Simulation of spontaneous G protein activation reveals a new intermediate driving GDP unbinding.

Xianqiang Sun,Gregory R Bowman,Sukrit Singh,Kendall J Blumer

doi:10.7554/elife.38465

Abstract

Activation of heterotrimeric G proteins is a key step in many signaling cascades. However, a complete mechanism for this process, which requires allosteric communication between binding sites that are ~30 Å apart, remains elusive. We construct an atomically detailed model of G protein activation by combining three powerful computational methods: metadynamics, Markov state models (MSMs), and CARDS analysis of correlated motions. We uncover a mechanism that is consistent with a wide variety of structural and biochemical data. Surprisingly, the rate-limiting step for GDP release correlates with tilting rather than translation of the GPCR-binding helix 5. β-Strands 1 - 3 and helix 1 emerge as hubs in the allosteric network that links conformational changes in the GPCR-binding site to disordering of the distal nucleotide-binding site and consequent GDP release. Our approach and insights provide foundations for understanding disease-implicated G protein mutants, illuminating slow events in allosteric networks, and examining unbinding processes with slow off-rates.

Highlights

Heterotrimeric G proteins are molecular switches that play pivotal roles in signaling processes from vision to olfaction and neurotransmission (Oldham and Hamm, 2006; Oldham and Hamm, 2008; Johnston and Siderovski, 2007)
These residues contact Ga in some guanosine diphosphate (GDP)-bound structures but not in G protein-coupled receptors (GPCRs)-bound structures, (Lambright et al, 1996; Noel et al, 1993) and removing these residues promotes GDP release due to a reduced GDP-binding affinity (Denker et al, 1992; Marin et al, 2002). Taken together, such evidence suggests that the last five residues of Gaq help stabilize the inactive state and that removing them would accelerate activation. In support of this hypothesis, we find that the energetic barrier to GDP release is lower in metadynamics simulations of the truncated variant than for full-length Gaq (Figure 2—figure supplement 1)
These simulations, and those described hereafter, were initiated from an X-ray structure of the Gaq heterotrimer bound to GDP and an inhibitor of nucleotide exchange (Nishimura et al, 2010); Gbg and the inhibitor were excluded from all simulations

Summary

Introduction

Heterotrimeric G proteins are molecular switches that play pivotal roles in signaling processes from vision to olfaction and neurotransmission (Oldham and Hamm, 2006; Oldham and Hamm, 2008; Johnston and Siderovski, 2007). A G protein adopts an inactive state in which guanosine diphosphate (GDP) binds between the Ras-like and helical domains of the a-subunit (Ga, Figure 1). Ga returns to the inactive state by hydrolyzing GTP to GDP and rebinding Gbg. Given the central role Ga plays, a common Ga numbering scheme (CGN) has been established to facilitate discussion of the activation mechanisms of different Ga homologs (Flock et al, 2015). S6 refers to b-strand 6 and s6h5 refers to the loop between S6 and H5

Methods

Results

Conclusion