Gustaf H. Carlson School of Chemistry and Biochemistry, Clark University, Worcester, MA 01610, USA
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Abstract |
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Keywords: calcium binding proteins/dynamics/EF-hand/gateway/modeling/parvalbumin
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Introduction |
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Parvalbumins share with the EF-hand protein superfamily a conserved architecture associated with the Ca2+ ion binding site (Kawasaki et al., 1998).The motif contains a central Ca2+ ion binding loop with the flanking helices positioned roughly perpendicular to each other. The Ca2+ ion binding loop consists of 12 sequential residues, in which those at positions 1, 3, 5, 7, 9 and 12 are involved in coordination to the cation. The cation is coordinated by the oxygen atoms at the axial positions x, y, z, y, x, z in a Cartesian coordinate system (Kretsinger and Nockolds, 1973
). In most of the EF-hand proteins, but not the CD site of parvalbumins, there is a coordinating water molecule at x. In all EF-hand proteins, there is a bidentate carboxyl group at z, consistent with a geometry best described as a `skewed pentagonal bipyramid' (Pauls et al., 1996
; Allouche et al., 1999
). The Ca2+ ion binding ligands in the functional CD and EF sites of SHPV-B are presented in Table I
.
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The central problem to be addressed in this computational study is the effect of selected `gateway' mutations (in the CD site) on the overall conformational stability of the CD helixloophelix motif in both the whole silver hake parvalbumin and in the isolated CD helixloophelix fragment of the whole protein. Of special importance in the current study is the effect that these mutations have on the integrity of the constellation of residues forming the CD Ca2+ ion coordination sphere. The naturally occurring residue at the CD site gateway position in the silver hake parvalbumin protein is glutamic acid (residue position 59). Our approach will be to generate mutant variants with arginine, cysteine or serine at this position and then to subject the variants to molecular dynamics simulations in order to assess their overall conformational stability and their ability to retain the integrity of their CD Ca2+ ion binding site. These three variants were selected for this study for a number of reasons. Arginine was selected because it has never been found at the gateway position of any EF-hand protein. It represents an extreme case of a non-conservative mutation. Cysteine was selected because this residue is observed least in natural EF-hand sites at the gateway position. Finally, serine was selected since it is the most common (by far), non-acidic residue substitution at this position in EF-hand proteins in general, but never in parvalbumins (Falke et al., 1994). Another important reason for selecting this set of variants is that all three were among a set of gateway variants that Drake and Falke (1996) attempted to generate experimentally in the EF-hand-containing galactose binding protein (GBP), using oligonucleotide-directed mutagenesis. Thirteen different GBP variants were made by Drake and Falke (1996): eight with one of the eight naturally occurring residues at the gateway position and five with residues not yet observed at this location (see Table II
). Of the 13 GBP variants produced, five were poorly expressed by Escherichia coli, suggesting that `certain substitutions can inhibit the synthesis of GBP or destabilize the folded protein in vivo'. The residue observed with the lowest frequency in natural EF-hand sequences (Cys, <1%), as well as four residues that have never been observed at the ninth EF loop position (Arg, Met, Leu, Val), each yielded undetectable levels of expression, probably because these side chains block Ca2+ binding or otherwise destabilize the EF-hand motif. The serine variant was well expressed although the mutant protein exhibited reduced thermal stability in the presence of saturating levels of Ca2+ ion. The binding affinity of GBP for Ca2+ ion was greatly increased when either Glu or Asp was substituted for the native Gln, with the largest effect coming from the longer Glu side chain which provides strong, direct metal coordination in many EF-hand binding loops, including the CD site of silver hake parvalbumin.
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Methods |
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The InsightII Biopolymer module was used to generate the arginine (E59R), cysteine (E59C) and serine (E59S) variants of the CD-hand in both SHPV-B and the CD fragment of SHPV-B. In order to mimic aqueous solvent conditions, all molecules to be subjected to simulations were soaked with a 15 Å shell of water molecules (typically ~15002500 water molecules depending on the size of the molecule). Simple molecular mechanics (MM) calculations preceded all molecular dynamics (MD) runs. After generating the `assembly' (protein plus water shell), the water shell was subjected to energy minimization using steepest descents to a maximum derivative <1.0 kcal/mol.Å and then by the conjugate gradient method until the maximum derivative was <0.1 kcal/mol.Å. Following this procedure, the remainder of the complex (i.e. the protein and the shell of water molecules) were energy minimized using steepest descents and conjugate gradient methods until the maximum derivative was <0.1 kcal/mol.Å for the entire assembly.
The kinetic energy for molecular dynamics simulation was provided by a thermal bath at a constant temperature of 300 K for 10 000 steps of 1.0 fs each. Non-bonded interactions were evaluated with a cutoff distance of 12.0 Å and a switch distance of 2.0 Å. Following thermal equilibration, the simulation was continued for 200300 ps with the output of binary data occurring every 1 ps during the trajectory and stored as a history file. The program also was set to take record graphic dynamic structures `snapshots' every 20 ps starting after the 10 ps thermal equilibration period.
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Results |
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Plots of total energy versus time for the molecular dynamics simulation of the non-mutated CD fragment in the presence and absence of Ca2+ ion are shown in Figure 1a and b, respectively. Both profiles show a plateau in the total energy versus time profile after only 50 ps, indicating that equilibrium has been attained in both simulations. The profiles for all of the other MD runs performed plateau in the same manner as these. In Figure 2
, dynamic structures obtained every 20 ps during the course of the MD simulations (of the non-mutated CD fragment with and without Ca2+ and the cysteine and arginine mutants of the CD fragment and whole SHPV-B protein) are superimposed. In these images, only the calcium ion-binding ligands and Ca2+ ions (where relevant) are displayed. Figure 2a
shows the results from the native CD fragment MD simulation. Note that the Ca2+ ligands are very well superimposed and tightly clustered in the native CD fragment (Figure 2a
), almost to the extent found in MD calculations on the whole protein (Richardson et al., 2000
). It should be noted, however, that even though the ligands are very well superimposed, the r.m.s. deviation for superpositioning of the backbone atoms of any two archive structures is relatively high (~3.54.0 Å), mostly due to fraying at the ends of the molecule. For the native CD fragment, KabschSander secondary structure calculations indicated that after the first 20 ps helix C shortens and helix D kinks in the CD-hand (data not shown). Figure 2b
shows the results of the CD fragment simulation in the absence of Ca2+ ion. Despite slightly less end-fraying in the apo-CD fragment, the r.m.s. deviation values for the superpositioning of any two archive structures is slightly higher (by a few tenths of an ångstrom) than that observed for the native CD fragment, with much of the contribution to the r.m.s. deviation coming from movement associated with the ligands which were previously bound to Ca2+. The ligands are fully allowed to move when the calcium is absent and they clearly fail to maintain their previous Ca2+ coordinating positions. During the apo-CD MD simulation, the helices remain long and unkinked after the first 20 ps.
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Now we will consider the consequences of mutating the naturally occurring glutamic acid residue at position 9 of the CD site (the gateway position) in the isolated CD fragment, as well as in the whole SHPV-B protein. Will residues that never occur (i.e. arginine) or occur only very rarely (i.e. cysteine) at the gateway position in EF-hand proteins cause more disruption in the Ca2+ ion coordination sphere than observed in Figures 2a and 3? What effect on Ca2+ binding will accompany a mutation to a residue that is common at the gateway position in EF-hand proteins (i.e. serine), but never in parvalbumins? Since the gateway mutation arginine was not expressed in GBP and Cys was very poorly expressed (Drake and Falke, 1996
), we decided to investigate the effects of these drastic mutations using a computational approach in parvalbumin. (Of course, the computational approach is the only approach available if the protein is not expressible.) As will be shown, the results are in line with Drake and Falke's work on GBP: the mutated proteins are not functional. The ninth position residue in the CD loop of SHPV-B is a glutamic acid residue (Glu59). This Glu residue is 80% conserved in nature for EF-hand proteins. [Very rarely (5.7% of sequences) Asp is found at this position in lieu of Glu.] Replacing Glu with either Arg or Cys is drastic. A medium-sized, negatively charged Ca2+ ion coordinating residue is changed into either a large, positively charged residue or a small, neutral residue, neither of which has the ability to coordinate Ca2+ ion. Figure 2c and d
show the superpositioning of the CD fragment Ca2+ ligands and the Ca2+ ions for the CD fragment mutants E59C and E59R. For both CD fragment mutants, the superpositioning of Ca2+ ligands and Ca2+ ions is significantly poorer than that observed for the non-mutated CD fragment in the presence of Ca2+ (Figure 2a
), especially for the Arg CD fragment mutant, although measurably better than that for the non-mutated apo-CD fragment (Figure 2b
), especially for the Cys CD fragment mutant.
Figure 2c and 2d show superpositions of just the CD Ca2+ ligands and the CD Ca2+ ions. Figure 4a and 4b
show the entire main-chain superpositions for the Cys and Arg CD fragment mutants, respectively. Figure 4a
reveals the overall tightness in both the main-chain and ligand regions of the CD fragment E59C mutant compared with the CD fragment E59R mutant (Figure 4b
). The original main-chain trace and positions of the ligands of the CD fragment E59C mutant are well retained whereas those of the CD-E59R mutant are only poorly superimposible. KabschSander secondary structure calculations indicate retention of two helices (helices C and D) in the CD fragment Arg mutant, although each helix is considerably smaller than that which occurs in the non-mutated CD fragment. Further note that significant fraying occurs at the ends of the fragments and that the Ca2+ ions are no longer tightly clustered. Clearly, the arginine substitution has caused a significant perturbation in the Ca2+ ion coordination sphere. The CD fragment Cys mutant is not as frayed at the ends of the molecule as the Arg mutant, but here the C helix is kinked to two helices. Figure 4d and e
show plots of number of helices and number of helical residues throughout the MD simulation for the Cys and Arg CD fragment mutants, respectively. The greater dynamics observed for the Arg mutant main chain also results in a substantial reduction in helicity.
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The CD Ca2+ ion-to-ligand distance trajectories for the four mutants (in the presence of Ca2+ ion) during the MD simulations are presented in Figure 5a f. Figure 5a and b
show plots for the CD fragment mutants E59C and E59R, respectively, while Figure 5c and e
are the corresponding plots for the whole SHPV-B mutants E59C and E59R. For comparison, EF Ca2+ ion-to-ligand distance trajectories in SHPV-B which has been mutated in the CD Ca2+ binding site at position 9 are shown in Figure 5d
(E59C) and f (E59R). The plots reveal that there are very significant changes in the binding affinity for Ca2+ in the mutated proteins. In the Cys mutant of the CD fragment, the Ca2+ is released after only ~50 ps of dynamics simulation, whereas this release takes ~100 ps for the CD fragment Arg mutant. Even the Asp53, Asp59 and Glu62 residues, which held the non-mutated CD fragment Ca2+ in place, move out to 610 Å from the Ca2+. The removal of the important gateway ligand at position 9 has left the remaining ligands unable to coordinate the Ca2+. The situation is improved when the AB and EF helixloophelix segments of the native protein are present to stabilize the mutated CD segment. In the Cys mutant of the whole protein, Phe57 (C=O) remains tightly bound to the Ca2+ until ~160 ps into the MD simulation. Asp51 and Asp53 remain close to the binding site. In the MD simulation on the native whole protein, Ser55 is the only ligand to leave the binding site during the first ~175 ps of the run (Richardson et al., 2000
). By ~175 ps, the Ca2+ in the E59C whole protein variant is no longer bound by any of the usual ligands, although there is evidence that it is retained by other ligands in the site. Similar results were observed with the whole protein Arg mutant. All of the ligands coordinate the Ca2+ for the first 25 ps, at which point all of the ligands, except Asp53 and Glu62, move out to ~6 Å from the Ca2+. The remaining CD loop Ca2+ ion ligands move to more than 4 Å by ~135 ps, thus eliminating any possibility for strong metal ion coordination. Thus both arginine and cysteine are not compatible with normal Ca2+ binding in CD site of SHPV-B, in agreement with the experiments of Drake and Falke (1996).
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The m.s.d. values are useful for tracking the overall movement of the Ca2+ ion during the MD simulations. Figure 6a presents Ca2+ m.s.d. traces for both the CD and EF Ca2+ ions for both the Cys CD and Arg CD mutants of whole SHPV-B. The m.s.d. trace for the CD Ca2+ in the Arg mutant lies far above that for all of the other m.s.d. traces. This is consistent with the results presented in Figure 2e and f
and Figure 5c and e
, which all indicated significantly more dynamic activity in the CD coordination sphere of the Arg mutant than in the Cys mutant.
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Since serine is the most common, non-acidic residue found at the gateway position in EF-hand protein and since Drake and Falke found that GBP with serine at the gateway position was very well expressed, a series of simulations were performed on SHPV-B with serine at the gateway position in the CD site. Since a parvalbumin sequence search indicated that serine never appears at the gateway position in any of the parvalbumin EF-hands (the PredictProtein Server, http://www.embl-heidel berg.de/predictprotein/predictprotein.html), we decided to examine the consequences of this substitution. Is there something unique about the helixloophelix sequences found in parvalbumin EF-hands? Or, is there something unique about the interaction of paired EF-hands in the parvalbumins that precludes incorporation of serine at the gateway position if high Ca2+ ion binding affinity is to be achieved? The MD simulations on the serine gateway variants of the whole SHPV-B protein and the isolated CD helixloophelix fragment have been designed to address these issues.
E59S whole SHPV-B MD simulations in the presence of Ca2+
Plots of ligand-to-calcium distances are presented in Figure 7 for all of the serine molecular variants examined. The CD and EF Ca2+ ion-to-ligand distance trajectories for the E59S whole protein variant (in the presence of Ca2+ ion) during 300 ps MD simulations are presented in Figure 7a and b
, respectively. Figure 7a
reveals that there are very significant changes in the binding affinity for Ca2+ in the CD site of the mutated protein. Shortly after 60 ps, all of the ligands of the CD Ca2+ ion leave the vicinity of the metal ion. The removal of the important gateway ligand (Glu59) at position 9 has left the remaining ligands unable to coordinate the Ca2+. The disruption of the CD coordination site has little immediate effect on the ligand positions of the EF Ca2+ ion, but after about 240 ps, the ligand coordination sphere becomes significantly disrupted. Thus, in parvalbumin, the Ser mutant proves to be no more stable than that of the Cys and Arg mutants: the binding site is again corrupted well before the end of the 300 ps MD simulation. These results are consistent with the experimental observation that no parvalbumin has yet been found with serine at the gateway position, even though it is a common gateway residue in other EF-hand proteins, such as in site 3 of calmodulin. In Figure 8
, dynamic structures obtained every 20 ps during the course of the MD simulations (of all the serine mutants) are superimposed. In these images, only the calcium ion-binding ligands and Ca2+ ions are displayed.
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E59S whole SHPV-B MD simulations in the presence of CD Ca2+ but with the EF Ca2+ absent
The CD Ca2+ ion-to-ligand distance trajectories for the E59S whole protein variant (in the presence of only the CD Ca2+ ion) during 300 ps MD simulations are presented in Figure 7c. Figure 7c
reveals a closer association between CD site ligands and the Ca2+ ion when compared with the two Ca2+ ion protein (Figure 7a
); however, the site clearly is far from functional: too many of the ligands are outside the optimum binding range. Early on in the MD run (at ~40 ps), five of the ligands of the CD Ca2+ ion are still within 2.53 Å of the calcium ion, but at ~100 ps, only three ligands remain. The first ligand to move out of the coordination sphere is Ser55, which occurred during the energy minimization (down to a maximum derivative of <0.1 kcal/Å) prior to the MD simulation. (The Ser59 mutant residue at position 9 has forced the Ser55 side chain out in its attempt to have its side chain ligated to the Ca2+ ion.) Asp51 and Glu62 are the next ligands to leave at about 40 ps. Phe57 moves away from the Ca2+ at ~110 ps, followed by Asp53 at ~140 ps. The only Ca2+ ion binding ligand remaining at the end of the 300 ps MD is Ser59, the site of mutation. Figure 8b
shows the superposition of the CD site ligands during the MD run on the E59S variant with Ca2+ ion only in the CD site. While the Ca2+ ion appears to be retained in the site longer than in the E59S variant with two bound Ca2+ ions, the coordination sphere is still lost by the end of the MD simulation. The EF site ligands also undergo a significant rearrangement, similar to that observed during the apo-CD MD simulation (not surprisingly, since Ca2+ ion is not present) during the MD run (data not shown). The m.s.d. measurements indicate that the CD Ca2+ ion in the E59S mutant with one bound Ca2+ ion is less mobile (at least for the initial 100 ps) than the CD Ca2+ ion in the E59S mutant with two bound Ca2+ ions (Figure 6b
). After ~100 ps, the Ca2+ ion in the E59S mutant with one bound Ca2+ ion moves a great distance both in and out of the Ca2+ ion binding site, to a final m.s.d. greater than that observed for the E59S mutant with two bound Ca2+ ions, but still less than the E59R mutant of the whole protein with two bound Ca2+ ions (Figure 6a
). In summary, the serine substitution of the important gateway ligand (Glu59) at position 9 in the one Ca2+ protein has not been as disruptive to the CD site as the serine substitution in the protein with two bound Ca2+ ions.
E59S CD SHPV-B MD simulations in the presence of Ca2+
Can the above-noted disruption of the CD site in the whole E59S protein be linked to the sequence within the CD helixloophelix unit? Early in this paper results were presented indicating that the cysteine variant of an isolated CD-hand was incapable of retaining the coordination site. The serine side chain is similar in size (but not in polarity) to the cysteine side chain. Would the serine variant behave similarly to the cysteine variant in an MD simulation? Figure 8c shows the superpositions of the CD site ligands during the 310 ps MD simulation on the E59S variant of the isolated CD helixloophelix unit. Note that the Ca2+ ligands are very well superimposed and tightly clustered, almost to the extent found in the native CD fragment (Figure 2a
). The r.m.s. deviation for superpositioning of the backbone atoms of any two archive structures is lower (to only 3.1 Å in the 310 ps MD run) than found in the native CD fragment as there is less fraying at the ends of the molecule. For the E59S variant of the CD fragment, KabschSander secondary structure calculations indicated that after the first 20 ps helix C is shortened and helix D kinks with only the last half of the helix retaining its helicity (Figure 4c
). The number of helical residues and number of helices for the CD-E59S mutant (Figure 4f
) is retained approximately equally to the native CD fragment (data not shown) and the CD-E59C mutant. The CD-E59R mutant, in contrast, has retained significantly less of its helical character throughout the MD simulation. Figure 8c
further reveals that all of the Ca2+ ions are rather tightly clustered. Figure 7d
presents Ca2+ ion-to-ligand distance trajectories during the MD simulation for the E59S variant of the CD fragment in the presence of Ca2+ ion. The plots reveal that there are no significant changes in this distance for Asp51, Asp53, Phe57 and Glu62 (bidentate) for the duration of the MD run. For these ligands the distances to the Ca2+ are almost constant, at 2.32.5 Å, over the time trajectory. Ser55 is found at a greater distance from the Ca2+, indicative of weaker coordination, which is consistent with the results of MD computations on the whole protein (Richardson et al., 2000
) and other variants containing the CD-hand (this work). The most deviant ligand is Ser59, which has replaced the Glu59 usually found in this site. After releasing the calcium at ~75 ps, Ser59 remains at a distance of about 6 Å. Surprisingly, even with the Ser mutation at position 9 of the loop, the other ligands are close enough to maintain this site since the coordination of only two ligands is lost in the MD. The C and D helices are able to accept the mutation and reorient so that the other ligands are able to retain coordination to the calcium when the CD site is excised from the whole protein. The m.s.d. results for the Ca2+ ion in the E59S mutant of the CD-hand fragment during the MD trajectory indicate that the CD Ca2+ has a smaller m.s.d. value at 125 ps than any of the other variants including the cysteine, arginine and serine variants of the whole protein (Figure 6a and b
). This is consistent with the tight clustering of the Ca2+ ions shown in Figure 8c
.
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Discussion |
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Arginine or cysteine at the gateway position in the CD site of parvalbumin
The molecular dynamics simulations performed on either the whole silver hake parvalbumin protein or the CD fragment of the protein, in which either an arginine or cysteine replaces the naturally occuring Ca2+ ion-binding gateway ligand (glutamic acid) severely disrupts all remaining Ca2+ ionligand interactions in the CD Ca2+ ion binding site (Figure 5a, b, c and e). The most important conclusion that can be drawn from these computational results is that neither arginine nor cysteine is predicted to be found at the CD site gateway position in parvalbumin. In the case of the cysteine computations, it should be noted that the disruption of the CD Ca2+ ionligand interactions occurs significantly earlier in the MD simulations for the CD fragment mutant than for the whole protein mutant (Figure 5a vs c
). The presence of interacting AB and EF regions results in a degree of stabilization of the mutated CD region in the whole protein computations.
For MD simulations on the whole protein, the arginine mutation induced considerably more disruption of the CD site coordination sphere and the disruption occurred significantly sooner in the MD run than for the cysteine mutation. Furthermore, the arginine substitution resulted in a more global effect. While the cysteine mutation in the CD site did not significantly disrupt the EF site structure, the arginine mutation in the CD site significantly disruped Ca2+ ionligand interactions in the remote EF site.
The overall results obtained in the arginine and cysteine variant MD simulations are completely consistent with the experimental results of Drake and Falke (1996), who observed undetectable levels of protein expression following both cysteine and arginine substitution at the gateway residue of the EF-hand protein GBP. Our results reveal that both of these substitutions are destabilizing influences in both the whole parvalbumin and in isolated helixloophelix fragments of the protein.
Serine at the gateway position in the CD site of parvalbumin
Serine is not an uncommon residue at the gateway position. The residue occurs with a frequency of about 20% at the gateway position in the EF-hand proteins surveyed by Drake and Falke (1996). However, serine has never been found at the gateway position in parvalbumins. Consistent with this observation, our molecular dynamics simulations indicated that both the CD and EF metal ion coordination sites in whole silver hake parvalbumin were severely disrupted when serine was incorporated into the CD site at the gateway position (Figure 7a and b). Interestingly, when the EF Ca2+ is removed from the serine whole protein mutant, the CD site retains structure much longer during the MD simulation (Figure 7c
). Removal of the EF Ca2+ ion has permitted the remaining ligands at the CD site to adjust, at least to some degree, permitting continued association of some of the ligands with the CD Ca2+ ion. The Ser59 residue actually remained within bonding distance of the Ca2+ ion for the entire MD simulation. (Presumably, the protein with only one bound Ca2+ ion is able to restructure globally to compensate for the presence of a shorter side-chain residue at the position 9 coordination site.) These results are in agreement with previous molecular dynamics simulations studies performed in our laboratory in which removal of the EF calcium in the native protein significantly increased the ordering of the CD site ligands (Richardson et al., 2000
). In that study, the Ca2+ is ligated by most of its usual calcium ion binding ligands throughout the 200 ps simulation; in fact, of all of the CD site ligands only Ser55 experienced a significant excursion away from the CD Ca2+ ion and this occurred at ~170 ps into the dynamics run. In conclusion, while some CD site stabilization is noted in the MD run on the serine variant containing only the CD Ca2+ ion, no claim can be made that a `functional' CD site is retained, hence these results remain consistent with the experimental observation that no parvalbumin has yet been found with serine at the gateway position.
The most intriguing and perhaps most biochemically relevant result of this study comes from the molecular dynamics simulation performed on the serine gateway variant of the isolated CD helixloophelix fragment. In marked contrast to the MD results obtained on the whole parvalbumin serine variant and the results obtained on both the arginine and cysteine variants of the CD helixloophelix fragment, the serine CD fragment variant exhibited remarkable stability during the entire molecular dynamics simulation, with clear retention of the Ca2+ binding site. These results indicate that there are no inherent problems (for Ca2+ ion binding) associated with the sequence of the CD helixloophelix fragment that precludes the incorporation of serine at the gateway position. The binding site loop does undergo some alteration, but the crystallographic ligands Asp51, Asp53, Phe57 and Glu62 are still able to coordinate the Ca2+. The previously liganding Glu59 is replaced by the liganding Ser59.
It has been proposed that Gly in position 6 of the EF-hand loop serves as an intra-loop hinge in order to enhance protein stability by allowing the unusual torsion angle requirements of the backbone fold at this loop position (Falke et al., 1994; Drake et al., 1997
). Monera et al. (1992) showed that hydrophobic residues are important in the formation of the two-site domain in troponin C and this hydrophobic association stabilizes Ca2+ ion binding. Are these facts somehow connected to what residues are permitted at the gateway position? Richardson et al. (2000) have proposed a possible `tension' between the two functional EF-hands in the silver hake parvalbumin (isoform B), which is relieved when the EF calcium is removed. Do we need the additional coordinating ligand at the gateway position to stabilize the CD cite (i.e. to overcome some repulsive interaction that would otherwise disrupt the structural integrity of the site), especially in the Ca2+-loaded protein? Our MD results our certainly consistent with this interpretation.
The results obtained in the serine variant MD simulations contrast with the experimental results of Drake and Falke (1996), who found that the GBP protein containing the serine substitution was well expressed. Our MD computations clearly reveal that serine is a destabilizing influence on the parvalbumin protein, which is consistent with the experimental observation that this residue has never been found at the gateway position in any parvalbumin.
Conclusion
This study demonstrates how incorporation of various amino acids into a binding site loop can dramatically affect (or not affect) binding site conformation. Parvalbumin, because of its globular shape and great stability, provides a good system for molecular modeling and molecular dynamics simulations. We feel that the loss of the CD site Ca2+ ion coordination sphere in the E59S serine mutant of the whole protein and the retention of this site in the isolated CD helixloophelix site (even though the site is not retained with either arginine or cysteine CD loop variants) is an important finding. It implies that there is a unique interaction between the partnered EF-hands in the parvalbumins that necessitates the presence of the rather large, negatively charged glutamic acid coordinating residue at the gateway position. Parvalbumins are unique in that they always contain an active coordinating ligand, almost always glutamic acid, in position 9 of the CD Ca2+ ion binding loop. GBP also has a coordinating ligand in the gateway position (the neutral Gln142 residue) but is somewhat different from the other EF-hand proteins in that it has only one calcium and one `EF-hand' segment, composed of a helixloopß-strand, in a much larger protein of 309 amino acids. Most other EF-hand proteins, including most calmodulins, troponin Cs, calbindins and the EF loop of parvalbumin have a non-functional (i.e. not directly involved in Ca2+ ion binding) gateway residue. Although an amino acid is in place that could bind a calcium ion (especially in the case of serine), traditionally there is a coordinating water molecule at this location. The cff91 forcefield and the program fdiscover, which we employed for the molecular dynamics simulations, are well suited to theoretical studies on globular Ca2+ ion binding proteins, such as parvalbumin. Since our data correlate well with studies in the literature on engineered proteins and all of our experimental evidence to date, we suggest that this computational approach can be used with confidence to study the effects of mutations prior to the cumbersome and time-consuming engineering in the laboratory or in lieu of inexpressible mutants in order to obtain data on the conformational effects of the mutations. Just within the EF-hand family of proteins, there have been a large number of engineered variants generated for study. It is our hope that insights gained through computational approaches will advance the understanding of the important structural attributes and functional basis for variation in EF-hand calcium binding proteins. Future studies should include the examination of the EF site of parvalbumin, in which a crystallographic water molecule is found to ligate the calcium in the gateway position instead of the glycine at position 9 in the loop. The results of studies at this site should be compared with those of calmodulin, calbindin or other helixloophelix Ca2+ binding proteins in which a water, rather than an amino acid residue, is responsible for coordinating the Ca2+ from the gateway position.
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Notes |
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Acknowledgments |
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References |
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Received June 24, 2000; revised October 12, 2000; accepted November 9, 2000.