McCullagh, M. | Search bioRxiv

bioRxiv2019

Motif V acts as a Regulator of Energy Transduction Between the Flavivirus NS3 ATPase and RNA Binding Cleft

The unwinding of double-stranded RNA intermediates is a critical component for the replication of flavivirus RNA genomes. This function is achieved by the C-terminal helicase domain of nonstructural protein 3 (NS3). As a member of the superfamily 2 (SF2) helicases, NS3 is known to require the binding and hydrolysis of ATP/NTP to translocate along and unwind double-stranded nucleic acids. However, the mechanism of energy transduction between the ATP and RNA binding pockets is not well understood. Previous molecular dynamics simulations published by our group have identified Motif V as a potential "communication hub" for this energy transduction pathway. In order to investigate the role of Motif V in this process, a study combining molecular dynamics, biochemistry and virology has been employed. Mutations of Motif V were tested in both replicon and recombinant protein systems to investigate viral genome replication, RNA binding affinity, ATP hydrolysis activity and helicase unwinding activity. Using these analyses, we found that T407A and S411A in Motif V demonstrated increased turnover rates, suggesting that the mutations causes the helicase to unwind dsRNA more quickly than WT. Additionally, simulations of each mutant were used to probe structural changes within NS3 caused by each mutation. These simulations indicate that Motif V controls communication between the ATP binding pocket and the helical gate. These data help define the linkage between ATP hydrolysis and helicase activity within NS3 and provide insight into the biophysical mechanisms for ATPase driven NS3 helicase function.

biochemistry↗

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↗