Rick L. Ornstein

Molecular dynamics simulations of a protein-protein dimer: particle-mesh Ewald electrostatic model yields far superior results to standard cutoff model.

In this article we present two 1000 ps molecular dynamics simulations on the rat micro-glutathione S-transferase dimeric enzyme in complex with the product 1-(S-glutathionyl)-2,4-dinitrobenzene, in a periodic box with explicit solvent molecules, and investigate the effect of long-range electrostatics models on the structure and dynamics of the dimer and its components. One simulation used the standard cutoff method (10A), whilst the other used the particle-mesh Ewald (PME) method. We monitored the root mean-square atomic deviation (RMSD) from the initial crystal structure to examine the convergence of both simulations, as well as several other structural parameters such as the distance between active sites, rigid body rotation between domains in subunits, radius of gyration, B-factors, number of hydrogen bonds and salt bridges and solvent-accessible surface area. For example, with the PME method, the dimer structure remains much closer to the initial crystallographic structure with an average RMSD of 1.3A +/- 0.1A and 1.0A +/- 0.1A for all heavy and backbone atoms, respectively, in the last 200 ps; the respective values for the cutoff simulation are 4.7A +/- 0.3A and 4.2A +/- 0.3A. The large deviations observed in the cutoff simulation severely affected the stability of the enzyme dimer and its complex with the bound product. This finding is contrary to that found in a similar study of the monomeric protein ubiquitin [Fox, T. & Kollman, P. A. Proteins Struct. Func. Genet. 25, 315-334 (1996)]. Unlike the earlier published work, the present study provides evidence that the standard cutoff method is not generally valid for the study of protein complexes, or their subunits.

Ab Initio Quantum Mechanics Analysis of Imidazole C-H…O Water Hydrogen Bonding and a Molecular Mechanics Forcefield Correction

While it is well established that classical hydrogen bonds play an important role in enzyme structure, function and dynamics, the role of weaker, but activated C-H donor hydrogen bonds is poorly understood. The most important such case involves histidine which often plays a direct role in enzyme catalysis and possesses the most acidic C-H donor group of the standard amino acids. In the present study, we obtained optimized geometries and hydrogen bond interaction energies for C-H...O hydrogen bonded complexes between methane, ethylene, benzene, acetylene, and imidazole with water at the MP2-FC/6-31++G(2d,2p) and MP2-FC/aug-cc-pVDZ/MP2-FC/6-31++G(2d,2p) levels of theory. A strong linear relationship is obtained between the stability of the various hydrogen bonded complexes and both separation distances for H...O and C----O. In general, these calculations indicate that C-H...O interactions can be classified as hydrogen bonding interactions, albeit significantly weaker than the classical hydrogen bonds, but significantly stronger than just van der Waals interactions. For instance, while the electronic energy of stabilization at the MP2-FC/aug-cc-pVDZ/MP2-FC/6-31++G(2d,2p) level of theory of a water O-H...O water hydrogen bond is 4.36 kcal/mol more stable than the methane C-H...O water interaction, the water-water hydrogen bond is only 2.06 kcal/mol more stable than the imidazole Ce-H...O water hydrogen bond. Neglecting this latter hydrogen bonding interaction is obviously unacceptable. We next compare the potential energy surfaces for the imidazole Ce-H...O water and imidazole Na-H...O hydrogen bonded complexes computed at the MP2/6-31++G(2d,2p) level of theory with the potential energy surface computed using the AMBER molecular mechanics program and forcefields. While the Weiner et al and Cornell et al AMBER forcefields reasonably account for the imidazole N-H...O water interaction, these forcefields do not adequately account for the imidazole Ce-H...O water hydrogen bond. A forcefield modification is offered that results in excellent agreement between the ab initio and molecular mechanics geometry and energy for this C-H...O hydrogen bonded complex.

DNA binding by TATA-box binding protein (TBP): a molecular dynamics computational study.

TATA-box binding protein (TBP) in a monomeric form and the complexes it forms with DNA have been elucidated with molecular dynamics simulations. Large TBP domain motions (bend and twist) are detected in the monomer as well as in the DNA complexes; these motions can be important for TBP binding of DNA. TBP interacts with guanine bases flanking the TATA element in the simulations of the complex; these interactions may explain the preference for guanine observed at these DNA positions. Side chains of some TBP residues at the binding interface display significant dynamic flexibility that results in flip-flop contacts involving multiple base pairs of the DNA. We discuss the possible functional significance of these observations.

A density functional theory investigation of metal ion binding sites in monosaccharides

Abstract The density functional theory method is used to study metal ion binding to simple carbohydrates such as cis -inositol and β- d -glucose. The complexes formed between Be 2+ , Mg 2+ , Ca 2+ , and Li + and cis -inositol and the complexes formed between Ca 2+ and β- d -glucose are optimized and metal binding sites are identified. There are two metal binding sites in cis -inositol and five in β- d -glucose. Our calculations demonstrate that smaller ions such as Be 2+ prefer to bind in the ax-ax-ax site of cis -inositol, while larger ions such Mg 2+ and Li + favor the ax-eq-ax site of cis -inositol. The preferred metal binding site in Ca 2+ - cis -inositol is defined by four hydroxyl groups instead of three (one equatorial and three axial OH). Among the five metal binding sites in β- d -glucose, the one defined by the ring oxygen atom, C1-OH and C6-OH is the most preferred site where Ca 2+ binds to three oxygen atoms. This tendency of metal ions to maximize interactions with carbohydrate ligands in the gas phase is in agreement with previous experimental and theoretical studies. The relevance of these metal-carbohydrate interactions in cell surface carbohydrate-binding proteins is also discussed. This study has demonstrated that the density functional theory method is a good method for identifying metal ion binding sites in carbohydrates.

Effect of Warmup Protocol and Sampling Time on Convergence of Molecular Dynamics Simulations of a DNA Dodecamer Using AMBER 4.1 and Particle-Mesh Ewald Method

This report describes one 3000 ps and two 1500 ps molecular dynamic simulations on a TATA box containing dodecamer DNA duplex in a periodic box of TIP3P water molecules, using the AMBER 4.1 implementation of the particle-mesh Ewald method. We compare the effect of warmup protocol and simulation time length on the root-mean square deviation (RMSD) parameter. For the longer simulation, the RMSD computed for the 500-1000 ps time interval is representative of longer time intervals, including 500-3000 ps. The various warmup protocols do not appear to have a significant effect on the simulation results. Based on the present results, DNA sequence-dependent differences in RMSD, or related properties, should exceed two standard deviations before being attributed to non-simulation factors, such as warmup protocol and sampling time effects; we recommend a minimum criterion of at least a three standard deviation difference with a sampling period of at least 500-1000 ps. In addition, while end effects appear negligible there is a consistent dependence of RMSD on DNA helix length.

Protein hinge bending as seen in molecular dynamics simulations of native and M61 mutant T4 lysozymes.

A dynamical model of interdomain hinge bending of T4 lysozyme in aqueous solution has been developed on the basis of molecular dynamics (MD) simulation. The MD model study provides a description of the conformational reorganization expected to occur for the protein in aqueous solution as compared to the crystalline environment. Three different 500 ps molecular dynamics simulations were calculated, each using a distinctly different crystal conformation of T4 lysozyme as the starting points of the MD simulations. Crystal structures of wild-type lysozyme and open and closed forms of M61 variant structures were analyzed in this study. Large-scale, molecular-conformational rearrangements were observed in all three simulations, and the largest structural change was found for the open form of the M61 allomorph. All three simulated proteins had closed relative to the wild-type crystal structure, and the closure of the jaws of the active site cleft occurred gradually over the time course of the trajectories. The time average MD structures, calculated over the final 50 ps of each trajectory, had all adapted to conformations more similar to each other than to their incipient crystal forms. Using a similar MD protocol on cytochrome P450BM-3 [M. D. Paulsen and R. L. Ornstein (1995) Proteins: Structure Function and Genetics, Vol. 27, pp. 237-243] we have found that the opposite type of motion relative to the starting crystal structure, that is, the open form of the crystal structure, had opened to a greater degree relative to the incipient crystal structure form. Therefore we do not believe that either result is merely a simulation artifact, but rather the protein dynamics are due to protein relaxation in the absence of crystal packing forces in the simulated solution environments.

Investigation of Domain Motions in Bacteriophage T4 Lysozyme

Hinge-bending in T4 lysozyme has been inferred from single amino acid mutant crystalline allomorphs by Matthews and coworkers. This raises an important question: are the different conformers in the unit cell artifacts of crystal packing forces, or do they represent different solution state structures? The objective of this theoretical study is to determine whether domain motions and hinge-bending could be simulated in T4 lysozyme using molecular dynamics. An analysis of a 400 ps molecular dynamics simulation of the 164 amino acid enzyme T4 lysozyme is presented. Molecular dynamics calculations were computed using the Discover software package (Biosym Technologies). All hydrogen atoms were modeled explicitly with the inclusion of all 152 crystallographic waters at a temperature of 300 K. The native T4 lysozyme molecular dynamics simulation demonstrated hinge-bending in the protein. Relative domain motions between the N-terminal and C-terminal domains were evident. The enzyme hinge bending sites resulted from small changes in backbone atom conformations over several residues rather than rotation about a single bound. Two hinge foci were found in the simulation. One locus comprises residues 8-14 near the C-terminal of the A helix; the other site, residues 77-83 near the C-terminal of the C helix. Comparison of several snapshot structures from the dynamics trajectory clearly illustrates domain motions between the two lysozyme lobes. Time correlated atomic motions in the protein were analyzed using a dynamical cross-correlation map. We found a high degree of correlated atomic motions in each of the domains and, to a lesser extent, anticorrelated motions between the two domains. We also found that the hairpin loop in the N-terminal lobe (residues 19-24) acted as a mobile flap and exhibited highly correlated dynamic motions across the cleft of the active site, especially with residue 142.

Molecular dynamics of subtilisin Carlsberg in aqueous and nonaqueous solutions

Crystal structures have recently appeared for the enzyme subtilisin Carlsberg in anhydrous acetonitrile and in water. To gain a mechanistic understanding of how the solvent environment affects protein structure and dynamics, we have performed molecular dynamics simulations on subtilisin Carlsberg in water and acetonitrile. We describe a 480 ps simulation of subtilisin in acetonitrile solution and a 450 ps simulation of subtilisin in water. Each simulation employed the all-atom AMBER force field. The calculated rms deviations, from their respective x-ray structures, were similar in each simulation, but ∼0.5 Å higher in the acetonitrile simulation. Only in the acetonitrile simulation does one helix undergo a reversible partial unwinding, which lasted for about 100 ps. The other secondary structure elements remain intact or undergo modest fluctuations. In the aqueous simulation, the calculated and experimental temperature factors agree very well. In the acetonitrile simulation, however, the calculated temperature factors are much higher than the experimental values. The larger rms deviation and thermal fluctuations noted in the acetonitrile simulation are consistent with the requirement for protein cross-linking in this crystal and a recent two-dimensional NH-exchange nmr study on horse heart cytochrome c in nonaqueous solution.

[38] Using molecular modeling and molecular dynamics simulation to predict P450 oxidation products

Publisher Summary This chapter focuses on the use of molecular modeling and molecular dynamics simulation to predict P450 oxidation products. The chapter overviews the application of computational methods to P450s and discusses the use of molecular dynamics simulations for the accurate assessment of oxidation specificity by P450 systems. It is observed that the substrates for the cytochromes P450I and P450II subfamilies cluster into two groups; substrates for the P450I subfamily are more planar in shape and exhibit smaller energy differences between the highest occupied and lowest unoccupied molecular orbitals (HOMO and LUMO, respectively) than the substrates for the P450II subfamily. Studies indicate that if due attention is given to ensure proper sampling of the important substrate conformations, accurate product profiles can be predicted for a number of reactions of P450, including hydroxylations, epoxidations, and sulfoxidations. Studies suggest that under appropriate conditions, many time-saving approximations such as the inclusion of little or no added explicit solvent or simulating the dynamics of only the substrate binding pocket can still lead to reliable results.

Molecular models of induced DNA premutational damage and mutational pathways for the carcinogen 4-nitroquinoline 1-oxide and its metabolites

The carcinogen 4-nitroquinoline 1-oxide (4NQO) and its metabolites undergo intercalative or covalent binding with DNA. Recent evidence indicates that the latter binding pattern is probably facilitated by an initial weaker intercalative interaction that can align potentially reactive sites on a 4NQO-metabolite and adjacent stacked bases. In the present study, we have proposed numerous possible covalent reaction products between 4NQO and its metabolites with DNA mini-helices based on chemical properties and key short-contacts after energy-minimization in 21 different intercalative-like complexes. It is known from numerous experimental studies that 90% of the quinoline-bound DNAs in vivo involve guanine with the remaining 10% apparently involving adenine residues. The results of the present study suggest that this trend is not due to the greater affinity of the quinolines for guanine, but instead results from secondary processes involving the preferential formation of apurinic sites at aralkyl-adenine residues over that of aralkyl-guanine residues. In addition, observed mutational patterns can be rationalized in terms of the proposed reaction-products. The role of DNA repair mechanisms in the removal and correction of the different proposed reaction products are discussed. The binding pattern of several other aromatic carcinogens are similar to those depicted in the present work for the 4NQO-metabolites; hence the present study may be of some general significance.