Structural basis for nucleotide exchange in heterotrimeric G proteins

Abstract

G protein-coupled receptors (GPCRs) relay diverse extracellular signals into cells by catalyzing nucleotide release from heterotrimeric G proteins, but the mechanism underlying this quintessential molecular signaling event has remained unclear. Here we use atomic-level simulations to elucidate the nucleotide-release mechanism. We find that the G protein α subunit Ras and helical domains-previously observed to separate widely upon receptor binding to expose the nucleotide-binding site-separate spontaneously and frequently even in the absence of a receptor. Domain separation is necessary but not sufficient for rapid nucleotide release. Rather, receptors catalyze nucleotide release by favoring an internal structural rearrangement of the Ras domain that weakens its nucleotide affinity. We use double electron-electron resonance spectroscopy and protein engineering to confirm predictions of our computationally determined mechanism.G protein-coupled receptors (GPCRs), which represent the largest class of drug targets, trigger cellular responses to external stimuli primarily by activating heterotrimeric G proteins: an activated GPCR, upon binding an inactive, GDP-bound G protein, dramatically accelerates GDP release, thus allowing GTP to bind spontaneously to the vacated nucleotide-binding site (1-2). This nucleotide exchange initiates G protein-mediated † To whom correspondence should be addressed. Ron.Dror@DEShawResearch.com, Telephone: (212) Fax: (212) 845-1981, David.Shaw@DEShawResearch.com, Telephone: (212) Fax: (212) Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript intracellular signaling. Despite breakthroughs in GPCR structure determination (3-5), key aspects of the molecular mechanism by which GPCRs accelerate GDP release remain unresolved.Heterotrimeric G proteins undergo a dramatic conformational change upon binding activated GPCRs (Fig. 1, A and B). Double electron-electron resonance (DEER) spectroscopy has demonstrated that the Ras and helical domains of the G protein α subunit (Gα), which tightly sandwich the nucleotide in all nucleotide-bound G protein crystal structures, separate by tens of angstroms upon GPCR binding and GDP release (6). A crystal structure of a GPCR-G protein complex (4), and accompanying deuterium exchange and electron microscopy data (7,8), confirmed this dramatic domain separation.These observations have raised several unresolved questions (4, 9). What is the role of domain separation in GDP release? Does a GPCR catalyze GDP release by forcing the domains to separate, or does the GPCR force out GDP, with the absence of GDP leading to subsequent domain separation? More generally, what is the structural mechanism by which a GPCR brings about GDP release?To address these questions, we performed atomic-level molecular dynamics (MD) simulations of heterotrimeric G proteins with and without bound GPCRs. We initiated simulations from crystal structures of nucleotide-bound G protein heterotrimers (in particular, G i (10) and a chimeric G t (11)), inc...

Capturing Invisible Motions in the Transition from Ground to Rare Excited States of T4 Lysozyme L99A

“…2), inspection of the back-calculated SHIFTXþ values for F114 13 C a shows that they recapitulate the experimentally derived differences between the ground and excited states over the entire Anton trajectory. Additionally, the back-calculated chemical shifts of the intermediate state and metastates are within error of the chemical shifts for the excited state and ground state, respectively.…”

“…In the following paragraphs, we describe the conformational differences that define each of these states as unique from the ground and excited states and show that these differences are manifested in the back-calculated chemical shifts, calculated using the SHIFTXþ algorithm (49). Interestingly, the back-calculated 13 C a chemical shifts of F114 in these metastates are within error of the back-calculated 13 C a chemical shifts in the ground state despite the wide range of conformations sampled in the four metastates.…”

“…2). Here, 13 Ca chemical shift predictions were calculated for each frame, then averaged over frames 1-12,569, representing the ground state, and frames 112,746-125,691, representing ES-Anton, of the Anton trajectory. A pH of 5.5 was input for the SHIFTX2 algorithm.…”

“…Additionally, with recent advances in specialized computational architectures like Anton, simulation timescales on the order of microseconds to milliseconds in a single trajectory are within reach (7,(12)(13)(14).…”

The GAPs, GEFs, GDIs and…now, GEMs: New kids on the heterotrimeric G protein signaling block

“…In the canonical scenario, activation of G proteins is triggered by agonist-bound GPCRs. By directly coupling to the Ga-subunit of the abg heterotrimer, [1][2][3][4] GPCRs induce a conformational change leading to loss of bound GDP and dissociation of Gbg subunits; in other words, GPCRs serve as guanine nucleotide exchange factors (GEFs; [ Fig. 1]).…”