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.
Involvement of the recoverin C-terminal segment in recognition of the target enzyme rhodopsin kinase
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Evgeni Yu. Zernii, Konstantin E. Komolov, Sergei E. Permyakov, Tatiana Kolpakova, Daniele Dell'orco, Annika Poetzsch, Ekaterina L. Knyazeva, Ilya I. Grigoriev, Eugene A. Permyakov, Ivan I. Senin, Pavel P. Philippov, Karl-Wilhelm Koch; Involvement of the recoverin C-terminal segment in recognition of the target enzyme rhodopsin kinase. Biochem J 15 April 2011; 435 (2): 441–450. doi: https://doi.org/10.1042/BJ20110013
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