Design of novel CV-N variants: Modulation of binding dynamics through distal mutations

Abstract

AbstractWe develop a computational approach to identify distal residues that allosterically modulate dynamics of binding sites by combining dynamic coupling with statistical analysis of co-evolution. Putative mutants of the predicted allosteric sites are subjected to Adaptive BP-Dock for binding analysis. Here, we apply this method to a small lectin, Cyanovirin-N (CV-N), that selectively binds to dimannose. Our method points out mutations on I34, that is 16Å away from binding pocket, can modulate binding. Experimental characterization confirms that I34K abolishes binding, while I34Y increases affinity. Increased affinity is not due to changes in the binding region, which are conserved in the crystal structure. However, ITC analysis reveals an opposite entropic contribution, suggesting that modulation of dynamics is responsible for the change in binding affinity. Our results point to a novel approach to identify and substitute distal sites by integrating evolutionary inference with protein dynamics in glycan-binding proteins to improve binding affinity.