Lethal potency and protein-binding affinity of Atxs, Atns, DPLA2 and their mutants. IC50 values (the concentration of competitor sPLA2 required to reduce the binding of 10 nM 125I-AtxC by 50%) were determined from competition binding experiments for binding to calmodulin (CaM), the mitochondrial receptor R25 and the neuronal M-type sPLA2 receptor, R180. #The recombinant toxin did not completely inhibit the binding of 125I-AtxC.
Secreted phospholipases A2 (sPLA2s) constitute a large family of interfacial enzymes that hydrolyze the
The most potent sPLA2 toxins display presynaptic (ß-)neurotoxicity by attacking the presynaptic site of neuromuscular junctions. The venom of the nose-horned viper,
In spite of numerous attempts to identify the surface residues of sPLA2s crucial for a particular pharmacological effect (i.e., the “ß-neurotoxic site” or the “anticoagulant site”) ‒ based initially on structural analysis, chemical modification and, later on, site-directed mutagenesis, the molecular basis of their toxicity has yet to be resolved (Kini, 2003; Križaj, 2011; Pungerčar & Križaj, 2007; Rouault et al., 2006). We have addressed this issue in studies based on protein engineering of the nosed-horned viper sPLA2s. These have resulted in more than fifty mutants and chimeric sPLA2 proteins that have been produced and characterized in terms of their biochemical and biological activities (most of them are shown in Table 1). The site-directed mutagenesis studies have provided answers to, or at least significantly improved, our knowledge concerning many important questions regarding the toxic and enzymatic activities of Atxs and other sPLA2s, such as:
Which regions of the molecule are important for the ß-neurotoxicity of Atxs and homologous toxic sPLA2s?
Which surface residues of Atxs are crucial for interaction with their high-affinity binding proteins? Is there a correlation between the affinity for binding to a particular protein target and the neurotoxic potency of Atxs, suggesting a role for the receptor protein in the process?
Which residues are responsible for interfacial membrane binding of Atxs? How do the interfacial membrane and kinetic properties of Atxs influence their toxic action? How do the enzymatic properties of Atxs compare with those of the best-studied mammalian sPLA2s?
What is the importance of the loss in evolution of enzymatic activity in the case of the AtnL and other myotoxic sPLA2-homologues?
The results obtained have contributed significantly to a better understanding of the molecular mechanisms of action of snake venom sPLA2s and provided clues to the action of the homologous groups of mammalian sPLA2s.
2. Search for the “neurotoxic site” and the role of enzymatic activity
The significant structural similarities of toxic and non-toxic sPLA2s, which differ in their pharmacological actions, have enticed a large number of researchers hoping to find the “holy grail” of sPLA2-toxin research – the toxic site. The site is presumed to comprise only a small number of crucial amino acid residues (Kini, 2003). To explain the wide range of pharmacological effects induced by snake venom sPLA2s, Kini & Evans (1989) proposed a model comprising specific “target sites” present on the surface of particular cell types. The target sites are proposed to be recognized by complementary “pharmacological sites” on the toxin molecule, these being structurally distinct from, and independent of, the “catalytic site.” Thus, high-affinity binding (at least in the nM range) of the toxin to specific target sites ensures that, upon entering the circulation, each toxin binds primarily to its proper target tissue. It is highly likely that the primary target, or acceptor, sites are proteins. This is because of the much lower affinity (mM–µM range) of sPLA2s for binding to the abundant zwitterionic phospholipid surfaces (i.e., cell membranes) (Bezzine et al., 2002; Petan et al., 2005; Singer et al., 2002) and, following enzyme adsorption to the membrane surface, indiscriminate binding to and hydrolysis of phospholipid molecules at the catalytic site. Therefore, separate pharmacological sites on an sPLA2 molecule recognizing different target binding sites should be the main structural determinants that differentiate their respective pharmacological actions, such as presynaptic or central neurotoxicity. However, according to the results of our mutagenesis structure-function studies of Atxs reviewed below, it is unlikely that there is a structurally distinct, single “presynaptic neurotoxic site” located in a specific part of the molecule, in contrast to the strict physical localization of the enzyme active site. Rather, different parts of the toxin molecule are likely to be involved in different stages of the complex multi-step process of neurotoxicity, all contributing to the final outcome. In this view, structurally different ß-ntxs may have different surface regions that bind to different (extra- and intracellular) targets, which are nevertheless involved in the same process, most probably the recycling of synaptic vesicles. However, they all share the nonspecific sPLA2 activity, i.e., the ability to bind and hydrolyze different phospholipid molecules embedded in membranes of various compositions ‒ an essential step in the complete, irreversible blockade of neuromuscular transmission. Therefore, at least in the case of ß-neurotoxic sPLA2s, the use of the term “presynaptic neurotoxic site” appears unsuitable for describing the multiple regions distributed on the surface of the sPLA2 molecule (Prijatelj et al., 2008).
Given the multi-step, and as yet incompletely known, molecular events leading to presynaptic neurotoxicity of sPLA2s, a simple correlation between their
3. Structural determinants of presynaptic neurotoxicity of sPLA2s
The subtlety of the structure-function relationship of sPLA2 neurotoxins is obvious on examination of the primary structures and toxicities of Atxs. The three sPLA2 toxins each consist of 122 amino acid residues and differ at only five positions (Križaj, 2011). AtxC may be considered as a natural double mutant (F124I/K128E) and AtxB as a triple mutant (Y115H/R118M/N119Y) of AtxA. Nevertheless, their lethal potencies in mice differ considerably. AtxA is the most lethal; and its protein isoforms, AtxC and AtxB, are 17- and 28-fold less potent, respectively (Thouin et al., 1982). The crystal structures of recombinant AtxA (PDB code 3G8G) and natural AtxC (PDB code 3G8H) demonstrate the absence of significant structural differences between the two toxins (Saul et al., 2010). There is only a minor conformational difference at positions 127 and 128 in the C-terminal region, caused by the charge-reversal substitution of Lys128 for Glu, which does not significantly influence the toxicity (Saul et al., 2010). An illustrative example of the subtle structure-function relationships of sPLA2s is the conversion, by a single mutation (F22Y), of the gene for bovine pancreatic group IB sPLA2 to a gene encoding a molecule able to compete with crotoxin, a ß-neurotoxic sPLA2 from the South American rattlesnake,
By substituting several basic residues in the C-terminal region (AtxANNTETE mutant: AtxA-K108N/K111N/K127T/K128E/E129T/K132E) and in the ß-structure region (AtxASSL mutant: AtxA-K74S/H76S/R77L) with acidic and non-ionic residues, we have shown, contrary to previous beliefs, that the basic character of Atxs, and probably of other ß-neurotoxic sPLA2s, is not obligatory for presynaptic toxicity (Ivanovski et al., 2004; Prijatelj et al., 2000; Table 1). According to our earlier structure-function analyses, the more than one order of magnitude lower toxicity of AtxC than that of AtxA is a consequence of the substitution of the aromatic Phe124 by Ile (Pungerčar et al., 1999). Furthermore, in accordance with the three substitutions responsible for the difference in toxicities of AtxA and AtxB, several other C-terminal residues of AtxA, namely the Tyr115/Ile116/ Arg118/Asn119 (YIRN) cluster, were shown to be important for the neurotoxicity of Atxs (Ivanovski et al., 2000). Thus, the lethal potency of the AtxA-Y115K/I116K/R118M/N119L (AtxAKKML) mutant was 290-fold lower than that of AtxA (Ivanovski et al., 2000 and Table 1).
The KKML cluster is present in the weakly neurotoxic DPLA2 from the venom of Russell’s viper,
Our next objective was to determine the contribution to the high neurotoxic potency of AtxA of the remaining nine N-terminal residues that differentiate it from DPLA2 (Met7, Gly11, Asn17, Pro18, Leu19, Thr20, Phe24, Val31 and Ser67). As described above, the importance of the aromatic Phe24 had already been established (Petan et al., 2002). In order to assess the importance of the remaining N-terminal residues, we first substituted Met7, Gly11 and Val31 in the highly toxic chimera AtxA/DPLA2YIRN by the corresponding residues present in DPLA2 ‒ lys, glu and trp, respectively. The mutant protein AtxAKEW/DPLA2YIRN displayed a 20-fold lower lethal potency than the AtxA/DPLA2YIRN chimera, suggesting involvement of the group of residues at positions 7, 11 and 31 in AtxA neurotoxicity. The lethality of the partial mutants, AtxAKE/DPLA2YIRN and AtxAW/DPLA2YIRN (Table 1), revealed that the contribution of the pair of residues in the N-terminal helix, Met7 and Gly11, to the neurotoxic potency of AtxA is substantially higher than that of Val31 ‒ in accordance with the negligible effect of the V31W mutation on the lethality of AtxA, despite its outstanding effect on enzymatic activity (Petan et al., 2005). Interestingly, the bulky Trp at position 31 of the AtxAV31W and AtxAW/DPLA2YIRN mutants had no significant impact on neurotoxicity, despite being spatially very close to Phe24 and also to the YIRN cluster. The collective contribution of the N-terminal residues Met7, Gly11 and Phe24 to the neurotoxicity of AtxA is very significant and similar to that of the YIRN cluster in the C-terminus. For example, the substitution of Phe24 with ser in AtxA caused an approximately 19-fold decrease in neurotoxicity (Petan et al., 2002), which is similar to the reduction seen after adding the N-terminal region of AtxAKEW, in which the residues Met7, Gly11 and Val31 were substituted with lys, glu and trp, respectively, to DPLA2YIRN, resulting in the AtxAKEW/DPLA2YIRN chimeric mutant. Therefore, it is highly likely that the remaining N-terminal residues differentiating AtxA and DPLA2, i.e., the Asn17/Pro18/Leu19/Thr20 cluster and Ser67, are not greatly involved in neurotoxicity.
Our structure-function studies of ß-neurotoxic sPLA2s (Ivanovski et al., 2000, 2004; Petan et al., 2002, 2005; Prijatelj et al., 2000, 2002, 2003, 2006b, 2008; Pungerčar et al., 1999) clearly show that different parts of the toxin molecule have separate roles in the distinct steps of the complex mechanism of presynaptic neurotoxicity (Pungerčar & Križaj, 2007). Most significantly, by selectively mutating parts of the DPLA2 molecule, we were able to map the residues that separate the weakly ß-neurotoxic sPLA2 from the 150-fold more potent AtxA (Prijatelj et al., 2008). In summary, the most important structural features responsible for the high neurotoxic potency of Atxs (Figure 2, A) are: the “upper” part of the molecule concentrated around the C-terminal region 115-119 (the YIRN cluster) and including the spatially close aromatic Phe124 and Phe24, and the N-terminal helix region with the Met7/Gly11 pair in the “lower right” part of the molecule.
4. Enzymatic activity of Atxs: factors influencing interfacial binding and hydrolysis of phospholipid membranes
Secreted PLA2s are prototypical interfacial enzymes (Berg et al., 2001). In order to gain access to their phospholipid substrate, sPLA2s first have to bind at the lipid/water membrane interface by their interfacial binding surface (IBS), a group of residues located on a relatively flat exposed region surrounding the entrance to the active site pocket (Lin et al., 1998; Ramirez & Jain, 1991). Only then, after the enzyme is bound to the surface, can binding of a single phospholipid molecule and its hydrolysis occur in the active site. The catalytic turnover cycle of sPLA2s includes a highly conserved His48‒Asp99 dyad and an activated water molecule that acts as a nucleophile during hydrolysis of the
The IBS of most sPLA2s comprises a ring of conserved hydrophobic residues, whereas, around them and on the edges of the IBS, some polar, basic and acidic residues are present (Petan et al., 2005; Snitko et al., 1997). Given that most sPLA2s have high activities on anionic membrane surfaces, it was long thought that electrostatic forces between cationic residues of the enzyme and anionic membrane phospholipids are crucial for the membrane binding and activity of sPLA2s (Scott et al., 1994). However, a number of studies have shown that, in fact, hydrophobic, aromatic and hydrogen-bonding interactions account for most of the binding energy, even to negatively charged membrane surfaces (Gelb et al., 1999; Ghomashchi et al., 1998; Lin et al., 1998). Nevertheless, it has been suggested that electrostatic interactions are responsible for proper orientation and initial association of sPLA2s with both anionic and zwitterionic membranes. They may significantly modulate the dynamics of establishing strong hydrophobic interactions, when aliphatic and aromatic residues on the IBS penetrate the membrane surface, needed to form a stable sPLA2-membrane complex (Beers et al., 2003; Petan et al., 2005; Prijatelj et al., 2008; Stahelin and Cho, 2001).
4.1. Basic structural, interfacial kinetic and binding properties of Atxs
Atxs are highly basic proteins (AtxA has a pI of 10.2, net charge +6), with structural features typical of group IIA sPLA2 enzymes (Figure 2, A): an N-terminal α-helix A (residues 1–14), a short α-helix B (residues 16–22), a Ca2+-binding loop (residues 25–35), a long α-helix C (residues 39–57), a loop preceding an antiparallel two-stranded ß-sheet (ß-structure; residues 75–78 and 81–84), a long α-helix D (antiparallel to helix C; residues 89–109) and a C-terminal extension (mostly disordered, with two short helical turns; residues 110–133). The active site cleft is buried at the end of a hydrophobic channel (formed by the residues Phe5, Gly6, Tyr22, Ser23, Cys29, Gly30, Ala102, Ala103, Phe106) leading to the highly conserved His48–Asp99 dyad located between the antiparallel α-helices C and D. The entrance to the catalytic site of all sPLA2s is surrounded by the IBS group of residues, forming a relatively flat surface on one side of the molecule that is responsible for membrane binding – the first and prerequisite step in sPLA2 catalytic action. The IBS of Atxs (Figure 2, B) is formed by Leu2, Leu3, Leu19, Thr20, Phe24, Val31, Ser67, Lys69, Thr70, Arg72, Arg118, Asn119, and Phe124 (Petan et al., 2005). The slight structural flexibility observed for the exposed side chains of residues in the IBS (e.g., Phe24 in Figure 2, B) is in keeping with the role of these residues in supporting optimal interactions of the molecule with the dynamic structure of phospholipid aggregates (Saul et al., 2010).
According to the enzymatic activities and membrane-binding affinities of Atxs determined on different phospholipid vesicles, it is clear that these snake venom sPLA2s are very effective in binding to and hydrolyzing different phospholipid membranes (Petan et al., 2005). This property exists despite the fact that they have evolved to be specific and potent neurotoxic molecules and despite the fact that they share striking structural and functional similarities with the mammalian (non-toxic) sPLA2s. The enzymatic activities of Atxs on vesicles composed of anionic phosphatidylglycerol (PG), which is, in general, the best sPLA2 substrate, are comparable to those displayed by the most potent mammalian sPLA2, the pancreatic group IB sPLA2, and 5-fold higher than those displayed by the mammalian group IIA sPLA2s on these vesicles (Singer et al., 2002). Atxs also have particularly high activities on phosphatidylserine (PS) vesicles, the main anionic phospholipid in eukaryotic membranes, well above the activities of the group IB and IIA sPLA2s that are the most active mammalian sPLA2s on these vesicles. Most importantly, the activities of Atxs were high also on pure zwitterionic phosphatidylcholine (PC) vesicles, much higher than that of the highly homologous and cationic hGIIA enzyme, which cannot bind to the PC-rich plasma membranes of mammalian cells (Beers et al., 2003; Bezzine et al., 2000, 2002; Birts et al., 2009; Singer et al., 2002). The hGIIA sPLA2 is well known for its preference for anionic phospholipid substrates and its negligible activity on PC-rich membrane surfaces (Beers et al., 2003; Bezzine et al., 2000, 2002). These properties strongly influence the physiological role of the hGIIA enzyme, for example, by enabling its high antibacterial concentrations in human tears without affecting the corneal epithelial cells (Birts et al., 2009). Although the activities of Atxs were lower than those displayed by group V and X sPLA2s, which are by far the most potent among the mammalian sPLA2s in hydrolyzing PC vesicles and releasing fatty acids from cell membranes (Bezzine et al., 2000, 2002; Singer et al., 2002), they were able to hydrolyze plasma membranes of different intact mammalian cells at a rate that correlated well with their specific activities on PC vesicles (Petan et al., 2005; Pražnikar et al., 2008). The high activity of Atxs on PC-rich vesicles is a consequence of the ability of these venom sPLA2s to bind well to such membrane surfaces, with affinities comparable to those of mammalian group V and X sPLA2s. Furthermore, unlike the neutral hGX and similarly to the highly cationic hGIIA enzyme, the presence of anionic phospholipids in the membrane surface greatly enhances the membrane-binding affinity of Atxs and consequently the rate of phospholipid hydrolysis (Bezzine et al., 2000, 2002; Petan et al., 2005, 2007). This property may have an important influence on both localization of the toxin to its target membrane and its enzymatic effectiveness
4.2. Role of different IBS residues in supporting interfacial binding and activity of Atxs
The presence of tryptophan on the IBS of different sPLA2s is a well-known determinant of their ability to bind with high affinity and hydrolyze PC-rich membranes, crucially influencing their biological roles. Its role has been highlighted in the cases of the hGV and hGX enzymes (Bezzine et al., 2002; Han et al., 1999), the acidic sPLA2 from
The role of aromatic residues in the interfacial binding of sPLA2s depends to a high degree on the nature of the residue itself, its position on the IBS and the orientation of its side-chain (Stahelin & Cho, 2001; Sumandea et al., 1999). This is evident from the fact that the substitution of Phe24 with Trp did not cause a substantial increase in enzymatic activity or interfacial binding affinity of AtxA (Petan et al., 2002, 2005) or hGIIA (Beers et al., 2003). Thus, both Phe and Trp can have similar roles in interfacial binding, despite the differences in their interactions with the membrane: the highly amphiphilic Trp favours partitioning in the interfacial phospholipid head group region of the bilayer (Yau et al., 1998), while the aromatic Phe penetrates deeper into the hydrophobic core of the phospholipid acyl chains (Stahelin & Cho, 2001; Sumandea et al., 1999). In the case of AtxA and hGIIA, Trp31 is obviously in a much better position to influence interfacial binding than is Trp24. Additionally, AtxA and AtxAF24W display higher activities and binding affinities than do the F24S, F24Y and F24N mutants on PC-rich vesicles containing anionic phospholipids. Although the structures of Trp and Phe differ significantly, both are obviously better suited to take advantage of the presence of anionic phospholipid in the interface than the polar Ser, Asn and even the aromatic Tyr. The importance of non-polar interactions in interfacial binding to negatively charged surfaces is also clear in the case of AtxB, AtxAKKML and AtxAV31W. These molecules already display very high activities on PC vesicles containing 10% PS (10% PS/PC vesicles), and they reach the level of maximal activity already on 30% PS/PC vesicles, indicating that the enzymes are fully bound to these vesicles (Petan et al., 2005). In general, we have observed the greatest increases in activity upon introduction of 10% anionic phospholipids (PS or PG) into PC vesicles for mutants that have numbers of hydrophobic and aromatic residues on their IBS similar to or higher than those in AtxA. Besides providing the basis for electrostatic interactions, the presence of anionic phospholipids in PC vesicles may facilitate non-polar interactions as a result of membrane perturbation (Buckland & Wilton, 2000).
Despite the remarkable impact of Trp at position 31 on interfacial binding of Atxs and other sPLA2s, such as hGIIA (Beers et al., 2003), our subsequent site-directed mutagenesis studies revealed that the positive influence of Trp on membrane binding of sPLA2s may be significantly diminished, depending on the delicate balance of contributions of each IBS residue to interfacial binding. While Atxs and hGIIA sPLA2 do not contain a Trp on their IBS, DPLA2, a weakly neurotoxic sPLA2 from Russell’s viper that differs from AtxA in only 22 residues, has a Trp residue at position 31 and yet displays interfacial binding and kinetic properties similar to those of AtxA (Petan et al., 2005; Prijatelj et al., 2003, 2008). Besides V31W, DPLA2 contains several other substitutions of equivalent AtxA IBS residues: L19I, T20P, F24S, S67N, T70S, R118M and N119L (Petan et al., 2005; Prijatelj et al., 2008). By analysing the properties of a range of AtxA/DPLA2 mutants and chimeras (Prijatelj et al., 2008), we were able to pinpoint the IBS residues that are crucial for the intriguing differences in interfacial binding and activity of AtxA and DPLA2. Not surprisingly, the introduction of Trp31 to the AtxA/DPLA2YIRN chimera (see Figure 1 for sequence data) caused a very significant 50-fold increase in the rate of hydrolysis of PC vesicles. However, the impact of the same Trp residue, when introduced along with two additional substitutions, producing the AtxAKEW/DPLA2YIRN mutant, was almost completely abolished, resulting in a low level of activity on PC vesicles, only 2 to 4-fold higher than those of AtxA/DPLA2YIRN, AtxA and DPLA2. These results clearly confirmed our earlier suggestion (Petan et al., 2005) that the well-known positive impact of Trp31 on the interfacial binding and enzymatic activity of mammalian and venom sPLA2s is, to a large extent, counterbalanced by the presence of Lys7 and Glu11 on the edges of the IBS in the case of DPLA2, instead of the hydrophobic Met7 and nonpolar Gly11 in AtxA. This is most probably a consequence of altered orientation of DPLA2 on the membrane, due to strong electrostatic interactions between Lys7 and Glu11 and the zwitterionic membrane surface, which prevents the productive interaction of Trp31, and thus DPLA2, with the interface. Similarly, two basic residues at nearly equivalent positions in the hGIIA enzyme, Arg7 and Lys10, were shown to have a negative influence on interfacial binding (Bezzine et al., 2002; Snitko et al., 1997). In agreement with this, we have shown that the substitution of Arg at position 72 of the IBS of Atxs and DPLA2 has a positive impact on interfacial binding and activity only upon introduction of a hydrophobic (Ile), but not a polar (Ser), acidic (Glu) or other basic (Lys) residue (Ivanovski et al., 2004). An additional, albeit smaller, negative effect on interfacial binding and enzymatic activity of DPLA2 is provided by the presence of the polar residue Ser24 instead of the aromatic and hydrophobic Phe in AtxA (Petan et al., 2002, 2005). However, there are several residues on the IBS of DPLA2, but not Atxs, that significantly improve its interfacial binding and activity. The YIRN cluster in the C-terminal part of AtxA, shown to be important for its neurotoxic effect (Ivanovski et al., 2000), contains the hydrophilic IBS residues Arg118 and Asn119, while the hydrophobic Tyr115 and Ile116 are not part of the presumed IBS. The introduction of the YIRN cluster into DPLA2, instead of its KKML cluster, or into the various AtxA/DPLA2 variants, resulted in a substantial decrease of the initial rates of hydrolysis of charge-neutral PC vesicles (Table 2). Therefore, the presence of a hydrophobic or aromatic IBS residue at positions 118 and 119 has a significant positive influence on interfacial binding to anionic and, particularly, zwitterionic phospholipid surfaces. Accordingly, this positive impact is evident in the case of Met118 and Leu119 in DPLA2 and the AtxAKKML mutant, as well as of Met118 and Tyr119 in AtxB, in contrast to the hydrophilic Arg118 and Asn119 in AtxA and mutants containing the YIRN cluster (Ivanovski et al., 2000; Petan et al., 2005; Prijatelj et al., 2003, 2008). Considering the similar activities on PC-rich vesicles displayed by AtxB and the AtxAKKML mutant, it is obvious that the roles of Tyr and Leu at position 119 are in fact very similar. This indicates that the removal of the polar cluster Arg118/Asn119 from AtxA has a greater positive effect on interfacial binding than the aromatic or hydrophobic nature of the substituting residue at position 119, which is in accordance with the negligible impact of the substitution Y119W on interfacial binding of hGIIA sPLA2 to 1,2-dioleoyl-
5. Interaction of Atxs with high-affinity binding proteins
Several high-affinity binding proteins for Atxs have been identified in porcine cerebral cortex. Two of these are membrane proteins of 25 kDa (R25) and 180 kDa (R180). R25, which is located in the mitochondrial membrane (Šribar et al., 2003a), binds only Atxs (Vučemilo et al., 1998), whereas R180, identified as the plasma membrane M-type sPLA2 receptor (sPLA2R), binds both toxic and non-toxic sPLA2s of groups IB and IIA (Čopič et al., 1999; Vardjan et al., 2001). Surprisingly, several cytosolic, high-affinity binding proteins for Atxs were identified as well ‒ calmodulin (CaM) (Šribar et al., 2001), the γ and ε isoforms of 14-3-3 proteins (Šribar et al., 2003b), and protein disulphide isomerase (Šribar et al., 2005). This suggests the possibility of not only intracellular, but also cytosolic, action of Atxs and other sPLA2s. They have since been shown to be surprisingly stable in the reducing environment of the eukaryotic cytosol. They are enzymatically active at low micromolar concentrations of Ca2+ ions, and they are also structurally stabilized and activated by binding to CaM (Kovačič et al., 2009, 2010; Petan et al., 2005; Petrovič et al., 2005). Atxs were also found to bind factor Xa (FXa) and display anticoagulant activity (Prijatelj et al., 2006a). Given that the topic of neuronal high-affinity binding proteins for Atxs and their putative roles in ß-neurotoxicity has been reviewed recently (Pungerčar & Križaj, 2007), here we only summarize the results obtained with a range of Atx mutants for binding to CaM, R25, FXa and the M-type sPLA2 receptor.
5.1. The C-terminal region of Atxs has a novel calmodulin binding motif
Our protein engineering and competition binding studies using a range of Atxs, DPLA2 and AtnI2 mutants and chimeras, as well as synthetic peptides corresponding to different regions of Atxs, ultimately resulted in the identification of a novel binding motif for the ubiquitous, highly conserved Ca2+-sensor CaM (Kovačič et al., 2010; Prijatelj et al., 2003). The C-terminal region of AtxA, with a distinctive hydrophobic patch within the region 107–125 surrounded by several basic residues, was identified as the most important element for binding to the N-terminal methionine-rich pocket of CaM in its Ca2+-induced dumbbell conformation. This work was based on the intriguing 150-fold lower lethal potency and the correspondingly 50-fold lower CaM-binding affinity of DPLA2 compared to AtxA (Prijatelj et al., 2003). Most notably, the DPLA2YIRN mutant, containing the C-terminal YIRN cluster of AtxA (see above and Figure 1), displayed a 7-fold increase in binding affinity for CaM, only 7-fold lower than that of AtxA. This result suggested a major role of this region for the interaction with CaM. On the other hand, introduction of the whole N-terminal half of AtxA to DPLA2 and DPLA2YIRN caused only a slight, 2 to 3-fold increase in binding affinities for CaM, suggesting that the N-terminal part of AtxA has only a supporting role in the binding and is probably located on the periphery of the CaM binding site (Prijatelj et al., 2008). Very recently, we confirmed unambiguously the crucial importance of the C-terminal region of Atxs in CaM binding by building several three-dimensional structural models of sPLA2–CaM complexes, based on AtxA–CaM interaction site mapping, our previous mutagenesis data and protein-docking algorithms (Kovačič et al., 2010). According to the model, the closed conformation of CaM forms a “clamp” around AtxA, with most of the C-terminal residues of AtxA in the region 108-131 being in direct contact with CaM (Figure 2, C). There are only a few additional, but important, contacts between the proteins, formed mainly by basic residues in α-helices C (Arg43 and Asn54) and D (Arg94, Lys108, Asn109 and Lys111) of AtxA. Importantly, the model was validated by superimposing the structures of several mammalian sPLA2s on that of AtxA in the complex and correctly predicting the favourable interactions of group V and X mammalian sPLA2s and CaM. The model also showed the basis for the unfavourable interactions, which prevented complex formation between the structurally very similar mammalian group IB and IIA sPLA2s and CaM. Formation of the complexes was confirmed by competition binding experiments and the augmentation of the enzymatic activity of those sPLA2s interacting with CaM. Most importantly, we have clearly demonstrated that CaM stabilizes and protects Atxs from denaturation in the reducing environment of the cell cytosol and acts as a non-essential activator of Atxs and of the mammalian group V and X sPLA2s. The fact that CaM-binding results in stabilization and augmentation of enzymatic activity of sPLA2s, ranging from the neurotoxic Atxs to the mammalian group V and X sPLA2s, suggests a novel role for CaM as a regulator of the intracellular actions of some toxic and non-toxic sPLA2s, which may be active even in the reducing environment of the cytosol (Kovačič et al., 2009, 2010). The significance of CaM–sPLA2 interactions in the pharmacological and (patho)physiological roles of sPLA2s remain to be confirmed.
5.2. The N- and C-terminal regions of Atxs are involved in binding to the neuronal M-type sPLA2 receptor (R180)
The M-type sPLA2 receptor, which was cloned and revealed as a member of the C-type lectin superfamily, has been well-characterized and is currently the best-known sPLA2 binding target. It was shown recently that the M-type receptor is involved in regulating cell senescence (Augert et al., 2009) and is the main antigen target in autoimmune human membranous nephropathy (Beck et al., 2009). However, the pathophysiological implications of the interaction between the M-type sPLA2 receptors and both toxic and non-toxic sPLA2s in general are intriguing and still unknown (Lambeau & Gelb, 2008; Pungerčar & Križaj, 2007; Rouault et al., 2006). Although it has been suggested that the neuronal M-type sPLA2 receptor located on the plasma membrane could be responsible for specific targeting and internalization of sPLA2 neurotoxins in presynaptic nerve terminals, the results of our mutagenesis studies have shown that it may not be involved in the neurotoxicity of Atxs (Prijatelj et al., 2006b). In order to prevent the interaction between AtxA and the receptor, we prepared three mutants of AtxA, containing either the 12-amino acid-long peptide ARIRARGSIEGR, named 12-AtxA, or one each of two variants of a shorter 5-amino acid peptide (ASIGQ and ASPGQ; named I-AtxA and P-AtxA, respectively) fused to the N-terminus of AtxA. These peptides are similar to the N-terminal propeptides present in proenzymes of mammalian group IB (e.g., DSGISPR in human pancreatic proPLA2) and group X sPLA2s (e.g., EASRILRVHRR in the human proenzyme), which do not bind to the M-type sPLA2 receptor (Hanasaki & Arita, 2002; Lambeau et al., 1995). The presence of the fusion peptides in AtxA indeed completely abolished the interaction of the toxin with the M-type receptor. However, the mutants displayed only one order of magnitude lower lethality in mice and were able to induce neurotoxic effects on a mouse phrenic nerve-hemidiaphragm preparation (see Table 1). Although the N-terminal fusion peptides acted effectively as “propeptides” for AtxA and lowered specific enzymatic activity to ~1% of the wild-type enzyme, their similar neurotoxic profiles on the neuromuscular junction indicate that minimal enzymatic activity suffices for presynaptic toxicity of sPLA2s. Additionally, antibodies targeting the sPLA2-binding C-type lectin-like domain 5 of the M-type sPLA2 receptor were unable to abolish the neurotoxic action of AtxA on the neuromuscular preparation. Thus, the interaction of AtxA with the neuronal M-type sPLA2 receptor, R180, is apparently not essential for its presynaptic neurotoxicity. Since we performed the binding experiments with the N-terminal fusion AtxA mutants, using porcine sPLA2 receptor, the question still remains as to whether the fusion peptides would also abolish the binding of AtxA to the mouse R180 receptor.
Nevertheless, our site-directed mutagenesis studies have provided important information about the parts of the AtxA molecule involved in binding to R180. Interestingly, the C-terminal YIRN cluster, which is essential for neurotoxicity and binding to CaM and R25, appears to be involved in binding to R180 as well. The first clues were provided by the lower affinity of the AtxAKKML mutant and DPLA2 for binding to R180 (Ivanovski et al., 2000). (Both contain KKML instead of the YIRN cluster.) The higher binding affinity of mutants containing the YIRN cluster (DPLA2YIRN or AtxA/DPLA2YIRN) confirmed that it is an important part of the AtxA/R180 interaction surface. However, other parts of the molecule also contribute considerably to the binding affinity of AtxA. Interestingly, the N-terminal part of AtxA in the chimera AtxA/DPLA2 did not increase the binding affinity of DPLA2 to R180. However, the introduction of three N-terminal DPLA2 residues (K, E and/or W) to AtxA/DPLA2YIRN caused an increase in the binding affinity for R180 up to the level of AtxA, confirming the expected involvement of the N-terminal part of the molecule in binding to R180 as well. Therefore, the interaction surface of AtxA with its neuronal M-type receptor, R180, extends from the C-terminal top of the molecule, across the area close to the Ca2+-binding loop and the IBS, reaching the N-terminal helix on the edges of the IBS.
5.3. Atxs bind to the mitochondrial receptor R25 through their C-terminal region
The C-terminal region of Atxs appears to be essential also for binding to the, as yet unidentified, mitochondrial membrane receptor R25. The affinities for binding of the chimeric mutants to R25 ranged from that of the non-toxic AtnI2, which does not bind to R25, to those of AtxA, AtnI2/AtxAK108N and AtnI2N24F/AtxAK108N, which were similar to that of AtxA (Prijatelj et al., 2002). Additionally, substitutions of Phe24 in the N-terminal part of AtxA had a negligible effect on its affinity for R25, but nevertheless indicated that this residue is in the vicinity of the toxin-receptor binding surface, most probably located in the C-terminal part of AtxA (Petan et al., 2002). Furthermore, while DPLA2 and the chimera AtxA/DPLA2 could not completely inhibit the binding of AtxC to R25 in competition binding experiments, the substitution of their KKML cluster with the YIRN cluster led to complete inhibition with a relatively high binding affinity. This implies that residues in the region 115–119 are very important for effective binding to R25, which is apparently present in multiple isoforms. However, the complete inhibition of binding of radiolabelled AtxC to R25 by the AtxAKKML mutant, in contrast to DPLA2 and AtxA/DPLA2, indicated that the interaction surface extends beyond the C-terminal region. Indeed there was a slight increase in binding affinity upon introduction of the N-terminal Lys7, Glu11 and/or Trp31 of DPLA2 to the AtxA/DPLA2YIRN chimera (K, E and/or W; Table 1). While our site-directed mutagenesis and competition binding studies have so far revealed a central role for the C-terminal residues of Atxs in high affinity binding to R25, the interaction surface probably extends beyond the C-terminal region.
5.4. Atxs exert anticoagulant effects by binding to factor Xa
In the analysis of non-neurotoxic effects of Atxs, our study of an array of site-directed mutants (Prijatelj et al., 2006a) showed that basic residues in the C-terminal and ß-structure regions are important for AtxA binding to human blood coagulation factor Xa (Figure 2, D). Hence, AtxA has anticoagulant activity. In addition, the 10-fold lower affinity of AtxC for factor Xa, in comparison with AtxA, is due to the (natural) charge-reversal substitution of Lys128 by Glu, leading to a small local conformational change in the C-terminal end of AtxC, which perturbs the interaction with factor Xa (Saul et al., 2010).
6. Restoration of enzymatic activity reduces the Ca2+-independent membrane damage induced by the myotoxic ammodytin L
Ammodytin L (AtnL) is a myotoxic and enzymatically inactive structural homologue of Atxs (Petan et al., 2007; Pungerčar et al., 1990). The main structural feature of snake venom sPLA2-like myotoxins is the substitution of the highly-conserved aspartic acid residue at position 49, effectively preventing Ca2+ binding and thus abolishing catalytic activity (Petan et al., 2007; Ward et al., 2002). The most common substitution is Lys, but AtnL is one of the two known Ser49 sPLA2 homologues. In addition to this replacement, several other substitutions of highly-conserved residues in the enzymatically inactive sPLA2s are found concentrated in the region of the Ca2+-binding loop, where the carbonyl oxygen atoms of Tyr28, Gly30 and Gly32 provide three additional coordinative bonds for Ca2+-binding. The myotoxic activity of the Lys49 sPLA2s, as well as of the Ser49-containing AtnL, is best characterized by their ability to induce myonecrosis
We recently described the first example of restoration of activity in AtnL or in any enzymatically inactive Ser49/Lys49 sPLA2 homologue (Petan et al., 2007). We prepared two enzymatically active quaternary mutants of AtnL (H28Y/L31V,W/N33G/S49D), differing at position 31. Although Asn33 is not directly involved in Ca2+ coordination, we suspected that it might have a negative impact on the optimal local structure of the Ca2+-binding loop. We therefore replaced it with a glycine residue, which is very often found at this position in catalytically active sPLA2s, as well as in Atxs. Val31 was present in the AtnLYVGD mutant in order to recreate the Ca2+-binding loop of Atxs, while the AtnLYWGD mutant had Trp31 in order to enhance its membrane binding affinity, and thus enzymatic activity, as previously shown in the case of the AtxAV31W mutant described above. The successful restoration of enzymatic activity by such a small number of substitutions indicates that, apart from the residues involved in Ca2+ coordination, the remainder of the substrate-binding and catalytic network of AtnL is very well conserved. Apparently, in evolution, Lys49 and Ser49 sPLA2 myotoxins have lost their Ca2+-binding ability and enzymatic activity through subtle changes in the Ca2+-binding network alone, without affecting the rest of the catalytic machinery. Our results strongly suggest that these changes were selected for their Ca2+-independent membrane-damaging ability and increase the specificity of their myotoxic activity. Although the restoration of enzymatic activity in AtnL increased its cytotoxic potency and lethality in general, it had a negative effect on its Ca2+-independent mechanism of membrane damage and on its ability to specifically target differentiated muscle cells
7. Future perspectives
Recently, we have been able to introduce a free cysteine residue into the molecule of AtxA by substituting Asn79 by Cys at the turn of the antiparallel ß-sheet. This mutation allowed for specific labelling of the neurotoxin molecule by either fluorescent or nanogold probes (Pražnikar et al., 2008, 2009). The conjugates were less toxic than the wild-type toxin, but retained its basic properties. They have proved to be a valuable tool in the study of presynaptic neurotoxicity. As an illustration, we have provided evidence that AtxA, a neurotoxic snake venom sPLA2, is indeed internalized into mammalian (mouse) motor nerve terminals (Logonder et al., 2009).
We believe that similar approaches can be used in further protein engineering of Atxs and also other ß-ntxs, especially in monitoring their trafficking and cellular action (enzymatic activity, binding to protein target molecules). This should finally lead to a complete understanding of the molecular mechanism of presynaptic toxicity of sPLA2 neurotoxins, which may also open the way for better medical treatment of snake envenomation. Moreover, similar research approaches can also be exploited in the studies of homologous mammalian sPLA2s whose biological roles are still to be clarified.
The authors wish to thank Dr Roger H. Pain for critical reading of the manuscript.
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