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Structural Flexibility of the Gαs Α-Helical Domain in the Β2

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Structural flexibility of the Gαs α-helical domain in the β2-adrenoceptor Gs complex Gerwin H. Westfielda,1, Søren G. F. Rasmussenb,c,1, Min Sua,1, Somnath Duttaa,1, Brian T. DeVreed, Ka Young Chungb, Diane Calinskid, Gisselle Velez-Ruizd, Austin N. Oleskiea, Els Pardone,f, Pil Seok Chaeg, Tong Liuh, Sheng Lih, Virgil L. Woods, Jr.h, Jan Steyaerte,f, Brian K. Kobilkab,2, Roger K. Sunaharad,2, and Georgios Skiniotisa,2 aLife Sciences Institute and Department of Biological Chemistry, dDepartment of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109; bDepartment of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305; cDepartment of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark; eStructural Biology Brussels and fVIB Department of Structural Biology, Vrije Universiteit Brussels, 1050 Brussels, Belgium; gDepartment of Chemistry, University of Wisconsin, Madison, WI 53706; and hDepartment of Chemistry, University of California at San Diego, La Jolla, CA 92093 Contributed by Brian K. Kobilka, August 19, 2011 (sent for review August 9, 2011) The active-state complex between an agonist-bound receptor and Results and Discussion a guanine nucleotide-free G protein represents the fundamental In a first step, we sought to examine the architecture of com- signaling assembly for the majority of hormone and neurotrans- plexes in the nucleotide-free state of Gαs. Before coupling with mitter signaling. We applied single-particle electron microscopy an agonist-bound receptor, the nucleotide binding pocket of the β (EM) analysis to examine the architecture of agonist-occupied 2- α-subunit of the Gαsβγ heterotrimer is occupied by GDP. Upon adrenoceptor (β AR) in complex with the heterotrimeric G protein 2 forming a complex with the β2AR, GDP dissociates, and the Gs (Gαsβγ). EM 2D averages and 3D reconstructions of the deter- resulting nucleotide-free β2AR-Gs complex is highly stable (2). gent-solubilized complex reveal an overall architecture that is in EM visualization of the nucleotide-free complex showed a very good agreement with the crystal structure of the active-state monodisperse particle population (Fig. 1A, and SI Appendix, Fig. α α ternary complex. Strikingly however, the -helical domain of G s S1). Reference-free alignment and classification of ∼17,000 fl appears highly exible in the absence of nucleotide. In contrast, particle projections revealed characteristic class averages with an the presence of the pyrophosphate mimic foscarnet (phosphono- overall density that is in very good agreement with the crystal PHARMACOLOGY formate), and also the presence of GDP, favor the stabilization of structure of the complex (2). Because of its shape, the complex α α the -helical domain on the Ras-like domain of G s. Molecular adsorbs on the carbon support with small variations (± 20°) of α modeling of the -helical domain in the 3D EM maps suggests mainly two diametrically opposite preferred orientations that that in its stabilized form it assumes a conformation reminiscent generate practically identical, mirror-related 2D projections (SI α γ to the one observed in the crystal structure of G s-GTP S. These Appendix, Figs. S2 and S3). α data argue that the -helical domain undergoes a nucleotide- The distinct features of the class averages in these preferred fl dependent transition from a exible to a conformationally orientations allowed us to assign the negative stain projection stabilized state. profiles from specific components of the complex (Fig. 1 B and C). A central oval density represents the β2AR in a detergent G protein-coupled receptor | negative stain electron microscopy | random micelle, with a small protruding density corresponding to T4 conical tilt lysozyme (T4L) that replaces the unstructured extracellular N terminus of the receptor and serves as an orienting landmark. he majority of hormones and neurotransmitters communi- This interpretation was confirmed by EM analysis of complexes Tcate information to cells via G protein-coupled receptors lacking T4L (SI Appendix, Fig. S4). Some class averages of the (GPCRs), which instigate intracellular signaling by activating T4L-β2AR-Gs complex do not reveal a density corresponding to their cognate heterotrimeric G proteins on the cytoplasmic side. the T4L. Besides the presence of a relatively flexible linker GPCRs constitute the largest family of membrane proteins and connecting T4L and the β2AR, this effect is mostly because T4L play essential roles in regulating every aspect of normal physi- lies at an angle to the longitudinal axis of the complex, as shown ology, thereby representing major pharmacological targets. De- in the X-ray structure (2). Because of this geometry, even a 10° spite a wealth of biochemical and biophysical studies on inactive variation in the way the particle adsorbs on the carbon support and active conformations of several heterotrimeric G proteins, drastically reduces the visibility of the T4L projection profile, as the molecular underpinnings of G-protein activation remain demonstrated by projection simulation experiments (SI Appen- fi elusive. The β2-adrenergic receptor (β2AR) and its complex with dix, Fig. S5). Thus, the visibility of the T4L projection pro le is heterotrimeric stimulatory G-protein Gs (Gαsβγ) represent an very sensitive to even limited out-of-plane particle tilting (e.g., ideal model system for the large family of GPCRs activated by because of particle “rock” and “roll” or because of variations in fl diffusible ligands. Agonist binding to the β2AR promotes inter- the atness of the carbon support). Because we observe a single actions with GDP-bound Gsαβγ heterotrimer, leading to the density corresponding to T4L, the detergent micelle contains β exchange of GDP for GTP, and the functional dissociation of Gs only a single copy of the 2AR, in agreement with the crystal fi into Gα-GTP and Gβγ subunits. To examine the architecture of structure. Therefore, the signi cant additional density around agonist occupied β2AR in complex with Gαsβγ under different conditions, we used electron microscopy (EM) and single-parti- cle analysis. Because of the limited size of the protein complex Author contributions: V.L.W., J.S., B.K.K., R.K.S., and G.S. designed research; G.H.W., S.G.F.R., ∼ M.S., S.D., B.T.D., K.Y.C., D.C., G.V.-R., A.N.O., E.P., P.S.C., T.L., S.L., and G.S. performed ( 148 kDa), we visualized specimens embedded in negative research; and B.K.K., R.K.S., and G.S. wrote the paper. fi stain, which provides suf cient contrast from relatively small The authors declare no conflict of interest. protein assemblies (1). This approach allowed us to obtain 2D 1G.H.W., S.G.F.R., M.S., and S.D. contributed equally to this work. projection averages and 3D reconstructions that provided new 2 β To whom correspondence may be addressed. E-mail: [email protected], sunahara@ insights into dynamic features of the 2AR-Gs complex, and umich.edu, or [email protected]. helped guide a successful approach to crystallize the complex This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. enabling a high-resolution structure (2). 1073/pnas.1113645108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1113645108 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 ABnucleotide-free T4L-β2AR-Gs C 63% 37% T4L T4L β2AR β2AR m m m m Gαs Gαs Gs-βγ Gs-βγ AH T4L T4L T4L T4L β2AR β2AR β2AR β2 AR m m m m mmmm Ras Ras Gαs Gαs α βγ Gαs Gs-βγ βγ G s Gs- Gs- Gs-βγ AH AH D nucleotide-free T4L-β2AR-Gs + Nb37 Fig. 1. Two-dimensional projection analysis of the T4L-β2AR-Gs complex in the nucleotide-free state. (A) Raw EM image of detergent-solubilized T4L-β2AR-Gs complex embedded in negative stain. (Scale bar, 50 nm.) (B) Representative EM class averages of the nucleotide-free complex with the projection profile of the AH domain not visible (Left), or visible on the Ras domain (Right, AH indicated by arrow). The cartoon models represent the conformations reflected by the EM averages, with the one on the left depicting the variable positioning of the AH domain, suggesting flexibility or multiple conformations (the position of the detergent micelle is indicated by gray shaded arcs and labeled with “m”). (Scale bar, 10 nm.) (C) Reprojections (Upper) of the crystal structure (2) (Lower) in the same overall orientation as B reveal the identity of each EM density component. The crystal structure on the Right shows the AH domain in the same position (relative to the Ras-like domain) as the one determined in the crystal structure of Gαs-GTPγS alone (4). (D) Representative class averages of nucleotide-free complex with nanobody Nb37 bound on the AH domain (arrows) reveal its flexibility. (Scale bar, 10 nm.) the receptor stems from the large micelle formed by the de- to the T4L domain, the projection profile of the AH domain in tergent (3). Diametrically opposite to the T4L domain, two main this position (SI Appendix, Fig. S3) is not sensitive to the rela- interacting densities representing the Gs trimer appear in close tively limited out-of-plane tilts (± 20°) of the preferred particle proximity to the receptor on its intracellular surface. One of the orientation on the carbon support (SI Appendix, Fig. S5). This two domains appears to extensively interact with the receptor EM analysis provided the initial evidence for a high degree of density, suggesting it corresponds to the Ras-like domain of Gαs, mobility of the AH domain relative to the Ras domain in the fi while its neighboring domain has a pro le consistent with the nucleotide-free β2AR-Gs complex.
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  • Hydrolysis of Guanosine Triphosphate (GTP) by the Ras·GAP Protein Complex: Reaction Mechanism and Kinetic Scheme † † ‡ § † ‡ Maria G

    Hydrolysis of Guanosine Triphosphate (GTP) by the Ras·GAP Protein Complex: Reaction Mechanism and Kinetic Scheme † † ‡ § † ‡ Maria G

    Article pubs.acs.org/JPCB Hydrolysis of Guanosine Triphosphate (GTP) by the Ras·GAP Protein Complex: Reaction Mechanism and Kinetic Scheme † † ‡ § † ‡ Maria G. Khrenova, Bella L. Grigorenko, , Anatoly B. Kolomeisky, and Alexander V. Nemukhin*, , † Chemistry Department, M.V. Lomonosov Moscow State University, Leninskie Gory 1/3, Moscow 119991, Russian Federation ‡ N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygina 4, Moscow 119334, Russian Federation § Department of Chemistry and Center for Theoretical Biological Physics, Rice University, Houston, Texas 77005, United States *S Supporting Information ABSTRACT: Molecular mechanisms of the hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate (Pi) by the Ras·GAP protein complex are fully investigated by using modern modeling tools. The previously hypothesized stages of the cleavage of the phosphorus−oxygen bond in GTP and the formation of the imide form of catalytic Gln61 from Ras upon creation of Pi are confirmed by using the higher-level quantum-based calculations. The steps of the enzyme regeneration are modeled for the first time, providing a comprehensive description of the catalytic cycle. It is found that for the · · · → · · · reaction Ras GAP GTP H2O Ras GAP GDP Pi, the highest barriers correspond to the process of regeneration of the active site but not to the process of substrate cleavage. The specific shape of the energy profile is responsible for an interesting kinetic mechanism of the GTP hydrolysis. The analysis of the process using the first-passage approach and consideration of kinetic equations suggest that the overall reaction rate is a result of the balance between relatively fast transitions and low probability of states from which these transitions are taking place.