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

Xin-Qiu Yao,Rabia U Malik,Nicholas W Griggs,Lars Skjærven,John R Traynor,Sivaraj Sivaramakrishnan,Barry J Grant

doi:10.1074/jbc.m115.702605

Abstract

G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle 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 that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gαi. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function.

Highlights

IntroductionImportant conformational transitions occurring at each stage of this regulated cycle have been characterized from extensive crystallographic studies
Network path analysis delineated the detailed mechanism of dynamic coupling and revealed residues predicted to be involved in mediating the distal (Ͼ30 Å) allosteric coupling of receptor, nucleotide, and helical domain (HD) interfaces
Experimental mutagenesis of a number of these sites together with in vitro cAMP and [35S]GTP␥S assays indicated that the signaling properties of G␣i can be modulated by these single point mutations that act allosterically

Summary

Introduction

Important conformational transitions occurring at each stage of this regulated cycle have been characterized from extensive crystallographic studies These include GDP, GTP analogue, G␤␥, GTPase-activating protein, GDI and most recently GPCR bound complex structures of G␣. A much larger (127°) clam-shell like displacement of the HD with respect to RasD was reported recently in the crystallographic structure of G␣s (the ␣ subunit of the stimulatory G protein for adenylyl cyclase) in complex with G␤␥ and the ␤2 adrenergic receptor [4] This conformational change, which effectively exposes the otherwise buried nucleotide binding site, has been linked to GPCR-mediated nucleotide exchange [4]. The novel L32A mutation (numbering based on the ␣ subunit of bovine transducin) was predicted to enhance domain opening and was found to increase nucleotide exchange rates, increase G protein activation, and decouple G protein from receptor activation leading to constitutive activity

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