We have made significant progress in several areas related to protein dynamics, folding, binding, and function. Multi-protein assemblies: We continued our development of coarse-grained models and effective energy functions to study the thermodynamic and structural properties of multiprotein complexes with relatively low binding affinity (Kd >1 micromolar). Folded protein domains are represented as rigid bodies. The interactions between the domains are treated at the residue level with amino-acid-dependent pair potentials and Debye-Huckel-type electrostatic interactions. Flexible linker peptides connecting rigid protein domains are represented as amino acid beads on a polymer with appropriate stretching, bending, and torsion-angle potentials. To enhance the sampling of multi-protein assemblies with long linkers or tethers that result in a network topology, we added the option of constructing Gaussian-chain type connections. With the validated model, in collaboration with the group of Dr. Hurley (NIDDK), we simulated the human ESCRT-0 complex comprised of Hrs and STAM proteins (Ren et al., Structure, 2009). ESCRT-0 helps sort ubiquitinated cell surface receptors to lysosomes for degradation. By combining information from previously solved domain structures and hydrodynamic measurements with our simulation model, we were able to build a structure for the complete ESCRT-0 complex. Our simulations of ESCRT-0 revealed a dynamic ensemble of conformations well suited for diverse functions. Simulation methodology: Molecular-dynamics (MD) simulations are widely used to study the structure and dynamics of bio-macromolecules. But despite their widespread use, two major issues are plaguing MD simulations: the accuracy of the underlying force fields describing the molecular interactions, and the efficiency of the computational sampling. We have made progress in both directions. Based on a careful validation of current force fields against experimental data on peptides, done in collaboration with Dr. Best (University of Cambridge), we could show that relatively minor corrections in the energy functions resulted in a dramatic improvement in the ability to produce correct secondary structure preferences in MD simulations (Best et al., J. Phys. Chem. B 2009). We also examined replica-exchange molecular dynamics, a widely used method to enhance the sampling of the conformation space of biomolecules (Rosta et al, J. Chem. Theory Comput. 2009) and could show that the use of certain thermostats can result in biased melting profiles for protein folding. Enzyme function. In collaboration with Dr. Gutkind (NIDCR) and Prof. Turjanski (Univ. Buenos Aires), we have studied the enzymatic function of mitogen-activated protein kinases (MAPK). MAPK signaling pathways play an essential role in the transduction of environmental stimuli to the nucleus, thereby regulating a variety of cellular processes, including cell proliferation, differentiation, and programmed cell death. We have modeled the interaction of the kinase ERK with a target peptide and analyzed the specificity toward Ser/Thr-Pro motifs. By using a quantum mechanics/molecular mechanics (QM/MM) approach, we identified a mechanism for the phosphoryl transfer reaction (Turjanski et al., J. Am. Chem. Soc. 2009). Our results suggest that (1) the proline residue has a role in both specificity and phosphor transfer efficiency, (2) the reaction occurs in one step, with ERK2 Asp147 acting as the catalytic base, (3) a conserved Lys in the kinase superfamily strongly stabilizes the transition state, and (4) the reaction mechanism is similar with either one or two Mg2+ ions in the active site. Taken together, our results provide a detailed description of the molecular events involved in the phosphorylation reaction catalyzed by MAPK and contribute to the general understanding of kinase activity.
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