 6) and involves both absolutely conserved and partially conserved residues from the N, G1, F, G2, and G3 boxes (Figure 4). Residues from the G1 and G3 boxes are involved in binding the adenosine moiety; those from the N and G2 boxes contact both the adenosine moiety and the triphosphates-Mg2+ moiety. The structure of
A long loop covering the ATP-binding site is called ATP-lid, which spans from the F box to the beginning of helix α4 and plays an important role in binding ATP. The ATP-lid is highly mobile; it has several glycine residues, including the conserved G2 box that contains three glycine residues (Figure 4). The bound ATP has extensive contacts with the ATP-lid residues, and thus ATP binding induces the closure of the ATP-lid. In the absence of ATP, this loop is partially disordered in crystal structures. Even in the presence of ATP, the ATP-lid shows high flexibility, indicated by high B-factors or in some cases is partially disordered in crystal structures [21-24]. The flexibility of the ATP-lid is important not only for binding ATP, but also for interacting with the DHp domain for the phosphotransfer reactions. The ATP-binding domain must adopt several positions relative to the DHp domain because the HK functions as an autokinase, phosphotransferase, or phosphatase in response to the environmental stimulates received. The flexibility of the ATP-lid allows the CA domain to bind to different regions of DHp depending on the conformation of the DHp domain and the status of the ATP-binding site of the CA domain.
2.5. Signal transduction mechanism from sensor domain to the kinase domain
Signals flow from the sensor domain to the kinase domain by conformational changes throughout the entire protein. Environmental cues received by the sensor domain cause a conformational change in the sensor domain. That conformational change is transmitted through the transmembrane helices and the HAMP domain to the kinase domain. Most of isolated sensor domains are monomers in solution. However, they are expected to form dimers in the context of the whole protein because the DHp and HAMP domains are homodimers. Therefore, ligand-induced dimerization cannot be a mechanism of signal transduction through the cell membrane. Instead, there are structural changes of the sensor domains upon ligand binding that could trigger rotation or piston-like translation movements of the TM helices [9, 25].
Comparison of structures of the sensor domain of CitA with and without citrate bound suggests a piston-type transmembrane signaling . The sensor domain of CitA has a central 5-stranded anti-parallel β-sheet flanked by α-helices on both sides (Figure 2). The β-sheet curls up to one side where citrate binds. There are two loops, referred to as major loop and minor loop, which cover the citrate-binding site. The major loop is disordered in the citrate-free structure and becomes ordered in the presence of citrate in the binding site. Binding of citrate also induces more bent of the central β-sheet. This results in lifting of the C-terminus of the domain relative to the N-terminal helix (Figure 7). The C-terminus of the domain connects to the second transmembrane helix (TM2). Therefore, binding of citrate induces a piston-type movement of TM2 relative to TM1. Similar piston-type movement has been proposed for NarX from
A rotation or screw-like motion of TM2 has been proposed as the signal transduction mechanism from the sensory rhodopsin into the cytosol for HtrII . Based on the structure of HtrII, the transmembrane helices of prototypical HKs are expected to form a four-helix bundle . A rotation or screw-like motion of the TM helices for propagating signal through membrane is compatible with that proposed for the HAMP domain, which is often found between TM2 and the DHp domain. Structural and biochemical studies suggest that HAMP domains are not rigid structures. They can alternate between different structures through helical rotation  or a shift in register of coiled-coils and rotation . Mutation of an alanine residue of the HAMP domain of Af1503 into larger side chain residues changes the helical packing gradually from complementary x-da to the canonical knobs-into-holes mode . Through these conformational changes, the HAMP domain passes the signals from the sensor domain to the downstream kinase domain.
Structures of chimeras of the Af1503 HAMP fused with the DHp domain of EnvZ show that conformational changes in the HAMP domain can trigger changes in the structure of the downstream DHp domain . Figure 8 shows three crystal structures of the chimeras. The HAMP domains preserve the same conformations as their corresponding isolated domains. Mutations of A291 into amino acids with larger hydrophobic side chains change the helical packing conformation. These conformational changes in the HAMP domain propagate to the DHp domain through the connecting helices. The helices linking the HAMP domain to the DHp domain do not have a strong hydrophobic core as in the four-helix bundles but are relatively flexible, showing variations among the structures with different mutations. Conformational variation is the most pronounced at the junction between the DHp four-helix bundle and the two-helix coiled-coil where there are charged residues (charged layer, Figure 8). The induced rotational movements in helix Dα1 gradually diminish at the conserved proline, which produces a kink in the helix. Helix Dα2 also changes conformation responding to changes of Dα1, and as a result the bundle radius differs above the proline layer among variant structures.
The structural changes in DHp induced by the HAMP domain are likely to alter the activities by regulating interactions between the DHp and CA domains. Despite the structural changes triggered by varying conformations in HAMP, the phosphorylation site histidine remains little perturbed, and thus altering accessibility of the conserved histidine is unlikely to be the mechanism of signal transmission. The lower half of the DHp helical bundle (below the proline layer) remains invariant among the structures. This region contains many residues important for binding the cognate RR and contributes the majority of the interface for RR binding in the HK-RR complex structures [19, 28]. Therefore, blocking direct binding of RR is unlikely to be a mechanism for signaling either. However, the region of DHp most variable among the structures (charged layer) plays an important role in interacting with the CA domain. In the structure of HK853-RR468 complex, residues F428 and L444 of the ATP-lid interact with an exposed hydrophobic core of DHp near the charged layer, and this interaction locks the ATP-binding domain in a kinase-inactive state . It is likely that conformational changes triggered by signals transmitted from the HAMP domain either promote this DHp-CA interaction (phosphatase state) or release the CA domain for it to phosphorylate the conserved histidine (kinase state).
Several structures of the entire kinase domain (DHp + CA) are available and give insights into the interactions between the two sub-domains. The crystal structure of the entire cytoplasmic portion of HK853 from
near the charged layer (Figures 8 and 9), and in the CA domain it involves the ATP-lid, helix α4, and residues at N-terminus of α2. The interactions are relatively weak, and thus the domain interface is dynamic, allowing the CA domain to move to other locations on DHp. The signals from the sensor domain control the interfacial stability through conformational changes of the helical bundle. The DHp-CA interface is destabilized for the kinase activity and stabilized for the phosphatase activity.
The crystal structure of HK853 complexed with RR468 reveals another binding interface between DHp and CA that is created by conformational changes in the helical packing . In this structure, the CA domain moves closer to H260 of the same subunit (Figure 9b). This suggests that the autophosphorylation occurs in
Structural plasticity of the HK proteins is essential to allow conformational changes in order to transmit signals received at the sensor domain to the kinase domain and switch its enzymatic activities. This structural plasticity is well demonstrated by the structures of the cytoplasmic domain of the
3. Structures of response regulators
Response regulators are simpler in structure than histidine kinases. The prototypical RR has two domains: a receiver domain that accepts a phosphoryl group from a cognate HK and an effector domain that generates the outputs of the signaling events. The structure and sequence of receiver domains are well conserved. In contrast, effector domains are variable, especially at the sequence level, reflecting their diverse output functions. The majority of RRs are transcription regulators with their effector domains as DNA-binding domains. A significant fraction of RRs have effector domains as enzymes. Many others have effector domains for binding RNA, ligands, or proteins to regulate bacterial cellular process at post-transcriptional and post-translational levels. There are also single domain RRs that have only the receiver domains such as CheY and Spo0F. RRs have been studied extensively for decades, and an extensive amount of structural data is available, as well as many well-written reviews . I will focus my efforts on transcription regulator RRs and will give a summary on how phosphorylation of the receiver domain controls the activities of the effector domain with respect to their structure-function relationship.
3.1. Receiver domain structures
Receiver domains have well-conserved sequences and structures, suggesting a common signaling mechanism by two component systems to pass signals from HKs to their cognate RRs. The receiver domain is also known as the phosphorylation domain or regulatory domain. There is an invariable aspartate residue in the receiver domain that accepts a phosphoryl group from a cognate HK. Phosphorylation results in conformational changes in the domain, which is transmitted to the effector domain to regulate its activities. Receiver domains have a conserved (βα)5 fold (Figures 10 and 11), in which alternating β-strands and α-helices in the sequence fold into a central five-stranded parallel β-sheet surrounded by helices α1 and α5 on one side and helices α2, α3, and α4 on the other side. The β-sheet consists of mainly hydrophobic side chains that form a hydrophobic core. The α-helices are amphipathic and pack against the central β-sheet with their hydrophobic face. Most of the conserved residues are at the C-terminal ends of strands β1, β3, and β4. Helix α1 is involved in binding to the DHp domain of HK . Although the structure and position of this helix is well conserved among RR structures, the amino acid sequence conservation is limited to the hydrophobic residues involved in packing against the hydrophobic core of the β-sheet (Figure 10). This helix is likely to play an important role in the specificity of HK-RR pairs.
The phosphorylation site is located at an acidic pocket near the C-termini of strands β1 and β3, where the conserved acidic side chains are clustered together. These acidic side chains are involved in binding a divalent cation, Mg2+ or Mn2+, that is essential for the phosphorylation reaction . An absolutely conserved aspartate at the end of the strand β3 is the phosphoacceptor. The loop β3-α3 forms a conserved structure that is held together by conserved residues: a Pro at 4 residues after the phosphorylation site aspartate and a Gly, which is at 4 residues after the Pro in sequence and the first residue of helix α3 (Figure 10).
The α4-β5-α5 face of the receiver domain has been proposed to be important for the function of RRs [32, 33]. Many RRs in the OmpR/PhoB subfamily are thought to be dimers in their active form. Structures of several RR receiver domains are found to form dimers through the α4-β5-α5 face when activated. Exposed side chains on this surface are primarily hydrophilic, suggesting that any interactions through this α4-β5-α5 face are likely to be dynamic. Comparison among receiver domain structures reveals that this face is considerably
variable. The sequence of helix α5 is well conserved among OmpR/PhoB subfamily RRs (Figure 10). Yet the length of this helix varies among known structures . In the structure of PrrA from
Activation of RRs by phosphorylation induces structural changes in the α4-β5-α5 face. This is accomplished by movements of two key residues, a conserved Thr/Ser at the C-terminus of β4 and a moderately conserved Tyr/Phe in the middle of β5 (Figure 11). These residues are referred to as switch residues. In the unphosphorylated RR, the side chain of Thr/Ser is oriented away from the phosphoacceptor, and that of Tyr/Phe extends outward toward the surface of the α4-β5-α5 face. Structures of activated receiver domains reveal that these side chains move in response to phosphorylation [36, 37]. All activated receiver domains of the OmpR/PhoB subfamily form a symmetric dimer through the same interface involving α4-β5-α5. Phosphorylation of the phosphoacceptor aspartate residue allows the side chain of the Thr/Ser residue to move closer to the phosphate group to make a favorable hydrogen bond. Repositioning the Thr/Ser side chain shifts the β4-α4 loop and helix α4, and makes the inward conformation of the Tyr/Phe residue more energetically favorable. Another conserved residue, a Lys in the β5-α5 loop, forms a salt bridge to the phosphoryl group and brings some modest shift in the β5-α5 loop . This Lys side chain has a salt bridge to the side chain of phosphoacceptor aspartate in the unphosphorylated structure PhoP from
3.2. Structures of the effector domains
Unlike the receiver domains, the effector domains are very diverse, reflecting the diversity of cellular processes that are controlled by two-component systems. Effector domains are also referred to as output domains. They can be DNA-binding, RNA-binding, ligand-binding, or transporter output domains, or enzymes . Except in archaea, which has almost 50% of RRs containing only the receiver domain, a great majority of bacterial RRs are transcription regulators that contain a C-terminal effector domain as a DNA-binding domain.
DNA-binding effector domains are grouped into several subfamilies based on predicted domain structures from amino acid sequences . The largest subfamily is the OmpR/PhoB group, whose structure has a winged helix-turn-helix (wHTH) motif. The second largest subfamily is the NarL/FixJ group, which has a helix-turn-helix (HTH) DNA-binding motif. Another subfamily LytR/AgrA is also fairly common. A structure of a member of this subfamily, LytTR from
The DNA-binding domain of the OmpR/PhoB subfamily has a conserved winged helix-turn-helix fold (Figure 12a). The structure starts with an N-terminal four-stranded anti-parallel β-sheet (β6 to β9), followed by three α-helices (α6 to α8) in the middle, and a β-hairpin (β11 and β12) at the C-terminus. A short strand (β10) between α6 and α7 assembles with the C-terminal β-hairpin to form a three-stranded anti-parallel β-sheet. The two β-sheets sandwich the three α-helices in the middle. The β-hairpin is called the wing, and α7, α8 and loop α7-α8 together constitute the helix-turn-helix motif. Helix α8 is the sequence recognition helix, which inserts into the major groove of DNA . The β-hairpin (wing) binds in the minor groove of DNA. Loop α7-α8 is termed transaction loop because it is essential for transcription activation.
The C-terminal DNA-binding domain of the NarL/FixJ subfamily is a compact bundle of 4 α-helices (Figure 12). The second and third helices (α8 and α9) form a helix-turn-helix motif that interacts with the major groove of DNA [41, 42]. Helix α9 is the recognition helix that inserts into major groove of DNA with side chains interacting with DNA bases. The proteins bind DNA as a symmetric dimer to recognize palindromic DNA repeats. Helix α10 and the loop α7-α8 form the protein dimer interface in the protein-DNA complex. Isolated DNA-binding domains, however, are found to be monomeric in solution when not binding to DNA .
3.3. Structures of full-length response regulators and regulation mechanism
Full-length structures are available for the OmpR/PhoB and NarL/FixJ subfamilies of response regulators. Even though most RRs have only two domains, full-length structures prove to be difficult to obtain, especially for the OmpR/PhoB subfamily. This is most likely due to the dynamic nature of interactions between the receiver and effector domains. The domains are individually folded, and their associations are transient if they do associate with each other. The dynamic and transient nature of domain interactions is important for the regulation of DNA-binding activities. Signals transmitted from the HK through phosphorylation of the receiver domains must be able to relay to the DNA-binding domain by conformational changes of the receiver domain. Phosphorylation promotes dimerization of the receiver domain and in some cases releases the blockage of the DNA-binding element of the receiver domain. So far, all available structures of full-length RRs are non-activated and without DNA bound.
The full-length NarL structure in its unphosphorylated form reveals that the receiver domain is associated with the DNA-binding domain through an interface involving the DNA-binding elements . The linker between the two domains forms a helix (α6) followed by a flexible loop that is disordered in the crystal structure (Figure 13). Helix α6 packs against α4 of the receiver domain and α10 of the effector domain. The domain interface also involves helices α8 and α9, locking the DNA-sequence-recognition helix α9 in a manner incapable of inserting into the DNA major groove. Phosphorylation is expected to dissociate the effector domain from the receiver domain, thus allowing it to bind DNA. Unphosphorylated NarL does not bind DNA in solution. NarL binds to inverted-repeat DNA sequences as a symmetric dimer, whose interface involves helix α10 and loop α7-α8 (Figure 12). Phosphorylation may also promote dimerization of the receiver domain and thus strengthens the NarL dimer.
Full-length structures of unphosphorylated StyR and DosR, which are members of the NarL/FixJ subfamily RRs, are also known (Figure 13). The receiver domain of StyR from
rearrangements that allows the receiver domain to form a dimer of (βα)5 fold and release the effector domain to bind DNA inverted repeats .
OmpR/PhoB subfamily RRs have been extensively studied in recent years. In addition to many isolated receiver and effector domain structures, there are six full-length RR structures available, DrrB  and DrrD  from
The structures of DrrD and DrrB both reveal monomers in crystal and have interactions between domains that do not preclude DNA binding (Figure 14). The interdomain interactions are relatively weak, suggesting that in solution the domains could be free and thus phosphorylation activation mechanism is likely through dimerization of the N-terminal receiver domain. The structures of PrrA and MtrA show more extensive interdomain interactions that involve the α4-β5-α5 face and block the recognition helix. However, the interfaces are polar in nature, and the open conformation is likely to exist in solution for both proteins. All these structural data suggest that phosphorylation is likely to shift the equilibrium toward dimerization as a mechanism to activate the effector domain.
The structure of PhoP from
4. Interactions between histidine kinases and response regulators
To complete the signaling events of two-component systems, histidine kinases, when activated, must associate with their cognate response regulators and transfer the phosphoryl group from the phosphorylated histidine to a conserved aspartate residue of RRs. Evidence suggests that HKs are also involved in dephosphorylation of RRs to turn off the signal cascade. The interactions between HK and RR must be specific because there is little cross-talk among TCS proteins. Yet, these interactions must be dynamic, as both proteins must adopt various conformations throughout the steps of the signaling event.
Several crystal structures of HK in complex with RR have been reported that offer insights into how HK and RR interact. A structure of the complex between HK853 and RR468 from
The RR binds to the DHp domain on the helical hairpin below the phosphorylation site histidine. Helix α1 of RR interacts with both helices of the helical hairpin through hydrophobic side chains to form a six-helix bundle. Each α1 interacts only with helices of the same subunit of DHp. Loop β5-α5 also makes contacts with the DHp domain. In the crystal structure, there is a sulfate ion bound between the phosphoacceptor aspartate of RR and the phosphorylation site histidine of DHp. Binding of the sulfate mimics phosphorylation and allows the switch residues of RR to adopt the active conformation. Consequently, both loops β3-α3 and β4-α4 are also in the active conformation. These two loops interact with the ATP-lid and loop α2-β2 of the CA domain. The interactions between HK853 and RR468 would preclude dimer formation of RR through the α4-β5-α5 face, despite that the RR is in an active conformation. RR468 is a single domain protein. The structure of activated RR468 with BeF3- shows changes in conformation of switch residues and loops β3-α3 and β4-α4, but it is a monomer . This is different from OmpR/PhoB subfamily RRs, whose activated form promotes dimerization, or NarL/FixJ family RRs, whose activated form releases the effector domain for binding DNA and might also promote dimerization. Despite these differences, binding interactions of α1 of RR with the helical hairpin of DHp is likely conserved among TCS proteins. The structure of HK853-RR468 complex is in a phosphatase-competent state because the RR is phosphorylated and the ATP-lid blocks access of the γ-phosphate of ATP by the histidine side chain. The structure indicates that the phosphorylation site histidine could play a role in the phosphatase activity by orienting a water molecule as a nucleophile.
A complex structure of ThkA/TrrA, also from
A structure of the Spo0F-Spo0B complex is related to HK-RR complexes in binding interactions and the mechanism of the catalyzed reactions. Spo0B is a phosphotransferase having a DHp domain similar to that of HKs, while Spo0F is a single domain RR [54, 55]. Spo0F is phosphorylated on its phosphoacceptor aspartate by sensor HK KinA. The phosphoryl group is then transferred to the phosphorylation site histidine on spo0B, which then transfers the phosphoryl group to the response regulator Spo0A. In the structure of the Spo0F-Spo0B complex, Spo0F binds to the four-helix bundle of Spo0B in a similar manner as the HK853-RR468 complex. Helix α1 of Spo0F packs against the helical hairpin region and makes up majority of the interface with DHp, confirming that helix α1 and the lower end of the DHp four-helix bundle as the binding interface between HK and RR.
5. Therapeutic potential against bacterial pathogens
Antibiotics resistance has been a major medical problem of modern medicine. Bacterial pathogens evolve mechanisms to become resistant to antibiotics, and strains resistant to multiple drugs or the last line of antibiotics, such as methicillin-resistant
Two component systems are major signaling proteins in bacteria. They are involved in every aspect of bacterial adaptation to their environments, including sensing the existence of antibiotics and regulating cellular responses to become drug resistant. Therefore, TCSs are potential drug targets for developing new antibiotics . Unlike conventional antibiotics that directly target the proteins involved in essential cellular activities, drugs inhibiting TCSs target the upstream functions that regulate these essential proteins. Thus anti-TCS drugs work in a manner different from conventional drugs and are likely to be effective against drug-resistant bacterial pathogens. Because TCS proteins are absent in animals, drugs targeting TCSs can potentially have less toxicity.
Recent advancements in structural and functional characterization of TCSs have identified several potential targets for developing new antibiotics . A PhoP-PhoQ system from
The author thanks Dr. Chou-Zen Giam, Dr. Linda Miallau, and Dr. Issar Smith for helpful comments and revisions of the manuscript. This work was supported by a grant GM079185 from the National Institute of Health of the United States.