Davidson, R. B. | bioRxiv Science

bioRxiv2020

RNA-dependent structures of the RNA-binding loop in the flavivirus NS3 helicase

The flavivirus NS3 protein is a helicase that has pivotal functions during the viral genome replication process, where it unwinds double-stranded RNA and translocates along the nucleic acid polymer in a nucleoside triphosphate hydrolysis-dependent mechanism. An increased interest in this enzyme as a potential target for development of antiviral therapeutics was sparked by the 2015 Zika virus epidemic in the Americas. Crystallographic and computational studies of the flavivirus NS3 helicase have identified the RNA-binding loop as an interesting structural element, which may function as an origin for the RNA-enhanced NTPase activity observed for this family of helicases. Microsecond-long unbiased molecular dynamics as well as extensive replica exchange umbrella sampling simulations of the Zika NS3 helicase have been performed to investigate the RNA-dependence of this loops structural conformations. Specifically, the effect of the bound single-stranded RNA (ssRNA) oligomer on the putative "open" and "closed" conformations of this loop are studied. In the Apo substrate state, the two structures are nearly isoergonic ({Delta}GO[->]C = -0.22 kcal mol-1), explaining the structural ambiguity observed in Apo NS3h crystal structures. The bound ssRNA is seen to stabilize the "open" conformation ({Delta}GO[->]C = 1.97 kcal mol-1) through direct protein-RNA interactions at the top of the loop. Interestingly, a small ssRNA oligomer bound over 13 [A] away from the loop is seen to affect the free energy surface to favor the "open" structure while minimizing barriers between the two states. The mechanism of the transition between "open" and "closed" states is characterized as are residues of importance for the RNA-binding loop structures. From these results, the loop is hypothesized to be a viable region in the protein for targeted small-molecule inhibition and mutagenesis studies, where stabilization of the "closed" RNA-binding loop will negatively impact RNA-binding and the RNA-enhanced NTPase activity.

biophysics↗

bioRxiv2019

The Effective Coarse-grained Hessian Dictates Allostery in IGPS

The long-ranged coupling between residues that gives rise to allostery in a protein is built up from short-ranged physical interactions. Computational tools used to predict this coupling and its functional relevance have relied on the application of graph theoretical metrics to residue-level correlations measured from all-atom molecular dynamics (aaMD) simulations. The short-ranged interactions that yield these long-ranged residue-level correlations are quantified by the effective coarse-grained Hessian. Here we compute an effective harmonic coarse-grained Hessian from aaMD simulations of a benchmark allosteric protein, IGPS, and demonstrate the improved locality of this graph Laplacian over two other connectivity matrices. Additionally, two centrality metrics are developed that indicate the direct and indirect importance of each residue at producing the covariance between the effector binding pocket and the active site. The residue importance indicated by these two metrics is corroborated by previous mutagenesis experiments and leads to unique functional insights; in contrast to previous computational analyses, our results suggest that fP76-hK181 is the most important contact for conveying direct allosteric paths across the HisF-HisH interface. The connectivity around fD98 is found to be important at affecting allostery through indirect means.

biophysics↗