Dynamic Coupling and Allosteric Networks in the Alpha Subunit of Heterotrimeric G Proteins

Abstract

Heterotrimeric G proteins operate as intracellular signaling hubs that couple the activation of cell surface receptors (GPCRs) to the regulation of a multitude of essential cellular processes. Important structural transitions occurring during G protein activation have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis and experimental mutagenesis of G[alpha]i that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide and helical domain interfaces. Most notably, we predict and experimentally characterize novel decoupling mutants that display enhanced helical domain opening, increased rates of nucleotide exchange and constitutive activity in the absence of receptor. In addition, our simulations reveal a previously unappreciated relationship between coupling of nucleotide binding site loops and alpha helical domain opening rate. In particular, our data indicate that a strongly coupled switch I and P-loop promote domain opening and subsequent nucleotide exchange. Collectively our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function.