title: Structural basis for calcium-induced inhibition of rhodopsin kinase by recoverin.
abstract: Recoverin, a member of the neuronal calcium sensor branch of the EF-hand superfamily, serves as a calcium sensor that regulates rhodopsin kinase (RK) activity in retinal rod cells. We report here the NMR structure of Ca(2+)-bound recoverin bound to a functional N-terminal fragment of rhodopsin kinase (residues 1-25, called RK25). The overall main-chain structure of recoverin in the complex is similar to structures of Ca(2+)-bound recoverin in the absence of target (<1.8A root-mean-square deviation). The first eight residues of recoverin at the N terminus are solvent-exposed, enabling the N-terminal myristoyl group to interact with target membranes, and Ca(2+) is bound at the second and third EF-hands of the protein. RK25 in the complex forms an amphipathic helix (residues 4-16). The hydrophobic face of the RK25 helix (Val-9, Val-10, Ala-11, Ala-14, and Phe-15) interacts with an exposed hydrophobic groove on the surface of recoverin lined by side-chain atoms of Trp-31, Phe-35, Phe-49, Ile-52, Tyr-53, Phe-56, Phe-57, Tyr-86, and Leu-90. Residues of recoverin that contact RK25 are highly conserved, suggesting a similar target binding site structure in all neuronal calcium sensor proteins. Site-specific mutagenesis and deletion analysis confirm that the hydrophobic residues at the interface are necessary and sufficient for binding. The recoverin-RK25 complex exhibits Ca(2+)-induced binding to rhodopsin immobilized on concanavalin-A resin. We propose that Ca(2+)-bound recoverin is bound between rhodopsin and RK in a ternary complex on rod outer segment disk membranes, thereby blocking RK interaction with rhodopsin at high Ca(2+).
authors.affiliation: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850, USA. ames@chem.ucdavis.edu
authors.forename: James B
authors.initials: JB
authors.lastname: Ames
authors.forename: Konstantin
authors.initials: K
authors.lastname: Levay
authors.forename: Jennifer N
authors.initials: JN
authors.lastname: Wingard
authors.forename: Jacqueline D
authors.initials: JD
authors.lastname: Lusin
authors.forename: Vladlen Z
authors.initials: VZ
authors.lastname: Slepak
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002417:Cattle; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000737:chemistry / Q000502:physiology*; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism; D011485:Protein Binding; D051603:Recoverin / Q000737:chemistry*; D012243:Rhodopsin / Q000737:chemistry
chemical_list: D051603:Recoverin; D012243:Rhodopsin; D051606:G-Protein-Coupled Receptor Kinase 1; D002118:Calcium