Structural Diversity in the RGS Domain and Its Interaction with Heterotrimeric G Protein ␣-Subunits
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Structural diversity in the RGS domain and its interaction with heterotrimeric G protein ␣-subunits Meera Soundararajan*, Francis S. Willard†, Adam J. Kimple†, Andrew P. Turnbull*, Linda J. Ball‡, Guillaume A. Schoch*, Carina Gileadi*, Oleg Y. Fedorov*, Elizabeth F. Dowler‡, Victoria A. Higman‡, Stephanie Q. Hutsell†, Michael Sundstro¨ m*§, Declan A. Doyle*¶, and David P. Siderovski†ʈ *Structural Genomics Consortium, Oxford University, Oxford OX3 7DQ, United Kingdom; †Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599; and ‡Leibniz-Institut fur¨ Molekulare Pharmakologie, Robert-Rössle Strasse 10, 13125 Berlin, Germany Communicated by Melvin I. Simon, California Institute of Technology, Pasadena, CA, February 14, 2008 (received for review December 18, 2007) Regulator of G protein signaling (RGS) proteins accelerate GTP RGS domain binding accelerates G␣-mediated GTP hydrolysis. hydrolysis by G␣ subunits and thus facilitate termination of sig- Thirty-seven proteins containing at least one region of homology to naling initiated by G protein-coupled receptors (GPCRs). RGS pro- the archetypal RGS domain fold are encoded by the human teins hold great promise as disease intervention points, given their genome (5), broadly classified into eight subfamilies. The R4 signature role as negative regulators of GPCRs—receptors to which (RGS1, -2, -3, -4, -5, -8, -13, -16, -18, -21) and RZ (RGS17, -19, -20) the largest fraction of approved medications are currently directed. subfamilies generally constitute little more than an RGS domain, RGS proteins share a hallmark RGS domain that interacts most although roles for short N-terminal extensions in membrane- avidly with G␣ when in its transition state for GTP hydrolysis; by targeting and receptor-selective functions have been described (8). binding and stabilizing switch regions I and II of G␣, RGS domain The R7 subfamily of RGS6, -7, -9, and -11 form dimers with G5 binding consequently accelerates G␣-mediated GTP hydrolysis. The (9) via a G␥-like domain N-terminal to their RGS domain. The R12 human genome encodes more than three dozen RGS domain- subfamily constitutes RGS10, -12, and -14, with the latter two containing proteins with varied G␣ substrate specificities. To fa- sharing additional domains reflecting unique roles as Ras/Raf/ cilitate their exploitation as drug-discovery targets, we have taken MAPK scaffolds (10). The other four subfamilies are signaling a systematic structural biology approach toward cataloging the regulators that have since become associated with the RGS protein structural diversity present among RGS domains and identifying superfamily (5, 11) upon discovery of more distantly related RGS ␣ molecular determinants of their differential G selectivities. Here, domains within them (i.e., Axin, Axil; RhoGEFs p115-RhoGEF, we determined 14 structures derived from NMR and x-ray crystal- LARG, and PDZ-RhoGEF; the sorting nexins SNX13, -14, and lography of members of the R4, R7, R12, and RZ subfamilies of RGS -25; the GPCR kinases GRK1–7). The RGS domains within the proteins, including 10 uncomplexed RGS domains and 4 RGS RhoGEF and GPCR kinase subfamilies have also been referred to ␣ domain/G complexes. Heterogeneity observed in the structural as rgRGS or RH domains, respectively (12, 13). architecture of the RGS domain, as well as in engagement of switch RGS proteins control the timing and duration of specific ␣ III and the all-helical domain of the G substrate, suggests that physiological processes that involve GPCR signaling. For exam- unique structural determinants specific to particular RGS pro- ␣ ple, RGS2-deficient mice exhibit heightened anxiety (14, 15) and tein/G pairings exist and could be used to achieve selective constitutive hypertension (16), the latter caused by loss of inhibition by small molecules. homeostatic control over vasoconstrictive hormonal signaling via Gq-coupled GPCRs in the vasculature (17). RGS2 is unique GTPase-accelerating proteins ͉ NMR structure ͉ RGS proteins ͉ x-ray among the R4 subfamily in its selectivity for G␣q in vitro (18), crystallography although in the context of intact cells or reconstituted receptor/ heterotrimer complexes, activity of RGS2 on G␣i-mediated protein-coupled receptors (GPCRs) are critical for many signaling is also seen (19, 20). Differences in G␣ selectivity lie, PHARMACOLOGY Gphysiological processes including vision, olfaction, neuro- at least in part, in heterogeneity within the structural determi- transmission, and the actions of many hormones (1). As such, nants of G␣ engagement by the RGS domain; initial evidence of GPCRs are the largest fraction of the ‘‘druggable proteome,’’ heterogeneity was seen in the structures of p115-RhoGEF and and their ligand-binding and signaling properties remain of considerable interest to academia and industry (2). GPCRs catalyze activation of heterotrimeric G proteins comprising a Author contributions: M. Soundararajan, F.S.W., A.J.K., D.A.D., and D.P.S. designed re- guanine nucleotide-binding G␣ subunit and an obligate G␥ search; M. Soundararajan, F.S.W., A.J.K., A.P.T., L.J.B., G.A.S., C.G., O.Y.F., E.F.D., V.A.H., dimer (3). Receptor–promoted activation of G␣␥ causes ex- and S.Q.H. performed research; F.S.W. and A.J.K. contributed new reagents/analytic tools; ␣ M. Soundararajan, F.S.W., A.J.K., A.P.T., and D.P.S. analyzed data; and M. Soundararajan, change of GDP for GTP by G and resultant dissociation of F.S.W., A.J.K., M. Sundstro¨m, and D.P.S. wrote the paper. ␥ ␣ ␥ G . GTP-bound G and freed G then regulate intracellular The authors declare no conflict of interest. effectors such as adenylyl cyclase, phospholipase C, ion channels, Data Deposition: The atomic coordinates and structure factors have been deposited in the RhoGEFs, and phosphodiesterases (1, 4). This ‘‘G protein cycle’’ Protein Data Bank, www.pdb.org [PDB ID codes 1ZV4, 2A72, 2AF0, 2BT2, 2BV1, 2ES0, 2GTP, is reset by the intrinsic GTP hydrolysis activity of G␣, producing 2IHB, 2IHD, 2IK8, and 2ODE (crystal structures); 2I59, 2JM5, 2JNU, and 2OWI (NMR struc- G␣⅐GDP that favors heterotrimer reformation and, conse- tures)]. quently, signal termination. Thus, a major determinant of the §Present Address: Novo Nordisk Foundation Center for Protein Research, University of duration and magnitude of GPCR signaling is the lifetime of G␣ Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen N, Denmark. in the GTP-bound state. ¶To whom correspondence may be addressed at: Old Road Campus Research Building, Roosevelt Drive, Oxford University, Oxford OX3 7DQ, United Kingdom. E-mail: Regulators of G protein signaling are GTPase-accelerating pro- [email protected]. teins (GAPs) for G␣ subunits and thus facilitate GPCR signal ʈTo whom correspondence may be addressed at: Department of Pharmacology, University termination (5). GAP activity is conferred by an RGS domain of North Carolina, Campus Box 7365, 1106 Mary Ellen Jones Building, Chapel Hill, NC 27599. present in one or more copies within members of this protein E-mail: [email protected]. superfamily (5). The archetypal RGS domain is composed of nine This article contains supporting information online at www.pnas.org/cgi/content/full/ ␣-helices (6) and binds most avidly to G␣ in the transition state for 0801508105/DCSupplemental. GTP hydrolysis (7); by stabilizing the flexible switch regions of G␣, © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801508105 PNAS ͉ April 29, 2008 ͉ vol. 105 ͉ no. 17 ͉ 6457–6462 Downloaded by guest on September 27, 2021 A B C D Fig. 1. Heterogeneity in the ␣V–␣VII regions of R12 subfamily RGS domains versus the canonical RGS domain fold of R4, R7, and RZ subfamily members. (A) Apo-RGS domains of R4 subfamily member RGS8 (green; PDB ID 2IHD), R7 subfamily member RGS9 (orange; PDB ID 1FQI), and RZ subfamily member RGS19 (gray; PDB ID 1CMZ) were aligned along helices ␣IV and ␣V and superimposed by using PyMOL. (B–D) Apo-RGS domains of RGS14 (B) (blue; PDB ID 2JNU), RGS10 from this study (C) (salmon; PDB ID 2I59), and RGS10 from Yokoyama et al. (D) (light purple; PDB ID 2DLR) are presented to highlight differences in the ␣V–␣VI–␣VII region. The heterogeneous ␣VI regions are specifically highlighted in cyan (B), red (C), and magenta (D), respectively. GRK2 bound to G␣13 and G␣q, respectively (12, 13), that exhibit the Protein Data Bank (PDB) by others (Table S2 and refs. 21, unique contacts not observed in the first resolved structures [i.e., 22, 27, and 28). Canonical RGS domains consist of a nine-helix RGS4/G␣i1 and RGS9/G␣t; (6, 21)]. To assess the structural bundle comprising two lobes formed by the ␣I, ␣II, ␣III, ␣VIII, diversity and G␣ selectivities of RGS domains, we have taken a and ␣IX helices and the ␣IV, ␣V, ␣VI, and ␣VII helices, systematic structural biology approach and present 14 structures respectively. The majority of apo-RGS domain structures we of RGS domains from the R4, RZ, R7, and R12 subfamilies, obtained conform to the structural archetype established by including four RGS domain/G␣ complexes, two of which involve RGS4 (6, 28). Crystal structures of RGS6 and RGS7 (PDB IDs ␣ G i3. In an accompanying work, Slep et al. (22) describe two 2ES0 and 2A72) are atypical, domain-swapped dimers. The additional structures of RGS16—uncomplexed and bound to significance of such dimerization is not known and likely arises ␣ G o. Detailed knowledge of RGS protein substrate specificity, because of crystal packing-induced interactions; an NMR solu- and the unique structural determinants underlying such speci- tion structure of RGS7 (PDB ID 2D9J) conforms to the canon- ficity, should greatly facilitate exploitation of these GPCR ical RGS domain structure. signaling regulators as drug targets (23). Substantial differences were observed between the R12 sub- Results and Discussion family RGS domain structures and the prototypical RGS do- mains of R4, R7, and RZ subfamily members (Fig.