Involvement of the recoverin C-terminal segment in recognition of the target enzyme rhodopsin kinase

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.