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

Abstract : 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.
Mots-clés : Life Sciences
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Submitted on : Thursday, March 31, 2011 - 2:52:43 AM
Last modification on : Thursday, March 31, 2011 - 2:52:43 AM
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Evgeni Zernii, Konstantin Komolov, Sergei Permyakov, Tatiana Kolpakova, Daniele Dell'Orco, et al.. Involvement of recoverin C-terminal segment in recognition of the target enzyme rhodopsin kinase. Biochemical Journal, Portland Press, 2011, 435 (2), pp.441-450. ⟨10.1042/BJ20110013⟩. ⟨hal-00581539⟩

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