title: Involvement of the recoverin C-terminal segment in recognition of the target enzyme rhodopsin kinase.
abstract: NCS (neuronal Ca2+ sensor) proteins belong to a family of calmodulin-related EF-hand Ca2+-binding proteins which, in spite of a high degree of structural similarity, are able to selectively recognize and regulate individual effector enzymes in a Ca2+-dependent manner. NCS proteins vary at their C-termini, which could therefore serve as structural control elements providing specific functions such as target recognition or Ca2+ sensitivity. Recoverin, an NCS protein operating in vision, regulates the activity of rhodopsin kinase, GRK1, in a Ca2+-dependent manner. In the present study, we investigated a series of recoverin forms that were mutated at the C-terminus. Using pull-down assays, surface plasmon resonance spectroscopy and rhodopsin phosphorylation assays, we demonstrated that truncation of recoverin at the C-terminus significantly reduced the affinity of recoverin for rhodopsin kinase. Site-directed mutagenesis of single amino acids in combination with structural analysis and computational modelling of the recoverin-kinase complex provided insight into the protein-protein interface between the kinase and the C-terminus of recoverin. Based on these results we suggest that Phe3 from the N-terminal helix of rhodopsin kinase and Lys192 from the C-terminal segment of recoverin form a cation-π interaction pair which is essential for target recognition by recoverin. Taken together, the results of the present study reveal a novel rhodopsin-kinase-binding site within the C-terminal region of recoverin, and highlights its significance for target recognition and regulation.
authors.affiliation: AN Belozersky Institute of Physico-Chemical Biology, MV Lomonosov Moscow State University, 119991 Moscow, Russia.
authors.forename: Evgeni Yu
authors.initials: EY
authors.lastname: Zernii
authors.forename: Konstantin E
authors.initials: KE
authors.lastname: Komolov
authors.forename: Sergei E
authors.initials: SE
authors.lastname: Permyakov
authors.forename: Tatiana
authors.initials: T
authors.lastname: Kolpakova
authors.forename: Daniele
authors.initials: D
authors.lastname: Dell'orco
authors.forename: Annika
authors.initials: A
authors.lastname: Poetzsch
authors.forename: Ekaterina L
authors.initials: EL
authors.lastname: Knyazeva
authors.forename: Ilya I
authors.initials: II
authors.lastname: Grigoriev
authors.forename: Eugene A
authors.initials: EA
authors.lastname: Permyakov
authors.forename: Ivan I
authors.initials: II
authors.lastname: Senin
authors.forename: Pavel P
authors.initials: PP
authors.lastname: Philippov
authors.forename: Karl-Wilhelm
authors.initials: KW
authors.lastname: Koch
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D054730:Protein Interaction Domains and Motifs / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D051603:Recoverin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: C067574:RCV1 protein, Bos taurus; D051603:Recoverin; D051606:G-Protein-Coupled Receptor Kinase 1