Molecular dynamics study of Ca2+ binding loop variants of parvalbumin with modifications at the `gateway' position

Kelly M. Elkins, Petia Z. Gatzeva-Topalova and Donald J. Nelson,1

Gustaf H. Carlson School of Chemistry and Biochemistry, Clark University, Worcester, MA 01610, USA


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The helix–loop–helix (i.e. EF-hand) Ca2+ ion binding motif is characteristic of a large family of high-affinity Ca2+ ion binding proteins, including the parvalbumins and calmodulins. In this paper we describe a set of molecular dynamics computations on the major parvalbumin from the silver hake (SHPV-B). In all variants examined, both whole protein and fragments thereof, the ninth loop residue in the Ca2+ binding coordination site in the CD helix–loop–helix region (the so-called `gateway' residue) has been mutated. The three gateway mutations examined are arginine, which has never been found at the gateway position of any EF-hand protein, cysteine, which is the residue observed least in natural EF-hand sites, and serine, which is the most common (by far) non-acidic residue substitution at this position in EF-hand proteins in general, but never in parvalbumins. Results of the molecular dynamics simulations indicate that all three modifications are disruptive to the integrity of the mutated Ca2+ binding site in the whole parvalbumin protein. In contrast, only the arginine and cysteine mutations are disruptive to the integrity of the mutated Ca2+ binding site in the CD fragment of the parvalbumin protein. Surprisingly, the serine variant of the CD helix–loop–helix fragment exhibited remarkable stability during the entire molecular dynamics simulation, with 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 helix–loop–helix fragment that precludes the incorporation of serine at the gateway position. Since the CD site is totally disrupted in the whole protein serine variant, this indicates that the Ca2+ ion binding deficiencies are most likely related to the unique interaction that exists between the paired EF-hands in the whole protein. Our theoretical results correlate well with previous studies on engineered EF-hand proteins and with all of our experimental evidence on the silver hake parvalbumin.

Keywords: calcium binding proteins/dynamics/EF-hand/gateway/modeling/parvalbumin


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Parvalbumins are among one of the 32 subclasses within the EF-hand superfamily of calcium binding proteins all sharing the common helix–loop–helix (EF-hand) motif (Kretsinger, 1975Go; Kretsinger and Nelson, 1976Go). Parvalbumins are small (10–12 kDa), acidic (pI 4.0–5.2), Ca2+ and Mg2+ binding proteins, particularly abundant in the muscle tissue of lower vertebrates. Proposed important functions for parvalbumins include involvement in the muscle relaxation/contraction cycle (Jiang et al., 1996Go), calcium buffering (Pauls et al., 1996Go) and signal transduction (Chard et al., 1993Go). Other roles proposed for parvalbumins include involvement in the triggering of gene expression (Chard et al., 1993Go), in cell division (Rasmussen and Means, 1989Go), in processes influencing cell shape and motility (Andressen et al., 1995Go) and in immune system development (Brewer et al., 1989Go). The X-ray crystal structure of silver hake parvalbumin, isoform B (SHPV-B), has been determined in our laboratory to 1.65 Å resolution and the coordinates have been deposited in the Protein Data Bank with the code 1bu3.pdb (Richardson et al., 2000Go).

Parvalbumins share with the EF-hand protein superfamily a conserved architecture associated with the Ca2+ ion binding site (Kawasaki et al., 1998Go).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, 1973Go). 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., 1996Go; Allouche et al., 1999Go). The Ca2+ ion binding ligands in the functional CD and EF sites of SHPV-B are presented in Table IGo.


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Table I. Ca2+ binding ligands in SHPV-B
 
Mutants of EF-hand proteins have been examined for many years. The `gateway' position (i.e. loop residue 9, the –x position) has proven especially interesting (Drake and Falke, 1996Go). Mutations at this site were first examined extensively by Williams et al. (1987) in their studies on oncomodulin. In a later study, Renner et al. (1993) proposed that the gateway residue may serve `to tune the kinetics of ion binding and release'. Different residues at this position in different EF-hand proteins may be utilized to `optimize the rates of Ca2+ binding and release' required for the specific function of the particular protein. Renner et al. (1993) and Falke et al. (1994) have suggested that both the size and the charge of the side chain at the gateway position may be critical for the kinetic tuning. The `gateway' may be viewed as the shortest pathway for a free metal ion to make its transit from solvent to the final coordination site. The effect on Ca2+ ion binding properties of having different residues at the gateway position has proven to be an intriguing problem without a clear solution. A wide range of residues have been identified at the gateway position in wild-type EF-hand proteins, including Asp, Asn, Met, Glu, Gln, Ser and Cys.

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 helix–loop–helix motif in both the whole silver hake parvalbumin and in the isolated CD helix–loop–helix 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., 1994Go). 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 IIGo). 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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Table II. Residues engineered at the `gateway' position by Drake and Falke (1996)
 
There are a number of precise questions asked by this study. The molecular dynamics simulations on the arginine variants (whole protein and CD helix–loop–helix fragment) will address why we never find this residue at the gateway position in any EF protein. The prediction is that the large, positively charged side chain would be most disruptive to the metal ion binding site which has evolved to bind the positively charged calcium ion. This is not a surprising prediction and these calculations will serve, in part, as a control. Drake and Falke (1996) were not able to express the arginine variant, and, as we shall see, the results of the MD simulations, entirely consistent with experiment, demonstrate the very disruptive nature of this mutation, both in the whole protein and in the isolated CD Ca2+ ion binding motif. The consequences of a glutamic acid to cysteine mutation at the gateway position are more difficult to predict. The cysteine side chain clearly cannot participate in Ca2+ coordination and in this sense the mutation might be considered drastic since we are losing a coordinating ligand; however, the side chain is relatively small and uncharged and might not be expected to be overly disruptive to the site. There are still plenty of ligands available for Ca2+ coordination. Why is cysteine so rare at the gateway position? Recall that Drake and Falke (1996) found very poor expression of the GBP cysteine variant. Our MD simulation results provide further evidence for the incompatibility of cysteine at the gateway position in both cysteine gateway variants examined, but especially in the isolated CD helix–loop–helix fragment, most probably due to the increased degrees of motional freedom exhibited by this species. Serine at the gateway position is particularly interesting, since it is a common residue at this position in EF-hand proteins (in general), but never in parvalbumins. (For example, serine is the gateway residue at the third Ca2+ ion coordination site in calmodulin.) What effect on Ca2+ binding might accompany the glutamic acid to serine change in our silver hake parvalbumin? Is the serine residue structurally incompatible in the unique parvalbumin Ca2+ binding loop (in our study the loop connecting the C helix with the D helix)? Or, alternatively, does the presence of serine at the gateway position interfere with the `EF-hand partnering' that exists at the interface of the CD and EF sites of parvalbumin molecules? These questions are explicitly addressed by performing MD simulations on both the whole protein serine gateway variant and the isolated CD helix–loop–helix serine gateway variant and, as will be shown, the results demonstrate that serine is not disruptive to an isolated EF-hand (on the contrary, an excellent complex is formed), but is disruptive in the whole protein, perhaps indicating a problem associated with the packaging of CD- and EF-hands into the whole protein, along with the necessity to have a Ca2+ ion coordinating residue at the gateway position in this site. We feel that computations on isolated helix–loop–helix motifs (as are performed in this study) are relevant, since the study of fragments of native proteins and designed synthetic peptides (both experimentally and computationally), have become very useful tools in revealing binding mechanisms and folding characteristics of native proteins. In a sense the present computational study complements a previous experimental study in which we characterized an enzymatically excised helix–loop–helix fragment of the silver hake parvalbumin (the EF-hand, residues 76–108) by NMR, circular dichroism and sedimentation equilibrium analysis (Revett et al. 1997Go). That study revealed that the EF-hand of SHPV-B binds divalent and trivalent cations, and, most importantly, adopts an {alpha}-helical conformation and participates in a monomer–dimer equilibrium in the presence of certain metal ions, such as La3+. This suggests a retention of native-like function, since the X-ray crystal structures of all parvalbumins examined to date indicate a precise mode of `packaging or partnering' of the CD- and EF-hands (they are related to one another in the whole protein by an approximate twofold axis of rotation and interact strongly).


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The computations described in this paper utilized the coordinates for silver hake parvalbumin isoform B (SHPV-B, 1bu3.pdb). Molecular dynamics simulations were performed on an Indy R5000 Silicon Graphics workstation, using the InsightII (v. 97.2) software package from Molecular Simulation (MSI) (San Diego, CA) and a Silicon Graphics O2 Workstation using InsightII (v. 98.0). The MSI program fdiscover was used for all molecular mechanics and molecular dynamics calculations, employing the second-generation cff91 force field (Maple et al., 1988Go, 1990Go, 1994Go).

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 ~1500–2500 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 200–300 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.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Non-mutated CD SHPV-B fragment MD simulations in the presence and absence of Ca2+

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 bGo, 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 2Go, 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 2aGo 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 2aGo), almost to the extent found in MD calculations on the whole protein (Richardson et al., 2000Go). 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 (~3.5–4.0 Å), mostly due to fraying at the ends of the molecule. For the native CD fragment, Kabsch–Sander 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 2bGo 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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Fig. 1. Plots of total energy versus time over the molecular dynamics trajectory for non-mutated CD fragment of SHPV-B with (a) and without (b) bound Ca2+ ion.

 


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Fig. 2. Superpositions of archive structures obtained during the molecular dynamics simulations, showing only the CD Ca2+ ion binding ligands and the CD Ca2+ ion. (a) Non-mutated CD fragment of SHPV-B with Ca2+ ion; (b) non-mutated CD fragment of SHPV-B without Ca2+ ion; (c) E59C variant of the CD fragment of SHPV-B with Ca2+ ion; (d) E59R variant of the CD fragment of SHPV-B with Ca2+ ion; (e) E59C variant of SHPV-B with Ca2+ ion; (f) E59R variant of SHPV-B with Ca2+ ion.

 
Figure 3Go presents Ca2+ ion-to-ligand distance trajectories during the MD simulation for the native, non-mutated CD fragment of SHPV-B in the presence of Ca2+ ion. While viewing the results on these distance trajectories for the isolated CD fragment, it is important to note that the Ca2+ ion-to-ligand distance trajectories for the CD ligands in the MD simulation for the native, whole SHPV-B protein indicated that all ligands, with the exception of the Ser55 side chain which made an excursion into the solvent early in the MD run but returned later, remained bound to the CD Ca2+ ion (Richardson et al., 2000Go). The absence of the AB and EF segments of the protein resulted in the loss of some of the crystallographic ligands; however, Figure 3Go reveals that there are still three residues (and five atoms within these residues) that remain within reasonably close distance to the CD Ca2+ ion during the entire course of the 300 ps MD run: Asp53 ({delta}1 oxygen), Glu59 ({varepsilon}1 and {varepsilon}2 oxygens) and Glu62 ({varepsilon}1 and {varepsilon}2 oxygens). For Glu59 ({varepsilon}2 oxygen) and Glu62 ({varepsilon}2 oxygen) these ligand-to-Ca2+ distances are almost constant, at 2.3–2.5 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., 2000Go) and other variants containing the CD-hand (this work). The most deviant ligand is Asp51, which, after releasing the calcium at ~50 ps, remains at a distance of almost 8 Å. Phe57, which coordinated the Ca2+ through its carbonyl function, also releases the Ca2+. Although three of the ligands are no longer coordinating the Ca2+, the other ligands are close enough to maintain this site. The mean square deviation (m.s.d.) and distance traveled results of the Ca2+ ion in the native CD-hand fragment during the MD trajectory indicate that the CD Ca2+ is both displaced less and travels less than in any of the mutants examined (data not shown). This is consistent with the tight clustering of the Ca2+ ions shown in Figure 2aGo, even though some of the Ca2+ ion ligands found in the whole protein have moved outside the primary coordination sphere in the CD fragment. The MD simulation on the non-mutated, isolated CD fragment predicts that metal ion should still bind to the peptide (consistent with experimental findings on an excised EF-hand from SHPV-B (Richardson et al., 2000Go), albeit with reduced affinity, and suggests that important stabilization influences are no longer in place in this unpartnered EF-hand.



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Fig. 3. Plots of Ca2+ ion-to-ligand distances in the CD site during the molecular dynamics simulation on the non-mutated CD fragment of SHPV-B.

 
MD simulations on non-conservative variants of whole SHPV-B and the CD fragment of SHPV-B in the presence of Ca2+: E59R CD SHPV-B, E59C CD SHPV-B, E59C whole SHPV-B and E59R whole SHPV-B

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 3GoGo? 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, 1996Go), 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 dGo 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 2aGo), especially for the Arg CD fragment mutant, although measurably better than that for the non-mutated apo-CD fragment (Figure 2bGo), especially for the Cys CD fragment mutant.

Figure 2c and 2dGo show superpositions of just the CD Ca2+ ligands and the CD Ca2+ ions. Figure 4a and 4bGo show the entire main-chain superpositions for the Cys and Arg CD fragment mutants, respectively. Figure 4aGo 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 4bGo). 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. Kabsch–Sander 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 eGo 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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Fig. 4. Secondary structure rendering of CD fragment variants of SHPV-B during the molecular dynamics simulations. (a) Superpositions of the archive structures of the E59C variant of the CD fragment of SHPV-B, showing the trace of the main chain and the Kabsch–Sander rendering of secondary structure for the 20 ps structure. (b) Superpositions of the archive structures for the E59R variant of the CD fragment of SHPV-B, showing the trace of the main chain and the Kabsch–Sander rendering of secondary structure for the 20 ps structure. (c) Superpositions of the archive structures for the E59S variant of the CD fragment of SHPV-B, showing the trace of the main chain and the Kabsch–Sander rendering of secondary structure for the 20 ps structure. (d) Bar graph showing the number of helical segments and the number of helical residues for the archive structures of the E59C variant of the CD fragment of SHPV-B. (e) Bar graph showing the number of helical segments and the number of helical residues for the archive structures of the E59R variant of the CD fragment of SHPV-B. (f) Bar graph showing the number of helical segments and the number of helical residues for the archive structures of the E59S variant of the CD fragment of SHPV-B.

 
Figure 2e and fGo show the superpositioning of the CD Ca2+ ligands and the Ca2+ ions for the whole protein mutants E59C and E59R, respectively. The CD Ca2+ ligands in Cys mutants of both the fragment and the whole protein retain positions that show less deviation than the Arg mutants from the ligand positions in the non-mutated, native molecules. There is also better superpositioning of the CD Ca2+ ligands in the Cys mutants of the fragment and the whole protein relative to that observed for the Arg mutants. Figure 2Go further reveals that the Ca2+ ions are less clustered and less centered in the ligand coordination sphere in the CD fragment mutants (Figure 2c and dGo) than in the whole protein mutants (Figures 2e and fGo). The constellation of residues forming the metal ion binding site in the whole protein, even in the presence of the mutations, are, perhaps not surprisingly, somewhat stabilized and constrained by the rest of the protein, minimizing the perturbation to the structural integrity of the CD site caused by the mutation. The r.m.s. deviation values obtained from the superpositions indicate that the mutants of the whole protein are structurally more stable than the CD fragment mutants. Superimposition of the tenth archive frame (200 ps) on to the first frame (20 ps) for the Cys and Arg mutants of the whole protein give r.m.s. deviation values of 1.47 and 2.41 Å, respectively. For the Cys and Arg mutants of the CD fragment, the r.m.s. deviation values are 3.35 and 3.87 Å, respectively.

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 –fGo. Figure 5a and bGo show plots for the CD fragment mutants E59C and E59R, respectively, while Figure 5c and eGo 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 5dGo (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 6–10 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 helix–loop–helix 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., 2000Go). 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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Fig. 5. Plots of Ca2+ ion-to-ligand distances in the CD and EF sites during the molecular dynamics simulations. (a) CD ligands in E59C variant of the CD fragment of SHPV-B; (b) CD ligands in E59R variant of the CD fragment of SHPV-B; (c) CD ligands in E59C variant of SHPV-B; (d) EF ligands in E59C variant of SHPV-B; (e) CD ligands in E59R variant of SHPV-B; (f) EF ligands in E59R variant of SHPV-B.

 
Since all members of the EF-hand superfamily of proteins possess at least two functional Ca2+ units, it is relevant to ask whether a mutation in the gateway residue in one site affects the Ca2+ interactions at other non-mutated sites in the protein. In our case, how does mutation of the gateway residue in the CD Ca2+ binding site of SHPV-B affect the ability of Ca2+ binding ligands in the EF site to interact with the EF Ca2+ ion? The EF Ca2+ ion-to-ligand distance trajectories for the two whole protein CD site mutants (in the presence of Ca2+ ion) during the MD simulations are presented in Figure 5d–fGo. It is clearly evident that the Cys mutation in the CD site has little effect on the ability of the EF ligands to bind the EF Ca2+ ion. In the SHPV-B:E59C mutant, with the exception of a brief excursion by Asp92 (which is the source of the highest B values for a single residue in the crystal structure of SHPV-B) late in the simulation, all of the ligands coordinate the EF Ca2+ from distances of 2.3–2.5 Å. The substitution of Arg in the CD site of the whole SHPV-B has caused significant disruption of the EF site ligands. Almost immediately, Asp92 and Asp94 move out to ~4 Å of the Ca2+, while the other ligands remain tightly bound. At ~100 ps, Asp92 again takes an additional excursion, this time out to a distance of ~9 Å, while the other ligands, with the exception of Glu101, all move out to ~4 Å from the Ca2+. Thus, the mutation to Cys produces only a local effect on calcium binding (at the CD site), whereas the Arg substitution produces a global effect.

The m.s.d. values are useful for tracking the overall movement of the Ca2+ ion during the MD simulations. Figure 6aGo 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 fGo and Figure 5c and eGo, 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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Fig. 6. (a) Plots of the CD Ca2+ and EF Ca2+ ion m.s.d. during the MD simulations on the E59C (CD, ; EF, {circ}) and E59R (CD, {triangleup}; EF, —) variants of whole SHPV-B. (b) Plots of the CD Ca2+ and EF Ca2+ ion m.s.d. during the MD simulations on the whole E59S (CD, —; EF, {circ}), whole E59S with only the CD calcium ion ({triangleup}) and the CD fragment E59S () variants of SHPV-B.

 
MD simulations on whole SHPV-B and the CD fragment of SHPV-B with serine at the gateway residue position

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 helix–loop–helix 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 helix–loop–helix 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 7Go 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 bGo, respectively. Figure 7aGo 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 8Go, 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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Fig. 7. Plots of Ca2+ ion-to-ligand distances in the CD and EF sites of silver hake parvalbumin during the molecular dynamics simulations. (a) CD ligands in mutated E59S variant of SHPV-B in which both CD and EF calcium ions are present; (b) EF ligands in mutated E59S variant of SHPV-B in which both CD and EF calcium ions are present; (c) CD ligands in mutated E59S variant of SHPV-B in the absence of the EF calcium; (d) CD ligands in mutated E59S variant of the CD fragment of SHPV-B.

 


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Fig. 8. Superpositions of archive structures obtained during the molecular dynamics simulations, showing only the CD Ca2+ ion binding ligands and the CD Ca2+ ion. (a) CD site of SHPV-B E59S mutant with both Ca2+ ions; (b) CD site of SHPV-B E59S mutant with only the CD Ca2+ ion; (c) CD site of SHPV-B CD fragment E59S mutant with the CD Ca2+ ion.

 
Note that the CD Ca2+ ligands are still somewhat superimposable and clustered (Figure 8aGo), although the CD Ca2+ clearly moves out of the coordination site. [The EF site is retained but appears to be strained in trying to retain the Ca2+ (data not shown).] Figure 6bGo shows that the EF Ca2+ is very stable and undergoes little displacement, as indicated by the calculated m.s.d. value of 6.5 Å2 after 150 ps of dynamics. The CD Ca2+ is much more mobile at the beginning of the MD, as is indicated by the m.s.d. calculation, although it seems to stabilize after about 100 ps (Figure 6bGo; see also Figure 8aGo). In order to address the issue of the origin of the disruption of the CD Ca2+ ion binding site (i.e. whether it is linked to the unique sequence of the CD helix–loop–helix motif or to interaction between paired motifs) molecular dynamics computations were performed on (1) the serine variant of the whole protein in which the EF Ca2+ ion has been removed (in order to see if any possible `tension' between the paired sites has been relieved) and (2) the serine variant of the isolated CD helix–loop–helix unit of the whole protein (in order to see if there are any inherent problems related to the sequence of the unit). The results are presented in the following two sections.

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 7cGo. Figure 7cGo reveals a closer association between CD site ligands and the Ca2+ ion when compared with the two Ca2+ ion protein (Figure 7aGo); 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 2.5–3 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 8bGo 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 6bGo). 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 6aGo). 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 helix–loop–helix 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 8cGo shows the superpositions of the CD site ligands during the 310 ps MD simulation on the E59S variant of the isolated CD helix–loop–helix unit. Note that the Ca2+ ligands are very well superimposed and tightly clustered, almost to the extent found in the native CD fragment (Figure 2aGo). 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, Kabsch–Sander 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 4cGo). The number of helical residues and number of helices for the CD-E59S mutant (Figure 4fGo) 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 8cGo further reveals that all of the Ca2+ ions are rather tightly clustered. Figure 7dGo 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 2.3–2.5 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., 2000Go) 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 bGo). This is consistent with the tight clustering of the Ca2+ ions shown in Figure 8cGo.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
A large number of molecular dynamics simulations are presented in this paper; however, in this section we will summarize and discuss further what we believe to be the most significant results of the entire study. The discussion will be split into two sections, the first dealing with the non-conservative gateway substitutions (Glu->Arg and Glu->Cys) and the second dealing with the more `conservative' gateway substitution (Glu->Ser).

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+ ion–ligand interactions in the CD Ca2+ ion binding site (Figure 5a, b, c and eGo). 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+ ion–ligand interactions occurs significantly earlier in the MD simulations for the CD fragment mutant than for the whole protein mutant (Figure 5a vs cGo). 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+ ion–ligand 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 helix–loop–helix 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 bGo). 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 7cGo). 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., 2000Go). 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 helix–loop–helix 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 helix–loop–helix 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 helix–loop–helix 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., 1994Go; Drake et al., 1997Go). 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 helix–loop–helix 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 helix–loop–ß-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 helix–loop–helix Ca2+ binding proteins in which a water, rather than an amino acid residue, is responsible for coordinating the Ca2+ from the gateway position.


    Notes
 
1 To whom correspondence should be addressed. E-mail: dnelson{at}clarku.edu Back


    Acknowledgments
 
This work was supported by instrument grants to D.J.N. from the National Science Foundation (DUE-9650859) and the Ira W.DeCamp Foundation. K.M.E. is a recipient of a Clare Booth Luce Fellowship (D.Nelson, PI).


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Received June 24, 2000; revised October 12, 2000; accepted November 9, 2000.





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