Theory and simulation of protein dynamics, folding, and function

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We have made significant progress in several areas related to protein dynamics, folding, binding, and function. Nucleic acid processing catalysis: With quantum mechanics / molecular mechanics (QM/MM) simulations we studied the cleavage of the ribonucleic acid (RNA) backbone in an RNA/DNA dimer (1). This reaction is catalyzed by ribonuclease H (RNase H), a prototypical member of a large family of enzymes that use two-metal catalysis to process nucleic acids. This family includes HIV reverse transcriptase, a primary target for antiretroviral drugs, with an RNase H domain that is essential for HIV viral replication. By calculating the multidimensional free energy surface for distances between pairs of atoms involved the reaction we could characterize the mechanism of the RNA cleavage reaction. In a first reaction step, a water molecule attacks the scissile phosphate, aided by magnesium ion A. This attack results in breaking of the bond between the phosphate and the ribose oxygen. In a second step, the reaction is completed by protonation of the ribose. We found that a neutral Asp132 provides a likely proton donor. Key steps in the reaction and its energetics are consistent with a broad range of experiments. Our calculations shed light on an important reaction in biology and may aid in the design of a new class of inhibitors of the RNase H functionality of retroviral reverse transcriptases such as HIV. Structure and dynamics of large multiprotein assemblies. We combined a coarse-grained simulation model and experiments to study the structures and motions of large multiprotein complexes and modular proteins with partially disordered segments. For this we developed an ensemble refinement approach that allows us to combine simulation ensembles and low-resolution x-ray scattering data (2). With this approach we were able to obtain detailed structures of ESCRT-III CHMP3, a key protein in the ESCRT membrane protein trafficking pathway (2). In collaboration with the experimental groups of Dr. Hurley and Dr. Eaton (both NIDDK, NIH), we also obtained the structure of ESCRT-I, another essential protein of the ESCRT pathway (3). Also in collaboration with Dr. Hurley, we could characterize the solution structure of protein kinase C (PKC) in the autoinhibited state (4). PKCs regulate a wide range of physiological functions that range from T cell recognition to cell proliferation and differentiation, and neuronal signaling. Together with the crystal structure of PKC in an intermediate state, this work provided new insights into the allosteric regulation of this important enzyme. Protein-protein interaction networks. With a theoretical model of protein-protein interaction networks, we could provide a possible physical explanation for the observation that multicellular organisms, from C. elegans to humans, have roughly the same number of protein encoding genes (5). Our calculations suggest that to prevent disease-causing nonspecific interactions between proteins, the proteome size is limited. By collective evolution of the amino-acid sequences of protein binding interfaces we estimated the degree of mis-binding as a function of the number of distinct proteins. We also showed that the need to optimize binding interfaces against misbinding may influence the topology of the protein-protein interaction network. 1. E. Rosta, M. Nowotny, W. Yang, G. Hummer, Catalytic mechanism of RNA backbone cleavage by ribonuclease H from quantum mechanics/molecular mechanics simulations, J. Am. Chem. Soc. 133 8934-8941 (2011). 2. B. Rozycki, Y. C. Kim, G. Hummer, SAXS ensemble refinement of ESCRT-III CHMP3 conformational transitions, Structure 19, 109-116 (2011). 3. E. Boura, B. Rozycki, D. Z. Herrick, H. S. Chung. J. Vecer, W. A. Eaton, D. Cafiso, G. Hummer, J. H. Hurley, Solution structure of the ESCRT-I complex by small angle x-ray scattering, EPR, and FRET spectroscopy, Proc. Natl. Acad. Sci. USA 108, 9437-9442 (2011). 4. T. A. Leonard, B. Rozycki, L. F. Saidi, G. Hummer, J. H. Hurley, Crystal structure and allosteric activation of protein kinase C βII, Cell 144, 55-66 (2011). 5. M. E. Johnson, G. Hummer, Nonspecific binding limits the number of proteins in a cell and shapes their interaction networks, Proc. Natl. Acad. Sci. USA 108, 603-608 (2011).