Molecular Dynamics

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DeMaria L. Giraldo J. and Wodak SJ.

Shift in Equilibrium Between Folded and Extended Conformations Contributes to Increased Rate of Catalysis of GpNp versus GpN in Barnase .

Proteins Structure Function and Bioinformatics Aug 1;56(2): 261-276
Govaerts C. Blanpain C. Deupi X. BalletS. Ballesteros J.A. Wodak S.J. Vassart G. Pardo L. &Parmentier M.

The TxP motif in the second transmembrane helix of CCR5: a structural determinant of chemokine-induced activation.

J. Biol. Chem. 276 (16) 13217-13225

CCR5 is a G-protein-coupled receptor activated by the chemokines RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein 1alpha and 1beta, and monocyte chemotactic protein 2 and is the main co-receptor for the macrophage-tropic human immunodeficiency virus strains. We have identified a sequence motif (TXP) in the second transmembrane helix of chemokine receptors and investigated its role by theoretical and experimental approaches. Molecular dynamics simulations of model alpha-helices in a nonpolar environment were used to show that a TXP motif strongly bends these helices, due to the coordinated action of the proline, which kinks the helix, and of the threonine, which further accentuates this structural deformation. Site-directed mutagenesis of the corresponding Pro and Thr residues in CCR5 allowed us to probe the consequences of these structural findings in the context of the whole receptor. The P84A mutation leads to a decreased binding affinity for chemokines and nearly abolishes the functional response of the receptor. In contrast, mutation of Thr-82(2.56) into Val, Ala, Cys, or Ser does not affect chemokine binding. However, the functional response was found to depend strongly on the nature of the substituted side chain. The rank order of impairment of receptor activation is P84A > T82V > T82A > T82C > T82S. This ranking of impairment parallels the bending of the alpha-helix observed in the molecular simulation study.

Van Belle D. De Maria L. Iurcu G. &Wodak S.J.

Pathways of ligand clearance in acetylcholinesterase by multiple copy sampling

J. Mol. Biol. 298(4): 705-26.

The clearance of seven different ligands from the deeply buried active-site of Torpedo californica acetylcholinesterase is investigated by combining multiple copy sampling molecular dynamics simulations, with the analysis of protein-ligand interactions, protein motion and the electrostatic potential sampled by the ligand copies along their journey outwards. The considered ligands are the cations ammonium, methylammonium, and tetramethylammonium, the hydrophobic methane and neopentane, and the anionic product acetate and its neutral form, acetic acid. We find that the pathways explored by the different ligands vary with ligand size and chemical properties. Very small ligands, such as ammonium and methane, exit through several routes. One involves the main exit through the mouth of the enzyme gorge, another is through the so-called back door near Trp84, and a third uses a side door at a direction of approximately 45 degrees to the main exit. The larger polar ligands, methylammonium and acetic acid, leave through the main exit, but the bulkiest, tetramethylammonium and neopentane, as well as the smaller acetate ion, remain trapped in the enzyme gorge during the time of the simulations. The pattern of protein-ligand contacts during the diffusion process is highly non-random and differs for different ligands. A majority is made with aromatic side-chains, but classical H-bonds are also formed. In the case of acetate, but not acetic acid, the anionic and neutral form, respectively, of one of the reaction products, specific electrostatic interactions with protein groups, seem to slow ligand motion and interfere with protein flexibility; protonation of the acetate ion is therefore suggested to facilitate clearance. The Poisson-Boltzmann formalism is used to compute the electrostatic potential of the thermally fluctuating acetylcholinesterase protein at positions actually visited by the diffusing ligand copies. Ligands of different charge and size are shown to sample somewhat different electrostatic potentials during their migration, because they explore different microscopic routes. The potential along the clearance route of a cation such as methylammonium displays two clear minima at the active and peripheral anionic site. We find moreover that the electrostatic energy barrier that the cation needs to overcome when moving between these two sites is small in both directions, being of the order of the ligand kinetic energy. The peripheral site thus appears to play a role in trapping inbound cationic ligands as well as in cation clearance, and hence in product release. Copyright 2000 Academic Press.

Oliveira I. & Wodak S.

Thermodynamics of cavity formation in waterand n-hexane using the widom particle insertion method

J. Chem. Phys. 111(18) 8576-8587
Pugliese L. Prevost M. and Wodak S.J.

Simulations of protein unfolding: insights into the hydration.

In Life Sciences of the Nato Science Series Vol. 305 Hydration Processes in Biology : Theoretical and Experimental Approaches 355-370
Giraldo J. Van Belle D. & Wodak S.J.

Conformational Analysis of GpA and GpAp in Aqueous Solution by Molecular Dynamics and Statistical

J. Mol. Biol. 283 863-882.

Barnase, an extracellular endoribonuclease from Bacillus amyloliquefaciens, hydrolyses single-stranded RNA. Its very low catalytic activity toward GpN dinucleotides, where N stands for any nucleoside, is markedly increased when a phosphate is added to the 3'-end, as in GpNp. Here we investigate the conformational properties of GpA and GpAp in solution, in order to determine whether differences in these properties may be related to the changes in enzymatic activity. Two independent 1.3 ns molecular dynamics trajectories are generated for each dinucleotide in the presence of explicit water molecules and counter ions. These trajectories are analysed by monitoring molecular properties, such as the solvent accessible surface area, the distance and orientation between the bases, the behaviour of torsion angles and formation of intramolecular H-bonds. To identify relevant correlations between these parameters, statistical techniques, comprising multiple regression, clustering and discriminant analysis are used. Results show that GpA has a significant propensity to form folded conformations (approximately 50%), fostered by a small number of intramolecular H-bonds, whereas GpAp remains essentially extended. The latter behaviour seems to be due to an H-bond between the terminal phosphate and adenosine ribose group, which restricts rotation about the adenine Agamma angle. We also find that GpA folding is induced by a concerted motion of specific torsion angles, which is closely coupled to the formation of a network of flexible hydrogen bonds. Finally, on the basis of an expression for barnase KM, which incorporates the folded/extended conformational equilibria of the dinucleotide substrates, it is argued that our findings on the differences between these equilibria, can qualitatively rationalize the experimentally measured differences in enzymatic properties. Copyright 1998 Academic Press.

Kocher J.-P.A. Prvost M. Wodak S.J. &Lee B.K.

Properties of the protein matrix revealed by the free energy of cavity formation.

Structure 4(12) 1517-1529

BACKGROUND: The classical picture of the hydrophobic stabilization of proteins invokes a resemblance between the protein interior and nonpolar solvents, but the extent to which this is the case has often been questioned. The protein interior is believed to be at least as tightly packed as organic crystals, and was shown to have very low compressibility. There is also evidence that these properties are not uniform throughout the protein, and conflicting views exist on the nature of sidechain packing and on its influence on the properties of the protein. RESULTS: In order to probe the physical properties of the protein, the free energy associated with the formation of empty cavities has been evaluated for two proteins: barnase and T4 lysozyme. To this end, the likelihood of encountering such cavities was computed from room temperature molecular dynamics trajectories of these proteins in water. The free energy was evaluated in each protein taken as a whole and in submolecular regions. The computed free energies yielded information on the manner in which empty space is distributed in the system, while the latter undergoes thermal motion, a property hitherto not analyzed in heterogeneous media such as proteins. Our results showed that the free energy of cavity formation is higher in proteins than in both water and hexane, providing direct evidence that the native protein medium differs in fundamental ways from the two liquids. Furthermore, although the packing density was found to be higher in nonpolar regions of the protein than in polar ones, the free energy cost of forming atomic size cavities is significantly lower in nonpolar regions, implying that these regions contain larger chunks of empty space, thereby increasing the likelihood of containing atomic size packing defects. These larger empty spaces occur preferentially where buried hydrophobic sidechains belonging to secondary structures meet one another. These particular locations also appear to be more compressible than other parts of the core or surface of the protein. CONCLUSIONS: The cavity free energy calculations described here provide a much more detailed physical picture of the protein matrix than volume and packing calculations. According to this picture, the packing of hydrophobic sidechains is tight in the interior of the protein, but far from uniform. In particular, the packing is tighter in regions where the backbone forms less regular hydrogen-bonding interactions than at interfaces between secondary structure elements, where such interactions are fully developed. This may have important implications on the role of sidechain packing in protein folding and stability.

Van Belle D. and Wodak S.J.

Extended Lagrangian formalism applied to temperature control and electronic polarization effects in molecular dynamics simulations

Computer Physics Communications 91 253-262
Pugliese L. Prvost M. and Wodak S.J.

Unfolding simulations of the 85-102 b-hairpin of barnase

J. Mol. Biol. 251(3) 432-447

Molecular dynamics simulations are used to investigate the unfolding reaction of an isolated beta-hairpin formed by residues 85 to 102 of barnase, a ribonuclease from Bacillus amyloliquefaciens. This peptide was considered following evidence from experimental studies that it may act as an initiation site for barnase folding by adopting a native-like conformation early during the folding process. Three successive molecular dynamics simulations of about 300 ps each were carried out for an all-atom model of the hairpin in water at 300 K, 450 K, and 600 K, respectively. A detailed analysis of all three simulations is presented. In particular we investigate the behavior of the backbone hydrogen bonds, and of hydrophobic interactions between side-chains, where distinction is made between contributions from native and non-native contacts, respectively. Furthermore, we investigate peptide water interactions and monitor the presence and size of empty cavities. The behavior of the hairpin in the three simulations, when considered sequentially, describes a process whereby a native-like conformation evolves to an unfolded state. Unfolding starts at the beginning of the 450 K simulation with the loss of two hydrogen bonds at the free hairpin extremities. At about the same time, the centrally located H-bonds are weakened and exchange more frequently with water, but the turn tightens up as the beta-sheet extends into the turn region. All this is accompanied by a volume expansion and the formation of a large hydrophobic side-chain cluster promoted by both native and highly fluctuating non-native apolar contacts involving residues 87 to 90 and 95 to 99. This collapsed but more loosely packed state, essentially stabilized by hydrophobic interactions, is stable throughout the entire 450 K simulation and for about 150 ps at 600 K, after which point it proceeds rapidly to completely denatured conformations. This behavior presents clear analogies with known features of the unfolding reaction of complete proteins. It may indicate that this beta-hairpin has a well-defined conformation on its own, which would be in agreement with its role as an initiation site for folding.

Wodak S.J. Van Belle D. and Prvost M.

Molecular dynamics and free energy calculations applied to the enzyme barnase and one of its stability mutants.

In J. Goodfellow (ed.) Computer modelling inmolecular biology VCH Weinheim 61-102
Lippens G. Van Belle D. Wodak S.J. andJeener J.

T1 relaxation time of water from a molecular dynamics simulation.

Mol. Phys. 80(6) 1469-1484
Lippens G. Hallenga K. Van Belle D. WodakS.J. Nirmala N.R. Hill P. Russell K.C. Smith D.D. and Hruby V.J.

Transfer NOE study of the conformation of oxytocin bound to bovine neurophysin I

Biochemistry 32(36) 9423-9434.

This study reports the structure of the peptide hormone oxytocin bound to its carrier protein, neurophysin I, obtained by nuclear magnetic resonance techniques. At the pH value of 2.1 in our experiments, the ligand is in fast exchange with its carrier protein, allowing the use of transfer-NOE methods. The number of distance constraints for the peptide being limited, considerable attention has been paid to an accurate distance determination. The resulting accurate distance limits were used as input for a distance geometry calculation followed by a restrained molecular dynamics run. Convergence to a well-defined family of structures for oxytocin in its bound state was reached. Both the backbone and the side-chain conformations differ between the bound form and the crystal structure of free oxytocin [Wood, S. P., et al. (1986) Science 232, 633]. These differences, as well as other structural features of the bound form, are discussed in terms of interactions made with the carrier protein. Transfer-NOE experiments at low peptide protein ratios provide direct experimental evidence for contacts between the oxytocin Tyr2 residue and an aromatic residue of neurophysin. The resonance assignments of the aromatic groups [Whittaker, B. A., et al. (1985) Biochemistry 24, 2782] together with the recently published X-ray structure of the neurophysin II protein complexed with a dipeptide [Chen et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4240] allow us to assign the aromatic signal on the protein to the neurophysin Phe22 residue.

Wodak S.J. Van Belle D. and Prvost M.

Molecular dynamics simulations of a protein-water system: analysis of structural and electrostatic properties.

In Cahiers IMABIO (eds. CNRS) Prdictionstructurale et dynamique molculaire des protines 7 39-43
Van Belle D. and Wodak S.J.

Molecular dynamics study of methane hydration and methane association in a polarizable water phase.

J. Am. Chem. Soc. 115(2) 647-652
Wodak S.J. Van Belle D. and Prvost M.

Molecular dynamics simulations and free energy calculations of barnase and two stability mutants.

In Proceedings of INSERM workshop 49Molecular modelling and graphics applied to structure-functionrelationships (Le Vsinet France November 1992)
Van Belle D. Froeyen M. and Wodak S.

Molecular dynamics simulation of polarizablewater by the extended Langrangian method.

Molecular Physics 77(2) 239-255
Karplus M. Prvost M. Tidor B. and Wodak S.

Simulation analysis of the stability mutants R96H of T4 lysozyme and I96A in barnases in Protein Conformation

CIBA. Foundation Symposium 161 Wiley-Chichester 63-74.

Free energy simulation methods are used to analyse the effects of the mutation Arg-96----His on the stability of bacteriophage T4 lysozyme and of Ile-96----Ala on the stability of barnase. By use of thermodynamic integration, the contributions of specific interactions to the free energy change are evaluated. It is shown that a number of contributions that stabilize the wild-type or the mutant partially cancel in the overall free energy difference; some of these involve the unfolded state. Comparison of the results with conclusions based on structural and thermodynamic data leads to new insights into the origin of the stability difference between wild-type and mutant proteins. For the charged-to-charged amino acid mutation in T4 lysozyme, the importance of the contributions of more distant residues, solvent water and the covalent linkage involving the mutated amino acid are of particular interest. Also, the analysis of the Arg-96 to His mutation with respect to the interactions with the C-terminal end of a helix (residues 82-90) indicates that the nearby carbonyl groups (Tyr-88 and Asp-89) make the dominant contribution, that the amide groups do not contribute significantly and that the helix dipole model is inappropriate for this case. For the non-polar-to-non-polar amino acid mutation in barnase, the solvent contribution is unimportant, and covalent terms are shown to be significant because they do not cancel between the folded and unfolded state.

Prvost M. Van Belle D. Lippens G. and WodakS.

Computer simulations of liquid water: Treatment of long-range interactions.

Molecular Physics 71 (3) 587-603.
Wodak S.J. Van Belle D. Froeyen M. andPrvost M.

Molecular dynamics simulations of a solvated protein: analysis of structural and electrostatic properties.

In Rivail (ed.) Modeling of MolecularStructures and Properties (Proceedings of an International Meeting Nancy France 11-15 September 1989). Studies in Physical andTheoretical Chemistry 71 491-513.