Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II

G protein-coupled receptors (GPCR) are seven transmembrane helix proteins that couple binding of extracellular ligands to conformational changes and activation of intracellular G proteins, GPCR kinases, and arrestins. Constitutively active mutants are ubiquitously found among GPCRs and increase the inherent basal activity of the receptor, which often correlates with a pathological outcome. Here, we have used the M257Y6.40 constitutively active mutant of the photoreceptor rhodopsin in combination with the specific binding of a C-terminal fragment from the G protein alpha subunit (GαCT) to trap a light activated state for crystallization. The structure of the M257Y/GαCT complex contains the agonist all-trans-retinal covalently bound to the native binding pocket and resembles the G protein binding metarhodopsin-II conformation obtained by the natural activation mechanism; i.e., illumination of the prebound chromophore 11-cis-retinal. The structure further suggests a molecular basis for the constitutive activity of 6.40 substitutions and the strong effect of the introduced tyrosine based on specific interactions with Y2235.58 in helix 5, Y3067.53 of the NPxxY motif and R1353.50 of the E(D)RY motif, highly conserved residues of the G protein binding site.

[1]  T. Okada,et al.  Local peptide movement in the photoreaction intermediate of rhodopsin , 2006, Proceedings of the National Academy of Sciences.

[2]  Oliver P. Ernst,et al.  Crystal structure of opsin in its G-protein-interacting conformation , 2008, Nature.

[3]  K. Palczewski,et al.  Crystal Structure of Rhodopsin: A G‐Protein‐Coupled Receptor , 2002, Chembiochem : a European journal of chemical biology.

[4]  Viktor Hornak,et al.  Helix Movement is Coupled to Displacement of the Second Extracellular Loop in Rhodopsin Activation , 2009, Nature Structural &Molecular Biology.

[5]  Krzysztof Palczewski,et al.  Role of the conserved NPxxY(x)5,6F motif in the rhodopsin ground state and during activation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[6]  D. Oprian,et al.  Mechanism of activation and inactivation of opsin: role of Glu113 and Lys296. , 1992, Biochemistry.

[7]  Xavier Deupi,et al.  The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex , 2009, Proceedings of the National Academy of Sciences.

[8]  Takahiro Yamashita,et al.  Covalent Bond between Ligand and Receptor Required for Efficient Activation in Rhodopsin* , 2009, The Journal of Biological Chemistry.

[9]  Oliver P. Ernst,et al.  Crystal structure of metarhodopsin II , 2011, Nature.

[10]  C. Altenbach,et al.  High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation , 2008, Proceedings of the National Academy of Sciences.

[11]  Bryan L Roth,et al.  Evidence for a Model of Agonist-induced Activation of 5-Hydroxytryptamine 2A Serotonin Receptors That Involves the Disruption of a Strong Ionic Interaction between Helices 3 and 6* 210 , 2002, The Journal of Biological Chemistry.

[12]  Oliver P. Ernst,et al.  A Ligand Channel through the G Protein Coupled Receptor Opsin , 2009, PloS one.

[13]  Roland Contreras,et al.  Structure and function in rhodopsin: High-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[14]  B. Kobilka,et al.  Energy landscapes as a tool to integrate GPCR structure, dynamics, and function. , 2010, Physiology.

[15]  Manfred Burghammer,et al.  Crystal structure of a thermally stable rhodopsin mutant. , 2007, Journal of molecular biology.

[16]  D. Oprian,et al.  Transducin activation by rhodopsin without a covalent bond to the 11-cis-retinal chromophore , 1991, Science.

[17]  C. Tate,et al.  Thermostabilisation of an Agonist-Bound Conformation of the Human Adenosine A2A Receptor , 2011, Journal of molecular biology.

[18]  T. Sakmar,et al.  Assays for activation of recombinant expressed opsins by all-trans-retinals. , 2000, Methods in enzymology.

[19]  Thomas Huber,et al.  Functional role of the "ionic lock"--an interhelical hydrogen-bond network in family A heptahelical receptors. , 2008, Journal of molecular biology.

[20]  T. Okada,et al.  Crystallographic analysis of primary visual photochemistry. , 2006, Angewandte Chemie.

[21]  Michael F. Brown,et al.  Two protonation switches control rhodopsin activation in membranes , 2008, Proceedings of the National Academy of Sciences.

[22]  E. Zaitseva,et al.  Structural Impact of the E113Q Counterion Mutation on the Activation and Deactivation Pathways of the G Protein-coupled Receptor Rhodopsin , 2008, Journal of molecular biology.

[23]  H. Khorana,et al.  Location of the Retinal Chromophore in the Activated State of Rhodopsin* , 2009, Journal of Biological Chemistry.

[24]  L. Pardo,et al.  Constitutively Active Mutants of the Histamine H1 Receptor Suggest a Conserved Hydrophobic Asparagine-Cage That Constrains the Activation of Class A G Protein-Coupled Receptors , 2008, Molecular Pharmacology.

[25]  D. Oprian,et al.  Effect of carboxylic acid side chains on the absorption maximum of visual pigments. , 1989, Science.

[26]  H Gobind Khorana,et al.  Structural origins of constitutive activation in rhodopsin: Role of the K296/E113 salt bridge. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Xavier Deupi,et al.  Structural insights into agonist-induced activation of G-protein-coupled receptors. , 2011, Current opinion in structural biology.

[28]  D. Oprian,et al.  Constitutively active mutants of rhodopsin , 1992, Neuron.

[29]  H. Hamm,et al.  Site of G protein binding to rhodopsin mapped with synthetic peptides from the alpha subunit. , 1988, Science.

[30]  Rob Leurs,et al.  Pharmacogenomic and structural analysis of constitutive g protein-coupled receptor activity. , 2007, Annual review of pharmacology and toxicology.

[31]  H. Seedorf,et al.  Conformational similarities in the beta-ionone ring region of the rhodopsin chromophore in its ground state and after photoactivation to the metarhodopsin-I intermediate. , 2003, Biochemistry.

[32]  T. Sakmar,et al.  Tracking G-protein-coupled receptor activation using genetically encoded infrared probes , 2010, Nature.

[33]  D. Oprian,et al.  An opsin mutant with increased thermal stability. , 2003, Biochemistry.

[34]  Gebhard F. X. Schertler,et al.  The structural basis of agonist-induced activation in constitutively active rhodopsin , 2011, Nature.

[35]  S. Rasmussen,et al.  Structure of a nanobody-stabilized active state of the β2 adrenoceptor , 2010, Nature.

[36]  Patrick Scheerer,et al.  Crystal structure of the ligand-free G-protein-coupled receptor opsin , 2008, Nature.

[37]  S. Rasmussen,et al.  Crystal Structure of the β2Adrenergic Receptor-Gs protein complex , 2011, Nature.

[38]  Manfred Burghammer,et al.  Structure of bovine rhodopsin in a trigonal crystal form. , 2003, Journal of molecular biology.

[39]  H. Hamm,et al.  Potent Peptide Analogues of a G Protein Receptor-binding Region Obtained with a Combinatorial Library (*) , 1996, The Journal of Biological Chemistry.

[40]  K. Prof,et al.  Crystal structure of rhodopsin: a G protein-coupled receptor. Palczewski K,*(1) kumasaka T, hori T, behnke CA, motoshima H, fox BA, trong IL, teller DC, okada T, stenkamp RE, yamamoto M, miyano M. Science 2000;289:739-745 , 2002, American journal of ophthalmology.

[41]  P. Reeves,et al.  Highly conserved tyrosine stabilizes the active state of rhodopsin , 2010, Proceedings of the National Academy of Sciences.

[42]  D. Oprian,et al.  Rhodopsin mutation G90D and a molecular mechanism for congenital night blindness , 1994, Nature.

[43]  H. Khorana,et al.  Requirement of Rigid-Body Motion of Transmembrane Helices for Light Activation of Rhodopsin , 1996, Science.