Effect of channel mutations on the uptake and release of the retinal ligand in opsin

In the retinal binding pocket of rhodopsin, a Schiff base links the retinal ligand covalently to the Lys296 side chain. Light transforms the inverse agonist 11-cis-retinal into the agonist all-trans-retinal, leading to the active Meta II state. Crystal structures of Meta II and the active conformation of the opsin apoprotein revealed two openings of the 7-transmembrane (TM) bundle towards the hydrophobic core of the membrane, one between TM1/TM7 and one between TM5/TM6, respectively. Computational analysis revealed a putative ligand channel connecting the openings and traversing the binding pocket. Identified constrictions within the channel motivated this study of 35 rhodopsin mutants in which single amino acids lining the channel were replaced. 11-cis-retinal uptake and all-trans-retinal release were measured using UV/visible and fluorescence spectroscopy. Most mutations slow or accelerate both uptake and release, often with opposite effects. Mutations closer to the Lys296 active site show larger effects. The nucleophile hydroxylamine accelerates retinal release 80 times but the action profile of the mutants remains very similar. The data show that the mutations do not probe local channel permeability but rather affect global protein dynamics, with the focal point in the ligand pocket. We propose a model for retinal/receptor interaction in which the active receptor conformation sets the open state of the channel for 11-cis-retinal and all-trans-retinal, with positioning of the ligand at the active site as the kinetic bottleneck. Although other G protein-coupled receptors lack the covalent link to the protein, the access of ligands to their binding pocket may follow similar schemes.

[1]  Mark R. Chance,et al.  Structural waters define a functional channel mediating activation of the GPCR, rhodopsin , 2009, Proceedings of the National Academy of Sciences.

[2]  T. Lamb,et al.  Dark adaptation and the retinoid cycle of vision , 2004, Progress in Retinal and Eye Research.

[3]  K. Palczewski,et al.  Ligand Channeling within a G-protein-coupled Receptor , 2003, Journal of Biological Chemistry.

[4]  D. C. Mitchell,et al.  Effect of protein hydration on receptor conformation: decreased levels of bound water promote metarhodopsin II formation. , 1999, Biochemistry.

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

[6]  Alan Grossfield,et al.  Internal hydration increases during activation of the G-protein-coupled receptor rhodopsin. , 2008, Journal of molecular biology.

[7]  Martin Heck,et al.  Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit , 2007, Proceedings of the National Academy of Sciences.

[8]  K. Palczewski,et al.  Role of Bulk Water in Hydrolysis of the Rhodopsin Chromophore* , 2011, The Journal of Biological Chemistry.

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

[10]  A. Gilman,et al.  The effect of activating ligands on the intrinsic fluorescence of guanine nucleotide-binding regulatory proteins. , 1987, The Journal of biological chemistry.

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

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

[13]  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.

[14]  T. Yoshizawa,et al.  Existence of a β-ionone ring-binding site in the rhodopsin molecule , 1975, Nature.

[15]  T. Oas,et al.  Conformational selection or induced fit: A flux description of reaction mechanism , 2009, Proceedings of the National Academy of Sciences.

[16]  M. Heck,et al.  Alkylated hydroxylamine derivatives eliminate peripheral retinylidene Schiff bases but cannot enter the retinal binding pocket of light-activated rhodopsin. , 2011, Biochemistry.

[17]  M. Cornwall,et al.  Role of Noncovalent Binding of 11-cis-Retinal to Opsin in Dark Adaptation of Rod and Cone Photoreceptors , 2001, Neuron.

[18]  P. Liebman,et al.  Temperature and pH dependence of the metarhodopsin I-metarhodopsin II kinetics and equilibria in bovine rod disk membrane suspensions. , 1984, Biochemistry.

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

[20]  D. Farrens,et al.  Role of the Retinal Hydrogen Bond Network in Rhodopsin Schiff Base Stability and Hydrolysis* , 2004, Journal of Biological Chemistry.

[21]  T. Yoshizawa,et al.  Existence of a beta-ionone ring-binding site in the rhodopsin molecule. , 1975, Nature.

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

[23]  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.

[24]  G. Wald,et al.  THE MOLAR EXTINCTION OF RHODOPSIN , 1953, The Journal of general physiology.

[25]  Leonardo Pardo,et al.  Molecular Basis of Ligand Dissociation in β-Adrenergic Receptors , 2011, PloS one.

[26]  K. Hofmann,et al.  Maximal Rate and Nucleotide Dependence of Rhodopsin-catalyzed Transducin Activation , 2001, The Journal of Biological Chemistry.

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

[28]  D. Oprian,et al.  Characterization of rhodopsin congenital night blindness mutant T94I. , 2003, Biochemistry.

[29]  D. Farrens,et al.  Engineering a functional blue-wavelength-shifted rhodopsin mutant. , 2001, Biochemistry.

[30]  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.

[31]  K. Hofmann,et al.  FORMATION OF THE STORAGE FORM, METARHODOPSIN III, FROM ACTIVE METARHODOPSIN II* , 2006 .

[32]  M. Cusanovich,et al.  Characterization of the recombination reaction of rhodopsin. , 1976, Biochemistry.

[33]  Matthias Elgeti,et al.  New Insights into Light-Induced Deactivation of Active Rhodopsin by SVD and Global Analysis of Time-Resolved UV/Vis- and FTIR-Data , 2008 .

[34]  T. Yoshizawa,et al.  Rhodopsin Regeneration is Accelerated via Noncovalent 11‐cis Retinal–Opsin Complex—A Role of Retinal Binding Pocket of Opsin † , 2008, Photochemistry and photobiology.

[35]  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.

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

[37]  Ting Wang,et al.  Retinal release from opsin in molecular dynamics simulations , 2011, Journal of molecular recognition : JMR.

[38]  K. Hofmann,et al.  Signaling States of Rhodopsin , 2001, The Journal of Biological Chemistry.

[39]  F. Malatesta The study of bimolecular reactions under non-pseudo-first order conditions. , 2005, Biophysical chemistry.

[40]  K. Hofmann,et al.  Interplay between hydroxylamine, metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes. , 1983, Biochimica et biophysica acta.

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

[42]  P. Scheerer,et al.  A G protein-coupled receptor at work: the rhodopsin model. , 2009, Trends in biochemical sciences.

[43]  R. Vogel,et al.  Conformations of the Active and Inactive States of Opsin* , 2001, The Journal of Biological Chemistry.

[44]  K. Foster,et al.  Transducin Activation by the Bovine Opsin Apoprotein (*) , 1995, The Journal of Biological Chemistry.

[45]  R. Crouch,et al.  11-cis- and all-trans-retinols can activate rod opsin: rational design of the visual cycle. , 2008, Biochemistry.

[46]  K. Hofmann,et al.  Secondary binding sites of retinoids in opsin: characterization and role in regeneration , 2003, Vision Research.

[47]  M. Sheves,et al.  Interactions of the beta-ionone ring with the protein in the visual pigment rhodopsin control the activation mechanism. An FTIR and fluorescence study on artificial vertebrate rhodopsins. , 1994, Biochemistry.

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

[49]  C. Cowan,et al.  A comparison of the efficiency of G protein activation by ligand-free and light-activated forms of rhodopsin. , 1997, Biophysical journal.

[50]  M. Engelhard,et al.  Interaction of a G protein-coupled receptor with a G protein-derived peptide induces structural changes in both peptide and receptor: a Fourier-transform infrared study using isotopically labeled peptides. , 2007, Journal of molecular biology.

[51]  Patrick Scheerer,et al.  Structural and kinetic modeling of an activating helix switch in the rhodopsin-transducin interface , 2009, Proceedings of the National Academy of Sciences.

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

[53]  D. Oprian,et al.  Slow binding of retinal to rhodopsin mutants G90D and T94D. , 2003, Biochemistry.

[54]  F. Siebert Application of FTIR Spectroscopy to the Investigation of Dark Structures and Photoreactions of Visual Pigments , 1995 .