ANALYSIS doi:10.1038/nature14663 Universal allosteric mechanism for Ga activation by GPCRs Tilman Flock1, Charles N. J. Ravarani1*, Dawei Sun2,3*, A. J. Venkatakrishnan1{, Melis Kayikci1, Christopher G. Tate1, Dmitry B. Veprintsev2,3 & M. Madan Babu1 G protein-coupled receptors (GPCRs) allosterically activate heterotrimeric G proteins and trigger GDP release. Given that there are 800 human GPCRs and 16 different Ga genes, this raises the question of whether a universal allosteric mechanism governs Ga activation. Here we show that different GPCRs interact with and activate Ga proteins through a highly conserved mechanism. Comparison of Ga with the small G protein Ras reveals how the evolution of short segments that undergo disorder-to-order transitions can decouple regions important for allosteric activation from receptor binding specificity. This might explain how the GPCR–Ga system diversified rapidly, while conserving the allosteric activation mechanism. proteins bind guanine nucleotides and act as molecular switches almost 30 A˚ away from the GDP binding region5 and allosterically trig- in a number of signalling pathways by interconverting between ger GDP release to activate them. 1,2 G a GDP-bound inactive and a GTP-bound active state . They The high-resolution structure of the Gas-bound b2-adrenergic recep- 3 5 consist of two major classes: monomeric small G proteins and hetero- tor (b2AR) provided crucial insights into the receptor–G protein inter- trimeric G proteins4. While small G proteins and the a-subunit (Ga)of face and conformational changes in Ga upon receptor binding6,7. Recent 6 8 heterotrimeric G proteins both contain a GTPase domain (G-domain), studies described dynamic regions in Gas and Gai , the importance of ˚ Ga contains an additional helical domain (H-domain) and also forms a displacement of helix 5 (H5) of Gas and Gat by up to 6 A into the complex with the Gb and Gc subunits. Although they undergo a similar receptor5, the extent of helical domain opening during GDP release9,10, 7,10 signalling cycle (Fig. 1), their activation differs in one important aspect. and identified residues that contribute to Gai activation . These stud- The guanine nucleotide exchange factors (GEFs) of small G proteins are ies focused on single, specific Ga proteins; however, in humans there are largely cytosolic proteins, whereas the GEFs of Ga proteins are usually 16 different Ga genes, with at least 21 isoforms4 that can be grouped into membrane-bound GPCRs. While GEFs of small G proteins interact four functional subfamilies (Gas,Gai,Gaq,Ga12), which each regulate 11 directly with the GDP binding region1,3, GPCRs bind to Ga at a site different signalling pathways . Although they belong to the same protein fold, they have diverged significantly in their sequence such that each Ga protein can be specifically activated by one or several of the ,800 human Pi 4 GAP-bound state Inactive state GPCRs . Thus, a fundamental question is whether there is a universal mechanism of allosteric activation that is conserved across all Ga protein GAP α GTPase domain γ β α 10 (G-domain) types . Allosteric communication in proteins is mediated through con- α-helical domain formational changes, which are facilitated by the re-organization of non- 40 structures (H-domain) 11 structures covalent contacts between residues. Thus, studying these contacts can GDP provide detailed insights into the mechanism of allostery12–16.Onthebasis of a comprehensive analysis, here we propose that GPCRs interact and Active state GEF-bound state Extracellular activate Ga subunits through a conserved mechanism. We describe molecular details of the key structural transitions and pinpoint residues Effector GPCR α GPCR that constitute the ‘common core’ of Ga activation. Intracellular γ β α Common Ga numbering and residue contact networks 25 structures α G We created a structural and sequence alignment of 80 Ga structures from diverse organisms and 973 sequences from 66 species that have a GTP 1 structure GPCR–G protein system (auto-activating plant Ga proteins were not considered; Methods). To enable the comparison of any residue/posi- Figure 1 | Ga signalling states and activation. Heterotrimeric G proteins (1) tion between different Ga proteins, we devised the common Ga num- release GDP upon binding to a guanine nucleotide exchange factor (GEF), bering (CGN) system (Fig. 2a). The CGN provides an ‘address’ for every which are G-protein-coupled receptors, (2) bind GTP and recruit downstream effectors, and (3) hydrolyse GTP, promoted by a GTPase activating protein residue in the DSP format, referring to: (1) the domain (D); (2) the (GAP), leading to (4) the inactive, GDP-bound state. Structures of the Ga consensus secondary structure (S); and (3) the position (P) within the subunit (blue) bound to GDP (Protein Data Bank (PDB) accession 1got; secondary structure element. For instance, phenylalanine 336 in Gai1 is G.H5.8 inactive state; top) and bound to the b2-AR (grey) (PDB 3sn6; active state; denoted as Phe336 as it is the eighth residue within the consensus bottom) are shown. helix H5 of the G-domain. The corresponding position in Gas2 is 1MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK. 2Laboratory of Biomolecular Research, Paul Scherrer Institut, 5232 Villigen, Switzerland. 3Department of Biology, ETH Zurich, 8039 Zurich, Switzerland. {Present address: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA. *These authors contributed equally to this work. 00 MONTH 2015 | VOL 000 | NATURE | 1 G2015 Macmillan Publishers Limited. All rights reserved RESEARCH ANALYSIS a α Secondary structure element (consensus) (e.g., H5) G s orthologue alignment GNAS2 α G G s2-chimp HN GNAL s Gα -rat ... ... ... s2 H2 GNAI2 omain S elementosition S1 GNAI1 Human Gα alignment Gα -cow D S P S5 s2 S3 GNAI3 Gα ... S4 s2 S6 H3 H4 e.g. S2 GNAT2 Gα H5 G i2 GNAT1 i ... α 973 sequences G s2: G-domain (G) GNAT3 H1 + f α GNAO 66 organisms G i2: GNAZ 16 orthologue alignments f ... HG GNAQ HF α α G 13 G 13: omain (e.g., G) GNA11 Gq 16 human Gα genes D HA GNA14 4 Gα subfamilies Common Gα HD GNA15 HE GNA12 G numbering GNA13 12 Position in the consensus HC H-domain (H) HB secondary structure element (SSE) mapped www.mrc-lmb.cam.ac.uk/CGN to the human reference alignment b PDB-11: 1zcb (Gα13) Residue contact network α G s2 for each of the 11 structures Gα PDB-2: 1gp2 (Gαi1) i2 PDB-1: 1got (Gαt1) PDB-11: 1got (Gα ) t1 Identify conserved ... Phe332 residues in all Gα s,i,q,13 Val Phe (561 complete sequences) Gα Ala 13 e.g. G.H1.12 e.g. G.H5.8 Inactive Ile 72 highly conserved residues ... His G.H5.8 G.H5.8 11 structures, G 11 His53 PDB-1: 3sn6 (Gαs2) Xaa Phe Xaa Phe s 1,246 contacts - 326 residues Use CGN to establish topological equivalence ... Xaa Xaa α Ile Ile PDB-1: 1zcb (G 13) Phe359 His His Glu Phe GEF-bound one structure, G G.H1.12 G.H1.12 Pro State consensus State consensus network between PDB-25: 4gnk (Gαq) Ile residue contact network universially conserved residues His 260 long-range contacts (535 total) 43 contacts PDB-2: 1gia (Gαi1) between 188 residues (289 total) 44 residues PDB-1: 1azt (G( αs2) His72 s,i,q 1,292 contacts - 318 residues Active 25 structures, G ... PDB-3: 3cx7 (Gα13) PDB-2: 4ekc (Gαq) PDB-1: 2gtp (Gαi1) i,q,13 α GAP-bound For example, residue interaction networks of 11 G For example, inactive state consensus network For example, inactive state consensus network 40 structures, G ... structures of the inactive state between residues conserved across all Gα subfamilies >100,000 (2,436 unique) residue contacts 457 unique long-range residue contacts 79 unique long-range residue contacts 80 structures between 386 positions between 247 shared positions between 70 universally conserved residues Figure 2 | The common Ga numbering (CGN) system and Ga conserved belonging to all the 4 subfamilies from 66 species allowed the identification of contact networks. a, Every position in a Ga is denoted by its domain (D), equivalent residues. An online webserver allows mapping of any Ga sequence the consensus secondary structure element where it is present (S), and position or structure to the CGN system (http://www.mrc-lmb.cam.ac.uk/CGN). (P) within the consensus SSE. Names of SSEs are shown in the cartoon; loops b, Computation of consensus residue contact networks between conserved are named with lowercase letters of the flanking SSEs (for example, h1ha; residues for different Ga signalling states. See Methods, Supplementary see Extended Data Fig. 1 for all SSEs). An alignment of all 973 Ga proteins Note and Supplementary Data. Phe376G.H5.8. Loops are labelled in lowercase letters of their flanking the Supplementary Note and Supplementary Information. Below, we secondary structure elements (SSE); for example, s6h5 refers to the loop describe the major findings pertaining to Ga activation. We first focus connecting strand S6 with helix H5 (see Extended Data Fig. 1, Methods on the GPCR–Ga interface and then describe the molecular details of and Supplementary Note). A CGN mapping webserver is available at how the non-covalent contacts are re-organized and propagated to the http://www.mrc-lmb.cam.ac.uk/CGN. GDP binding pocket, leading to GDP release. The Ga structures were assigned to the four major signalling states (Fig. 1) and the non-covalent contacts between residues were calculated GPCR–Ga protein interface for each structure. We computed the consensus non-covalent residue Analysis of the buried surface area (BSA) and residue contacts between 5 ˚ 2 contacts from all Ga structures of the same signalling state.