Involvement of recoverin C-terminal segment in recognition of the target enzyme rhodopsin kinase
Résumé
Neuronal calcium sensor (NCS) 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 that could therefore serve as structural control elements providing specific functions like target recognition or Ca2+-sensitivity. Recoverin, an NCS protein operating in vision, regulates the activity of rhodopsin kinase, GRK1, in a Ca2+-dependent manner. We investigated a series of recoverin forms that were mutated at the C-terminus. Using pull down assay, surface plasmon resonance spectroscopy and rhodopsin phosphorylation assay, 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 modeling of recoverin-kinase complex provided insight in the protein-protein interface between kinase and the C-terminus of recoverin. Based on these data we suggest that F3 from the N-terminal helix of rhodopsin kinase and K192 from the C-terminal segment of recoverin form a cation-π interaction pair which is essential for target recognition by recoverin. Taken together, our study revealed a novel rhodopsin kinase binding site within C-terminal region of recoverin and highlights its significance for target recognition and regulation.
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