_______________________________________________________________________________

Molecular dynamics simulations of domain motions of substrate-free S-adenosyl-L-homocysteine hydrolase.

C. Hu, J. Fang, R. T. Borchardt, R. L. Schowen and K. Kuczera.

Proteins, 71:131-143 (2008).

S-Adenosyl-L-homocysteine hydrolase (SAHH) plays a pivotal role in regulating intracellular methylation reactions. The homotetrameric enzyme exists in an open conformation in absence of substrate. Enzyme:inhibitor complexes crystallize in the closed conformation, in which the ligands are engulfed by the protein due to a ca. 18o reorientation of domains within each of the four subunits. We present a microscopic description of the structure and dynamics of the substrate-free, NAD+-bound SAHH in solution, based on a 15 ns molecular dynamics simulation using explicit solvation. In the 15 ns trajectory, the substrate-binnding domains (SBDs) and cofactor-binding domains (CBDs) within each subunit retained internal structures similar to the crystal conformation. At the tetrameric level, the CBDs of the four subunits formed a relatively immoble core, with conformation similar to that found in the crystal and fluctuations comparable to x-ray temperature factors. The SBDs, located at the tetramer exterior, performed large amplitude rigid-body reorientations in solution, which consisted of two components, faster, 20-50 ps rotations, corresponding to diffusion in a cone of 3-4 deg and slower, 8-23 ns rotations, corresponding to diffusion in a cone of 14-22 deg . The latter time scale, amplitude and type of motion are in accord with fluorescence anisotropy decay data, which detected a 10-20 ns domain reorientation of ca. 20 deg amplitude in the substrate-free enzyme. Interestingly, the SBD reorientations involved roughly equal contributions from motions along and perpendicular to the direction of the open-to-closed transition. Using a continuum electrostatics and buried surface area model, we analyzed the average interactions between the protein and its NAD+ cofactors. Surprisingly, the enzyme:cofactor electrostatic interactions were unfavorable, and the buried surafce area term was the driving force for complex formation. Overall, our simulation results complement the existing experimental data, providing an improved understanding of SAHH structure, dynamics and cofactor binding in solution. We will employ these insights into the catalytic mechanism to develop SAHH inhibitors that will be specific and potent antiparasitic agents.

Structure of SAHH. Average reorientation of the four SBS from MD.

_______________________________________________________________________________

Phospholamban Dynamics in Complex Environments: Effects of CMAP Correction and Electrostatic Cutoffs.

Y. Houndonougbo, K. Kuczera and G. S. Jas.

J. Biomol. Struct. Dyn., 26: 17-34 (2008).

We have performed a series of 5-20ns molecular dynamics simulations to investigate the influence of environment and force field on the microscopic structure and dynamics of phospholamban (PLB), a small integral membrane protein. Effect of the environment is studied by simulations in methanol, water and DPPC bilayer, and the influence of force field modification followed by using three variants of the CHARMM22 force field, the standard parameters with nonbonded cutoffs, a version with cutoffs and improved description of protein backbone tortions (CMAP correction), and one with both CMAP correction and Ewald treatment of electrostatics. The simulations show that two main features of PLB structure and dynamics are present under all studied conditions: existence of two well-defined helical domains at the N- and C-termini, and large-amplitude rigid-body motions of these domains. The average interhelix angle of PLB was sensitive to the environment. In the methanol and water solution trajectories, the two helical domains tended to adopt a “closed” orientation, with the interhelix angle below 90 deg, while in the lipid bilayer the domains tend to be in an open conformation, with the interhelix angle above 90 deg. Within each studied environment, simulations employing different force field models provided qualitatively similar description of PLB structure and dynamics. The only significant discrepancy was the presence of -helical hydrogen bonds in trajectories generated with the standard CHARMM22 force field. Trajectories generated with CMAP correction, with both cutoff and Ewald electrostatics, exhibited predominantly -helical and some 310-helical hydrogen bonding interactions, and no -helical hydrogen bonding, in accord with NMR data. Thus, our results indicate that models including CMAP, with both cutoff and Ewald electrostatics, provide the most realistic description of PLB structure and dynamics. Many features observed in the simulations are in a good agreement with the experimental measurements. These include the mostly helical secondary structure of the protein, including the range explored by the interhelix angle in methanol, as well as the interhelix distance and C-terminal helix orientation in the DPPC bilayer. The observed effect of opening up of the PLB interhelix angle in the lipid environment relative to solution is also qualitatively reproduced in the simulations as well as the more rigid and compact structure of the C-terminal domain in the membrane relative to solution.

NMR structure of phoshlamban: 1N7L model 1. PLB in DPPC bilayer with water and Na+/Cl- ions

_______________________________________________________________________________

Go back to main abstracts page

or go on to 2007 abstracts