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Molecular Architecture of G O and the Structural Basis for RGS16

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Molecular Architecture of G O and the Structural Basis for RGS16 Molecular architecture of G␣o and the structural basis for RGS16-mediated deactivation Kevin C. Slep*†, Michele A. Kercher‡§, Thomas Wieland¶ʈ, Ching-Kang Chen¶, Melvin I. Simon¶**, and Paul B. Sigler‡§†† *Department of Biology, University of North Carolina, Chapel Hill, NC 27599; ‡Department of Molecular Biophysics and Biochemistry, and §Howard Hughes Medical Institute, Yale University, New Haven, CT 06511; and ¶Division of Biology, California Institute of Technology, Mail Code 147-75, Pasadena, CA 91125 Contributed by Melvin I. Simon, February 16, 2008 (sent for review December 18, 2007) Heterotrimeric G proteins relay extracellular cues from heptahelical (11). With the number of RGS proteins greatly exceeding the transmembrane receptors to downstream effector molecules. number of G␣ subunits, RGS proteins are likely to be finely tuned, Composed of an ␣ subunit with intrinsic GTPase activity and a ␤␥ titrated, and localized to regulate specific signaling pathways within heterodimer, the trimeric complex dissociates upon receptor- aG␣ subunit’s repertoire of effector targets. To mediate the mediated nucleotide exchange on the ␣ subunit, enabling each specificity and fidelity requisite for accurate signal transmission, the component to engage downstream effector targets for either G␣ subunit and its binding partners (GPCR, G␤␥, RGS, and activation or inhibition as dictated in a particular pathway. To effector) must have specific reciprocating molecular determinants mitigate excessive effector engagement and concomitant signal to minimize the convergence of independent signaling pathways. transmission, the G␣ subunit’s intrinsic activation timer (the rate of Thus, a comprehensive understanding of the molecular basis for G␣ GTP hydrolysis) is regulated spatially and temporally by a class of engagement with its activators, regulators, and effectors is critical GTPase accelerating proteins (GAPs) known as the regulator of G for elucidating specificity determinants. RGS subfamilies pair with protein signaling (RGS) family. The array of G protein-coupled distinct cognate G␣ substrates via unique stereochemical binding receptors, G␣ subunits, RGS proteins and downstream effectors in determinants. The RGS subfamilies R4, R7, and R12 engage G␣i/o mammalian systems is vast. Understanding the molecular deter- (3, 12, 13); the R4 subfamily also engages G␣q (14); the rgRGS minants of specificity is critical for a comprehensive mapping of the subfamily engages G␣12/13 (15); and the RZ subfamily engages G␣z G protein system. Here, we present the 2.9 Å crystal structure of the and G␣i (3, 16, 17). It is noted that G␣–RGS interaction modes enigmatic, neuronal G protein G␣o in the GTP hydrolytic transition observed in R4, R7 and R12 (5, 7) [see accompanying article by state, complexed with RGS16. Comparison with the 1.89 Å struc- Soundararajan et al. (18)] contrast with the unique interlocking ture of apo-RGS16, also presented here, reveals plasticity upon G␣o geometry observed between p115RhoGEF rgRGS and the G␣13/i1 binding, the determinants for GAP activity, and the structurally chimera (19) and the effector-like binding mode observed between unique features of G␣o that likely distinguish it physiologically GRK2 and G␣q (20). from other members of the larger G␣i family, affording insight to G␣o is a member of the G␣i family, which includes G␣i1-3 and receptor, GAP and effector specificity. G␣t. Although G␣o is the most abundant G␣ subunit in the human brain, little is known about the pathways it is involved in, G protein ͉ GAP ͉ RGS in stark contrast to our understanding of G␣i1-3 and G␣t. Much of the work on G␣o to date has implicated a role for its cognate ␤␥ 2ϩ any extracellular cues ranging from photons to neurotrans- G subunit in the activation of Ca channels (21). Additional ␣ Mmitters are detected with high specificity by G protein- evidence points to G o involvement in signaling from A1- coupled receptors that in turn elicit an intracellular response by adenosine receptors (22), dopamine D2 receptors (D2R) (23) ␮ ␣ promoting GTP exchange on the ␣ subunit of a heterotrimeric and -opioid signaling (24). A G o effector molecule has been ␣ reported, GRIN1, which promotes growth cone neurite exten- G protein. The heterotrimeric G protein, composed of an ␣ subunit exhibiting endogenous GTPase activity and a het- sion in the mammalian brain (25). The implicated role of G o as ␤␥ a mediator and regulator of core neurological and cognitive erodimeric subunit, dissociates, enabling each component to ␣ activate downstream effectors until GTP is hydrolyzed on the ␣ GPCR-coupled pathways positions G o as a prime target for subunit and the heterotrimeric complex reforms. The ␣ subunit’s pharmaceutical intervention. A molecular understanding of endogenous GTP hydrolysis rate is relatively slow, therefore the downstream signaling components is a key step toward identi- cell uses GTPase accelerating proteins (GAPs) to increase the fying potential therapeutic points of drug intervention used rate to suit the time scale and magnitude needed for a specific physiological response. Author contributions: K.C.S., M.A.K., and P.B.S. designed research; K.C.S. and M.A.K. per- BIOCHEMISTRY The regulators of G protein signaling (RGS) proteins are a class formed research; T.W., C.-K.C., and M.I.S. contributed new reagents/analytic tools; K.C.S., of heterotrimeric G protein GAP first identified in Saccharomyces M.A.K., and P.B.S. analyzed data; and K.C.S. and M.I.S. wrote the paper. cerevisiae (Sst2) and Caenorhabditis elegans (Egl10) (1, 2). Studies, The authors declare no conflict of interest. both biochemical and structural, have shown an overall preference Data Deposition: The atomic coordinates have been deposited in the Protein Data Bank, for RGS domains to bind G␣ subunits in their transition state www.pdb.org (PDB ID codes 3C7L and 3C7K). Ϫ (mimicked by the analog GDP⅐AlF4 ) and to accelerate GTPase †To whom correspondence may be addressed at: Department of Biology, CB# 3280, 402 activity by stabilizing the transition state of hydrolysis, thereby Fordham Hall, University of North Carolina, Chapel Hill, NC 27599-3280. E-mail: optimizing the endogenous GTPase activity of the G␣ subunit [email protected]. ʈ without directly contributing to the hydrolytic mechanism (3–5). Present Address: Institute of Experimental and Clinical Pharmacology and Toxicology, Medical Faculty Mannheim, University of Heidelberg, Maybachstrasse 14, D-68169 RGS proteins serve to quench the G protein signal temporally and Mannheim, Germany. spatially, either independently, or coupled (in cis or in trans)toan **To whom correspondence may be addressed at: Division of Biology, California Institute effector (6, 7). Thirty-seven RGS proteins have been identified in of Technology, Mail Code 147-75, 1200 East California Boulevard, Pasadena, CA 91125. the human genome, cataloged into eight subfamilies based on the E-mail: [email protected]. protein family the RGS domain resides delineated as RGS sub- ††Deceased January 11, 2000. families R4, R7, R12, RZ, and RhoGEF RGS (rgRGS); G protein- This article contains supporting information online at www.pnas.org/cgi/content/full/ coupled receptor kinases (GRKs); sorting nexins; and Axin (8–10). 0801569105/DCSupplemental. Each member displays a unique expression and localization pattern © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801569105 PNAS ͉ April 29, 2008 ͉ vol. 105 ͉ no. 17 ͉ 6243–6248 Downloaded by guest on September 28, 2021 Ϫ Ϫ Fig. 1. Structure of the G␣o⅐GDP⅐AlF4 ⅐RGS16 complex. The G␣o⅐GDP⅐AlF4 ⅐RGS16 complex is represented in ribbons format. RGS16 is indicated in gray; G␣o GTPase domain is in green with nucleotide-dependent switch regions highlighted in orange; G␣o helical domain is in purple. Structure at Right is rotated 90° about the y axis relative to the structure at Left. alone or in combination with GPCR-targeted drugs to minimize 7). The two subdomains are united by a conserved hydropho- side effects (26). bic interface that constitutes the structure’s core. RGS16 is a Here, we report the first crystal structure determination of conserved member of the R4 RGS subfamily and retains G␣o, presented in the transition state of hydrolysis, complexed central conserved elements that define RGS domains except with the R4 subfamily RGS protein RGS16 (27). G␣o displays a for one component, Thr-158, which replaces the conserved number of unique determinants that likely affords it specificity isoleucine or valine found in the equivalent position across all in receptor, RGS, and effector engagement. We discuss the other RGS members (Fig. S1). Thr-158 is buried in the conserved nature of the RGS GAP mechanism, structural hydrophobic core between the two subdomains. It enhances aspects that confer specificity, and the plasticity of the RGS stabilization through a hydrogen bond to the residue 154 domain required to engage and maintain binding during the backbone carbonyl and engages the side chain of Phe-93 in a ␣ transformation of G o from the GTP-bound state to the tran- unique geometry independently confirmed by Soundararajan ␣ ⅐ ⅐ Ϫ⅐ sition state by comparing the G o GDP AlF4 RGS16 complex et al. (18). to our independently determined structure of apo-RGS16, also ␣ presented here. The G –RGS interactions noted in our deter- The RGS16 GAP Mechanism. The RGS16 RGS domain engages the ␣ mination of G o complexed to RGS16 dovetails with specificity G␣ switch regions via a conserved interface that buttresses their findings presented in an accompanying article by Soundararajan transition state conformation. The interaction is structurally et al. (18), facilitating the first examination of an RGS protein similar to that observed in G␣ ⅐RGS4 and G␣ ⅐RGS9 struc- ␣ i1 i1/t complexed to different G subunits. tures (5, 7). Although the total group of interactions between the ␣ Results and Discussion RGS domain and the G switch regions is not identical between ␣ ⅐ ␣ these pairs, key conserved RGS residues are used in identical Architecture of the G o RGS16 Complex.
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