Assessing and refining molecular dynamics simulations of proteins with nuclear magnetic resonance data

The sophistication of the force fields, algorithms and hardware used for molecular dynamics (MD) simulations of proteins is continuously increasing. No matter how advanced the methodology, however, it is essential to evaluate the appropriateness of the structures sampled in a simulation by comparison with quantitative experimental data. Solution nuclear magnetic resonance (NMR) data are particularly useful for checking the quality of protein simulations, as they provide both structural and dynamic information on a variety of temporal and spatial scales. Here, various features and implications of using NMR data to validate and bias MD simulations are outlined, including an overview of the different types of NMR data that report directly on structural properties and of relevant simulation techniques. The focus throughout is on how to properly account for conformational averaging, particularly within the context of the assumptions inherent in the relationships that link NMR data to structural properties.     

Solution conformations of a trimannoside from nuclear magnetic resonance and molecular dynamics simulations

N-linked oligosaccharides often act as ligands for receptor proteins in a variety of cell recognition processes. Knowledge of the solution conformations, as well as protein-bound conformations, of these oligosaccharides is required to understand these important interactions. In this paper we present a model for the solution conformations sampled by a simple trimannoside, methyl 3, 6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, which contains two of the most commonly found glycosidic linkages in N-linked oligosaccharides. This model was derived from simulated annealing protocols incorporating distance restraints extracted from NOESY spectra along with torsional restraints computed from three-bond (1)H-(13)C coupling constants measured across the glycosidic bonds. The model was refined in light of unrestrained molecular dynamics simulations conducted in the presence of solvent water. The resulting model depicts a molecule undergoing conformational averaging in solution, adopting four major and two minor conformations. The four major conformations arise from a pair of two-state transitions, one each at the alpha(1-->3) and alpha(1-->6) linkages, whereas the minor conformations result from an additional transition of the alpha(1-->6) linkage. Our data also suggest that the alpha(1-->3) transition is fast and changes the molecular shape slightly, whereas the alpha(1-->6) is much slower and alters the molecular shape dramatically.     

A magnetization-transfer nuclear magnetic resonance study of the folding of staphylococcal nuclease

The equilibrium between alternative folded states of a globular protein, staphylococcal nuclease, has been investigated by using 1H NMR. Magnetization-transfer experiments have revealed the existence of a related structural heterogeneity of the unfolded state, and quantitative analysis of a series of these experiments has permitted the kinetics of folding and interconversion of the different states to be explored. A model based on cis/trans isomerism at the peptide bond preceding Pro-117 has been developed to account for the results. This model, recently supported by a protein-engineering experiment [Evans et al. (1987) Nature (London) 329, 266], has been used to interpret the kinetic data, providing insight into the nature of the folding processes. The predominance of the cis-proline form in the folded state is shown to derive from a large favorable enthalpy term resulting from more effective overall folding interactions. The kinetics of folding and isomerization are shown to occur on similar time scales, such that more than one pathway between two states may be significant. It has been possible, however, to compare the direct folding and unfolding rates within the cis- and trans-proline-containing populations, with results suggesting that the specific stabilization of the cis peptide bond is effective only at a late stage in the folding process.     

NMR assignments of the four histidines of staphylococcal nuclease in native and denatured states

NMR signals from all four histidine ring C epsilon protons and three of the four histidine C delta protons in the protein staphylococcal nuclease have been assigned by comparing spectra of the wild-type (Foggi strain) protein to spectra of three variants that each lack a different histidine residue. All proteins studied were cloned and overproduced in Escherichia coli. The NMR spectra of the three mutant proteins (H8R, H46Y, and H124L) used to make these assignments were similar to one another and to those of the wild type, except for signals from the mutated residues. The pKa values of those histidines conserved between the wild type and the mutants remained essentially unchanged. Multiple histidine C epsilon proton resonances due to non-native forms of nuclease were observed in both thermally induced and acid-induced unfolding. Residue-specific assignments of H epsilon protons in the thermally denatured forms of the mutant H46Y were obtained from connectivities to the native state by saturation transfer.     

Volumetric and spectroscopic characterizations of the native and acid-induced denatured states of staphylococcal nuclease

We have characterized the acid-induced denaturation of staphylococcal nuclease (SNase) at different urea concentrations by a combination of ultrasonic velocimetry, high precision densimetry, and CD spectroscopy. Our CD spectroscopic results suggest that, at low salt and acidic pH, the protein is unfolded with disrupted secondary and tertiary structures. Furthermore, as judged by far UV CD spectra, the protein is further unfolded at acidic pH upon the addition of urea up to the concentration of 1.5 M. The midpoint of the transition shifts to more neutral pH values and the cooperativity of the transition decreases as the acid-induced denaturation of SNase occurs at higher urea concentrations. We find that the change in volume, Deltav, accompanying the acid-induced denaturation of SNase increases from -0.013 cm(3) g(-1) (-218 cm(3) mol(-1)) in the absence of urea to 0.011 cm(3) g(-1) (185 cm(3) mol(-1)) at 1.5 M urea. At all urea concentrations, the partial specific adiabatic compressibility, k(o)(s), of the protein decreases upon its unfolding with the values of Deltak(o)(s) equal to -6.3x10(-6) (-0.106 cm(3) mol(-1) bar(-1)), -4.5x10(-6) (-0.076 cm(3) mol(-1) bar(-1)), -4.6x10(-6) (-0.077 cm(3) mol(-1) bar(-1)), and -3.8x10(-6) (-0.064 cm(3) mol(-1) bar(-1)) cm(3) g(-1) bar(-1) at urea concentrations of 0, 0.5, 1.0, and 1.5 M, respectively. In general, our volumetric results suggest that the acid-induced denatured state of SNase is only partially unfolded with the solvent-exposed surface area equal to 70-80 % of that expected for the fully extended conformation.     

Conformation of a trimannoside bound to mannose-binding protein by nuclear magnetic resonance and molecular dynamics simulations

A model of the carbohydrate recognition domain of the serum form of mannose-binding protein (MBP) from rat complexed with methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside is presented. Allowed conformations for the bound sugar were derived from simulated annealing protocols incorporating distance restraints computed from transferred NOESY spectra. The resulting sugar conformations were then modeled into the MBP binding site, and these models of the complex were refined using molecular dynamics (MD) simulations in the presence of solvent water. These studies indicate that only one of the two major conformations of the alpha(1-->6) linkage found in solution is significantly populated in the bound state (omega = 60 degrees ), whereas the alpha(1-->3) linkage samples at least two states, similar to its behavior in free solution. The bound conformation allows direct hydrogen bonds to form between the sugar and K182 of MBP, in addition to other water-mediated hydrogen bonds. Estimates of binding constants of candidate complexes based on changes in solvent-accessible surface areas upon binding support the NMR and MD results. These estimates further suggest that the enthalpic gains of the additional sugar-MBP interactions in a trisaccharide as opposed to a monosaccharide are offset by entropic penalties, offering an explanation for previous binding data.     

Multiple loop conformations of peptides predicted by molecular dynamics simulations are compatible with nuclear magnetic resonance

The affinity and selectivity of protein-protein interactions can be fine-tuned by varying the size, flexibility, and amino acid composition of involved surface loops. As a model for such surface loops, we study the conformational landscape of an octapeptide, whose flexibility is chemically steered by a covalent ring closure integrating an azobenzene dye into and by a disulfide bridge additionally constraining the peptide backbone. Because the covalently integrated azobenzene dyes can be switched by light between a bent cis state and an elongated trans state, six cyclic peptide models of strongly different flexibilities are obtained. The conformational states of these peptide models are sampled by NMR and by unconstrained molecular dynamics (MD) simulations. Prototypical conformations and the free-energy landscapes in the high-dimensional space spanned by the phi/psi angles at the peptide backbone are obtained by clustering techniques from the MD trajectories. Multiple open-loop conformations are shown to be predicted by MD particularly in the very flexible cases and are shown to comply with the NMR data despite the fact that such open-loop conformations are missing in the refined NMR structures.     

Analysis of long-range interactions in a model denatured state of staphylococcal nuclease based on correlated changes in backbone dynamics.

An expanded, highly dynamic denatured state of staphylococcal nuclease exhibits a native-like topology in the apparent absence of tight packing and fixed hydrogen bonds (Gillespie JR, Shortle D, 1997, J Mol Biol 268:158-169, 170-184). To address the physical basis of the long-range spatial ordering of this molecule, we probe the effects of perturbations of the sequence and solution conditions on the local chain dynamics of a denatured 101-residue fragment that is missing the first three beta strands. Structural interactions between chain segments are inferred from correlated changes in the motional behavior of residues monitored by 15N NMR relaxation measurements. Restoration of the sequence corresponding to the first three beta strands significantly increases the average order of all chain segments that form the five strand beta barrel including loops but has no effect on the carboxy terminal 30 residues. Addition of the denaturing salt sodium perchlorate enhances ordering over the entire sequence of this fragment. Analysis of seven different substitution mutants points to a complex set of interactions between the hydrophobic segment corresponding to beta strand 5 and the remainder of the chain. General patterns in the data suggest there is a hierarchy of native-like interactions that occur transiently in the denatured state and are consistent with the overall topology of the denatured state ensemble being determined by many coupled local interactions rather than a few highly specific long-range interactions.     

Effects of denaturants and substitutions of hydrophobic residues on backbone dynamics of denatured staphylococcal nuclease

Analysis of residual dipolar couplings (RDCs) in the Delta131Delta fragment of staphylococcal nuclease has demonstrated that its ensemble-averaged structure is resistant to perturbations such as high concentrations of urea, low pH, and substitution of hydrophobic residues, suggesting that its residual structure is encoded by local side-chain/backbone interactions. In the present study, the effects of these same perturbations on the backbone dynamics of Delta131Delta were examined through (1)H-(15)N relaxation methods. Unlike the global structure reported by RDCs, the transverse relaxation rates R(2) were quite sensitive to denaturing conditions. At pH 5.2, Delta131Delta exhibits an uneven R(2) profile with several characteristic peaks involving hydrophobic chain segments. Protonation of carboxyl side chains by lowering the pH reduces the values of R(2) along the entire chain, yet these characteristic peaks remain. In contrast, high concentrations of urea or the substitution of 10 hydrophobic residues eliminates these peaks and reduces the R(2) values by a greater amount. The combination of low pH and high urea leads to further decreases in R(2). These denaturant-induced increases in backbone mobility are also reflected in decreases in (15)N NOEs and in relaxation interference parameters, with the former reporting an increase in fast motions and the latter a decrease in slow motions. Comparison between the changes in chain dynamics and the corresponding changes in Stokes radius and the patterns of RDCs suggests that regional variations in backbone dynamics in denatured nuclease arise primarily from local contacts between hydrophobic side chains and local interactions involving charged carboxyl groups.     

Dynamic aspects of antibody:oligosaccharide complexes characterized by molecular dynamics simulations and saturation transfer difference nuclear magnetic resonance

Carbohydrates are likely to maintain significant conformational flexibility in antibody (Ab):carbohydrate complexes. As demonstrated herein for the protective monoclonal Ab (mAb) F22-4 recognizing the Shigella flexneri 2a O-antigen (O-Ag) and numerous synthetic oligosaccharide fragments thereof, the combination of molecular dynamics simulations and nuclear magnetic resonance saturation transfer difference experiments, supported by physicochemical analysis, allows us to determine the binding epitope and its various contributions to affinity without using any modified oligosaccharides. Moreover, the methods used provide insights into ligand flexibility in the complex, thus enabling a better understanding of the Ab affinities observed for a representative set of synthetic O-Ag fragments. Additionally, these complementary pieces of information give evidence to the ability of the studied mAb to recognize internal as well as terminal epitopes of its cognate polysaccharide antigen. Hence, we show that an appropriate combination of computational and experimental methods provides a basis to explore carbohydrate functional mimicry and receptor binding. The strategy may facilitate the design of either ligands or carbohydrate recognition domains, according to needed improvements of the natural carbohydrate:receptor properties.     

Robustness of the long-range structure in denatured staphylococcal nuclease to changes in amino acid sequence

A nativelike low-resolution structure has been shown to persist in the Delta 131 Delta denatured fragment of staphylococcal nuclease, even in the presence of 8 M urea. In this report, the physical-chemical basis of this structure is addressed by monitoring changes in structure reflected in residual dipolar couplings and diffusion coefficients as a function of changes in amino acid sequence. Ten large hydrophobic residues, previously shown to play dominant roles in the stability of the native state, are replaced with polar residues of similar shape. Modest increases in the Stokes radius determined by NMR methods result from replacement of five isoleucine/valine residues with threonine, one leucine with glutamine, and oxidation of four methionines to the sulfoxides. Yet in the presence of all ten hydrophobic to polar substitutions and 8 M urea, the NMR signature of a native-like topology is still largely intact. In addition, removal of 30 residues from either the N-terminus (which deletes a three-strand beta meander) or C-terminus (a long extended segment and the final alpha helix) produces only very small changes in long-range structure. These data indicate that both the general shape of the denatured state and the angular relationships of individual bond angles to the axes describing the spatial distribution of the protein chain are insensitive to large changes in the amino acid sequence, a finding consistent with the conclusion that the long-range structure of denatured proteins is encoded primarily by local steric interactions between side chains and the polypeptide backbone.     

Nucleotide-dependent conformational changes in HisP: molecular dynamics simulations of an ABC transporter nucleotide-binding domain

ATP-binding cassette (ABC) transporters mediate the movement of molecules across cell membranes in both prokaryotes and eukaryotes. In ABC transporters, solute translocation occurs after ATP is either bound or hydrolyzed at the intracellular nucleotide-binding domains (NBDs). Molecular dynamics (MD) simulations have been employed to study the interactions of nucleotide with NBD. The results of extended (approximately 20 ns) MD simulations of HisP (total simulation time approximately 80 ns), the NBD of the histidine transporter HisQMP2J from Salmonella typhimurium, are presented. Analysis of the MD trajectories reveals conformational changes within HisP that are dependent on the presence of ATP in the binding pocket of the protein, and are sensitive to the presence/absence of Mg ions bound to the ATP. These changes are predominantly confined to the alpha-helical subdomain of HisP. Specifically there is a rotation of three alpha-helices within the subdomain, and a movement of the signature sequence toward the bound nucleotide. In addition, there is considerable conformational flexibility in a conserved glutamine-containing loop, which is situated at the interface between the alpha-helical subdomain and the F1-like subdomain. These results support the mechanism for ATP-induced conformational transitions derived from the crystal structures of other NBDs.     

Hydrogen-1 nuclear magnetic resonance studies of staphylococcal nuclease variant H124L: pH dependence of histidines and tyrosines

The pH dependence of the 1H NMR spectrum of staphylococcal nuclease H124L was investigated as a function of the binding of Ca2+, the ion required for enzymatic activity, and deoxythymidine-3',5'-diphosphate (pdTp), a competitive inhibitor. The protein studied was the product of a cloned gene expressed in Escherichia coli which yields a protein having a sequence identical to that of the nuclease isolated from the V8 strain of Staphylococcus aureus. Of the observable ring protons of the three histidine residues, only the C delta 1H of His46 shows a large chemical shift perturbation on formation of the ternary complex, (nuclease H124L).pdTp.Ca2+. The pKa of His46 is lowered by 0.2 pH unit in the binary complex. All seven tyrosines titrate with normal pKa values between 9 and 11 in the unligated nuclease. In the ternary complex, however, the pKa values of Tyr85 and Tyr93 increase above pH 11.0. The chemical shift perturbations of the ring protons of the Tyr27, Tyr85, Tyr113, and Tyr115 were observed between pH 4 and 6; these spectral perturbations are attributed to interactions with carboxylate groups. Binding Ca2+ alone acted opposite to the perturbation in Tyr113 and Tyr115. Ca2+ binding leads to deshielding the ring protons of Tyr113, but this effect is removed in the ternary complex. Binding of pdTp and Ca2+ stabilizes the protein against high pH denaturation up to pH 11.5.     

Nucleotide-mediated conformational changes of monomeric actin and Arp3 studied by molecular dynamics simulations

Members of the actin family of proteins exhibit different biochemical properties when ATP, ADP-P_i, ADP, or no nucleotide is bound. We used molecular dynamics simulations to study the effect of nucleotides on the behavior of actin and actin-related protein 3 (Arp3). In all of the actin simulations, the nucleotide cleft stayed closed, as in most crystal structures. ADP was much more mobile within the cleft than ATP, despite the fact that both nucleotides adopt identical conformations in actin crystal structures. The nucleotide cleft of Arp3 opened in most simulations with ATP, ADP, and no bound nucleotide. Deletion of a C-terminal region of Arp3 that extends beyond the conserved actin sequence reduced the tendency of the Arp3 cleft to open. When the Arp3 cleft opened, we observed multiple instances of partial release of the nucleotide. Cleft opening in Arp3 also allowed us to observe correlated movements of the phosphate clamp, cleft mouth, and barbed-end groove, providing a way for changes in the nucleotide state to be relayed to other parts of Arp3. The DNase binding loop of actin was highly flexible regardless of the nucleotide state. The conformation of Ser14/Thr14 in the P1 loop was sensitive to the presence of the γ-phosphate, but other changes observed in crystal structures were not correlated with the nucleotide state on nanosecond timescales. The divalent cation occupied three positions in the nucleotide cleft, one of which was not previously observed in actin or Arp2/3 complex structures. In sum, these simulations show that subtle differences in structures of actin family proteins have profound effects on their nucleotide-driven behavior.     

Insights on pH-dependent conformational changes of mosquito odorant binding proteins by molecular dynamics simulations

Chemical recognition plays an important role for the survival and reproduction of many insect species. Odorant binding proteins (OBPs) are the primary components of the insect olfactory mechanism and have been documented to play an important role in the host-seeking mechanism of mosquitoes. They are “transport proteins” believed to transport odorant molecules from the external environment to their respective membrane targets, the olfactory receptors. The mechanism by which this transport occurs in mosquitoes remains a conundrum in this field. Nevertheless, OBPs have proved to be amenable to conformational changes mediated by a pH change in other insect species. In this paper, the effect of pH on the conformational flexibility of mosquito OBPs is assessed computationally using molecular dynamics simulations of a mosquito OBP “CquiOBP1” bound to its pheromone 3OG (PDB ID: 3OGN). Conformational twist of a loop, driven by a set of well-characterized changes in intramolecular interactions of the loop, is demonstrated. The concomitant (i) closure of what is believed to be the entrance of the binding pocket, (ii) expansion of what could be an exit site, and (iii) migration of the ligand towards this putative exit site provide preliminary insights into the mechanism of ligand binding and release of these proteins in mosquitoes. The correlation of our results with previous experimental observations based on NMR studies help us provide a cardinal illustration on one of the probable dynamics and mechanism by which certain mosquito OBPs could deliver their ligand to their membrane-bound receptors at specific pH conditions.     

A model of the changes in denatured state structure underlying m value effects in staphylococcal nuclease.

Hydrogen exchange kinetics were measured on the native states of wild type staphylococcal nuclease and four mutants with values of mGuHCl (defined as dDeltaG/d[guanidine hydrochloride]) ranging from 0.8 to 1.4 of the wild type value. Residues within the five-strand beta-barrel of wild type and E75A and D77A, two mutants with reduced values of m GuHCl, were significantly more protected from exchange than expected on the basis of global stability as measured by fluorescence. In contrast, mutants V23A and M26G with elevated values of mGuHCl approach a flat profile of more or less constant protection independent of position in the structure. Differences in exchange protection between the C-terminus and the beta-barrel region correlate with mGuHCl, suggesting that a residual barrel-like structure becomes more highly populated in the denatured states of m- mutants and less populated in m+ mutants. Variations in the population of such a molten globule-like structure would account for the large changes in solvent accessible surface area of the denatured state thought to underlie m value effects.