Electrostatic networks for characterization of allosteric pathways: Allosteric paths in Cas9 apo, DNA- and RNA-bound forms.

Abstract

Allostery is a fundamental process by which biological macromolecules transmit the effect of a local perturbation at one site to a distal, functional site, allowing for regulation of activity. The long-range coupling between residues that gives rise to allostery in a protein is built up from short-range electrostatic and hydrophobic interactions. These are arguably the largest determinants of protein structure and are essential regulators of protein function. We introduce an effective coulombic electrostatic coupling network obtained from the analysis of molecular dynamics simulations of Cas9 in its apo, DNA- and RNA-bound forms. We characterize key electrostatic events that determine its functional activity and targeting precision. We demonstrate the locality of the electrostatic-interaction network over other connectivity matrices as validated through direct comparisons to NMR measurements. We define an electrostatic-based centrality metric that allows us to pinpoint relevant donor-acceptor pairs that promote charge displacements that modulate the cross-interaction between the PAM-interacting region and catalytic domains. We determine key amino acid residues central to the network, allowing us to identify a circular allosteric pathway that channels perturbations from the PAM-interacting domain to the HNH and RuvCII domains, and then back to the PAM-contacting region. The connectivity around HNH is important for controlling the directionality of signal transfer from and towards the PAM-interacting domain. The effective coulombic electrostatic coupling network makes it possible to elucidate allosteric pathways and provides valuable interpretations of experimental measurements.