Potential influence of Asp in the Ca2+ coordination position 5 of parvalbumin on the calcium-binding affinity: a computational study.

Parvalbumins (PV) are calcium-binding proteins, all sharing the common helix-loop-helix (EF-hand) motif. This motif contains a central twelve-residue Ca(2+)-binding loop with the flanking helices positioned roughly perpendicular to each other. The precise role of these coordination residues has been the subject of intense studies. In this work, we focus on the coordination position 5 in the CD Ca(2+)-binding site of silver hake parvalbumin isoform B (SHPV-B). The most common residue at site 5 of calcium-binding loop in canonical EF-hands is Asp [B.J. Marsden, G.S. Shaw, B.D. Sykes, Biochem. Cell Biol. 68 (1990) 587-601], but in the CD site of PV, this position is almost always serine (Ser). The substitution of Ser with Asp will add the 5th carboxylate residue in the CD coordination sphere. However, as predicted by the acid pair hypothesis, the Ca(2+)-binding affinity would be maximized in an EF-hand motif that has four carboxylate ligands paired along the +/-x, and +/-z-axes [R.E. Reid, R.S. Hodges, J. Theor. Biol. 84 (1980) 401-444]. Molecular dynamics simulations and free energy calculations were employed to investigate the influence of Ser to Asp mutation at position 5 on calcium-binding affinity. We found that the Asp variant exhibited remarkable stability during the entire molecular dynamics simulation, with not only the retention of the Ca(2+)-binding site, but also increased compactness in the coordination sphere. The S55D fragment also accommodated Ca(2+) well. We conclude that the reason why Asp which is the most common residue at site 5 of calcium-binding loop in canonical EF-hands has never been identified at this position experimentally for PVs might be related to its physiological functions.

Molecular dynamics simulations of complexes between wild-type and mutant anthrax protective antigen variants and a model anthrax toxin receptor.

Bacillus anthracis, a spore-forming infectious bacterium, produces a toxin consisting of three proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). LF and EF possess intracellular enzymatic functions, the net effect of which is to severely compromise host innate immunity. During an anthrax infection PA plays the critical role of facilitating entry of both EF and LF toxins into host cell cytoplasm. Crystal structures of all three of the anthrax toxins have been determined, as well as the crystal structure of the (human) von Willebrand factor A (integrin VWA/I domain) -- an anthrax toxin receptor. A theoretical structure of the complex between VWA/I and PA has also been reported. Here we report on the results of 1,000 psec molecular dynamics (MD) simulations carried out on complexes between the Anthrax Protective Antigen Domain 4 (PA-D4) and the von Willebrand Factor A (VWA/I). MD simulations (using Insight II software) were carried out for complexes containing wild-type (WT) PA-D4, as well as for complexes containing three different mutants of PA-D4, one containing three substitutions in the PA-D4 "small loop" (residues 679-693) (D683A/L685E/Y688C), one containing a single substitution at a key site at the PA-D4 -- receptor interface (K679A) and another containing a deletion of eleven residues at the C-terminus of PA (Delta724-735). All three sets of PA mutations have been shown experimentally to result in serious deficiencies in PA function. Our MD results are consistent with these findings. Major disruptions in interactions were observed between the mutant PA-D4 domains and the anthrax receptor during the MD simulations. Many secondary structural features in PA-D4 are also severely compromised when VWA complexes with mutant variants of PA-D4 are subjected to MD simulations. These MD simulation results clearly indicate the importance of the mutated PA-D4 residues in both the "small loop" and at the carboxyl terminus in maintaining a PA conformation that is capable of effective interaction with the anthrax toxin receptor.

Fluorescence study of the interaction of Suwannee River fulvic acid with metal ions and Al3+-metal ion competition.

In this study emission and synchronous-scan fluorescence spectroscopy have been used to investigate the interaction of the class A (oxygen seeking 'hard acid') metal Al(3+), with Suwannee River fulvic acid (SRFA), as well as competition between Al(3+) and several other metal ions (Ca(2+), Mg(2+), Cu(2+), Pd(2+), La(3+), Tb(3+) and Fe(3+)) for binding sites on SRFA. Of the four metal ions possessing very similar (and relatively low) ionic indices (Ca(2+), Mg(2+), Cu(2+) and Pd(2+)) only the latter two paramagnetic ions significantly quenched SRFA fluorescence emission intensity. Of the four metal ions possessing very similar (and relatively low) covalent indices (Ca(2+), Mg(2+), La(3+) and Tb(3+)) only the last paramagnetic ion (Tb(3+)) significantly quenched SRFA fluorescence. None of these metals was able to significantly compete with SRFA-bound Al(3+).Fe(3+), which differs substantially from all of the other metals examined in this study in that it possesses a relatively high ionic index (but not as high as Al(3+)) and a relatively low covalent index (but not as low as Al(3+)), was able not only to quench SRFA fluorescence but also to compete (at least to some extent) with SRFA-bound Al(3+). Synchronous-scan fluorescence SRFA spectra taken in the absence and presence of Fe(3+) and/or Al(3+) support the view that these two metal ions can compete for sites on SRFA. The results of these fluorescence experiments further confirm the Al(3+), and metal ions that have electronic properties somewhat similar to Al(3+) (such as Fe(3+)) are somewhat unique in their ability to interact strongly with binding sites on fulvic acids.

MD simulations of anthrax edema factor: calmodulin complexes with mutations in the edema factor "switch a" region and docking of 3'-deoxy ATP into the adenylyl cyclase active site of wild-type and mutant edema factor variants.

Bacillus anthracis, a spore-forming infectious bacterium, produces an exotoxin, called the edema factor (EF), that functions in part by disrupting internal signalling pathways. When complexed with human host cell calmodulin (CaM), EF becomes an active adenylyl cyclase, producing the internal signal substance cyclic-AMP in an uncontrolled fashion. Recently, the crystal structures for uncomplexed EF and EF:CaM complexes in the presence and absence of a substrate analog (3'-deoxy-ATP), were reported. EF mutational studies have implicated a number of residues important in CaM binding and/or in the generation of the adenylyl cyclase active site, formed by the movements of the EF switch A, B and C regions upon CaM binding. Here we report on the results of molecular dynamics (MD) simulations on two EF:CaM complexes, one containing wild-type EF and the other containing EF in which a cluster of residues in the switch A region (L523, K525, Q526 and V529) have been mutated to alanine. The switch A mutations cause a large increase in the flexibility of the switch C region, the rupture of a number of EF-CaM interactions, an expansion of the carboxyl-terminal domain of CaM, and a change in the Ca(2+) ion binding abilities of the CaM that is in complex with EF. The results indicate the importance of the mutated switch A residues in maintaining a compact EF:CaM complex that appears to be a prerequisite for the generation of a fully-functional adenylyl cyclase active site. The effects of mutating key residues (K346, K353, H577, E588, D590 and N639) in the active site region of EF (to alanine) on the ability of EF to bind the 3'-deoxy-ATP substrate analog were also examined. Active-site residue substitutions at positions 583 (N583A) and 577 (H577A) were found to be particularly disruptive for the placement of the adenine ring moiety into the position found in the x-ray crystal structure of the ligand-protein complex.

Fluorescence characterization of the interaction of Al3+ and Pd2+ with Suwannee River fulvic acid in the absence and presence of the herbicide 2,4-dichlorophenoxyacetic acid.

In an effort to understand the role of environmental metal ions in the interaction of charged pesticides with humic substances, a fluorescence study of the interaction of the widely-used herbicide 2,4-dichlorophenoxyacetic acid (DCPAA) with Al(3+) and Pd(2+) and Suwannee River fulvic acid (SRFA) was undertaken. Initial fluorescence experiments on binary solutions clearly indicated that both Al(3+) and Pd(2+) strongly interact with both SRFA and DCPAA when alone in solution with the metal ion. Titrations of SRFA with Al(3+) at pH values of 4.0, 3.0 and 2.0 revealed decreased degrees of fluorescence emission enhancement (at lambda(emission, max)=424 nm) with decreasing pH, consistent with the expected loss of rigidity in the SRFA-Al(3+) complexes formed as pH is lowered. In contrast, titrations of SRFA with Pd(2+) at all of these pH values resulted in significant fluorescence quenching. Al(3+) additions to solutions of DCPAA at pH values above the pK(a) (2.64) of DCPAA resulted primarily in significant changes in the wavelength of maximum emission (without significant quenching or enhancement of emission intensity), while Pd(2+) additions to DCPAA solutions resulted primarily in very significant fluorescence quenching. The DCPAA fluorescence results strongly support the formation of an Al(3+)-DCPAA complex at pH values above the pK(a) of DCPAA. The fluorescence results obtained for solutions of Pd(2+) and DCPAA are best explained by a collisional quenching mechanism, that is, energy transfer from excited DCPAA molecules to Pd(2+) following the collision of these two species in solution. Excitation-emission matrix plots obtained on ternary solutions (at environmentally-relevant pH 4.0) containing SRFA, DCPAA and metal ions (i.e., either Al(3+) or Pd(2+)) provides evidence (especially for systems containing Al(3+)) for the existence of ternary complexes between fulvic acid species, the herbicide DCPAA and metal ion, suggesting (at least at pH 4.0, where the predominant DCPAA species is negatively-charged) that metal ions may function to "bridge" negatively-charged fulvic acids to negatively-charged pesticides.

Fold recognition, homology modeling, docking simulations, kinetics analysis and mutagenesis of ATP/CTP:tRNA nucleotidyltransferase from Methanococcus jannaschii.

ATP/CTP:tRNA nucleotidyltransferases (NTases) and poly(A) polymerases (PAPs) belong to the same superfamily and their catalytic domains are remotely related. Based on the results of fold-recognition analysis and comparison of secondary structure patterns, we predicted that these two NTase families share three domains, corresponding to "palm," "fingers," and "fingernails" in the PAP crystal structure. A homology model of tRNA NTase from Methanococcus jannaschii was constructed. Energy minimization calculations of enzyme-nucleotide complexes and computer-aided docking of nucleotides onto the enzyme's surface were carried out to explore possible ATP and CTP binding sites. Theoretical models were used to guide experimental analysis. Recombinant His-tagged enzyme was expressed in Escherichia coli, and kinetic properties were characterized. The apparent K(M) for CTP was determined to be 38 microM, and the apparent K(M) for ATP was 21 microM. Three mutations of basic amino acids to alanine were created in a highly conserved region predicted to be in the vicinity of the nucleotide binding site. A deletion was also constructed to remove the C-terminal structural domain defined by the model; it retained about 1% of wild type enzymatic activity using CTP as co-substrate, confirming that detectable catalytic activity is exhibited by the N-terminal domain, as defined by the model. Our results suggest a mechanism of differential ATP and CTP binding, which explains how the tRNA NTase, having only one catalytic site, utilizes different nucleotide triphosphates depending on the nature of the tRNA substrate.

The three-dimensional model of Dictyostelium discoideum racE based on the human rhoA-GDP crystal structure.

The three-dimensional structure of racE was modeled using several homologous small G proteins, and the best model obtained using the human rhoA as modeling template is reported. The three-dimensional fold of the racE model is remarkably similar to the cellular form of human ras p21 crystal structure. Its secondary structure consists of six alpha-helices, six beta-strands and three 3(10) helices. The model retains its secondary structure after a 300 K, 300 ps molecular dynamics (MD) simulation. Important domains of the protein include its effector loop (residues 34-46), the insertion domain (residues 121-136), and the polybasic motif (between 210 and 220) not modeled in the current structure. The effector loop is inherently flexible and the structure docked with GDP exhibits the effector loop moving significantly closer to the nucleotide binding pocket, forming a tighter complex with the bound GDP. The mobility of the effector loop is conferred by a single residue 'hinge' point at residue 34Asp, also allowing the Switch I region, immediately preceding the effector loop, to be equally mobile. In comparison, the Switch II region shows average mobility. The insertion domain is highly flexible, with the insertion taking the form of a helical domain, with several charged residues forming a complex charged interface over the entire insertion region. While the GDP moiety is loosely held in the active site, the metal cation is extensively co-ordinated. The critical residue 38Thr exhibits high mobility, and is seen interacting directly with the metal ion at a distance of 2.64 A, and indirectly via an intervening water molecule. 64Gln, a key residue involved in GTP hydrolysis in ras, is seen facing the beta-phosphate group and the metal ion. Certain residues (i.e. 51Asn, 38Thr and 65Glu) exhibit unique characteristics and these residues, together with 158Val, may play important roles in the maintenance of the protein's integrity and function. There is strong consensus of secondary structural elements between models generated using various templates, such as h-rac1, h-rhoA and h-cdc42 bound to RhoGDI, all sharing only 50-55% sequence identity with racE, which suggests that this model is in all probability an accurate prediction of the true tertiary structure of racE.

Molecular dynamics study of Ca(2+) binding loop variants of silver hake parvalbumin with aspartic acid at the "gateway" position.

The helix-loop-helix (i.e., EF-hand) Ca(2+) ion binding motif is characteristic of a large family of high-affinity calcium ion binding proteins, including the parvalbumins, oncomodulins and calmodulins. In this work we describe a set of molecular dynamics computations on the major parvalbumin from the silver hake (SHPV-B) and on functional fragments of this protein, consisting of the first four helical regions (the ABCD fragment), and the internal helix-loop- helix region (the CD fragment). In both whole protein and protein fragments (i.e., ABCD and CD fragments), the 9th loop residue in the calcium ion binding site in the CD helix-loop-helix region (the so-called "gateway" position) has been mutated from glutamic acid to aspartic acid. Aspartic acid is one of the most common residues found at the gateway position in other (non-parvalbumin) EF- hand proteins, but has never been found at the gateway position of any parvalbumin. (Interestingly, aspartic acid does occur at the gateway position in the closely related rat and human oncomodulins.) Consistent with experimental observations, the results of our molecular dynamics simulations show that incorporation of aspartic acid at the gateway position is very disruptive to the structural integrity of the calcium ion coordination site in the whole protein. The aspartic acid mutation is somewhat less disruptive to the calcium ion coordination sites in the two parvalbumin fragments (i.e., the ABCD and CD fragments), presumably due to the higher degree of motional freedom allowable in these protein fragments. One problem associated with the E59D whole protein variant is a prohibitively close approach of the aspartate carboxyl group to the CD calcium ion observed in the energy-minimized (pre-molecular dynamics) structure. This steric situation does not emerge during energy-minimization of the wild-type protein. The damage to the structural integrity of the calcium ion coordination site in the whole protein E59D variant is not relieved during the molecular dynamics simulation. In fact, during the course of the 300 picosecond simulation, all of the calcium ion ligands leave the primary coordination sphere. In addition, the conserved hydrogen- bonds (in the short beta-sheet structure) that links the CD site to the symmetry-related EF site (in the non-mutated whole protein) is also somewhat disrupted in the E59D whole protein variant. These results suggest that the Ca(2+) ion binding deficiencies in the CD loop are related, at least in part, to the unique interaction that exists between the paired CD and 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 whole silver hake parvalbumin and enzymatically-generated parvalbumin fragments.