Networks of Dynamic Allostery Regulate Enzyme Function

Many protein systems rely on coupled dynamic networks to allosterically regulate function. However, the broad conformational space sampled by non-coherently dynamic systems has precluded detailed analysis of their communication mechanisms. Here, we have developed a methodology that combines the high sensitivity afforded by nuclear magnetic resonance relaxation techniques and single-site multiple mutations, termed RASSMM, to identify two allosterically coupled dynamic networks within the non-coherently dynamic enzyme cyclophilin A. Using this methodology, we discovered two key hotspot residues, Val6 and Val29, that communicate through these networks, the mutation of which altered active-site dynamics, modulating enzymatic turnover of multiple substrates. Finally, we utilized molecular dynamics simulations to identify the mechanism by which one of these hotspots is coupled to the larger dynamic networks. These studies confirm a link between enzyme dynamics and the catalytic cycle of cyclophilin A and demonstrate how dynamic allostery may be engineered to tune enzyme function. •The RASSMM approach allows identification and modulation of allosteric networks•Two distinct networks of dynamic allostery are identified within cyclophilin A•Active-site conformational sampling modulates enzymatic function in cyclophilin A•Non-coherent interactions mediate communication within larger dynamics networks Allosteric communication in many enzymatic systems is mediated by altering inherent conformational sampling. Holliday et al. introduce a new experimental approach to identifying residues that act as dynamic allosteric regulators in such systems and utilize this approach to define allosteric networks that modulate function in the enzyme cyclophilin A.