ИСТИНА

Neuronal calcium sensors (NCSs) are the family of EF-hand Са2+-binding proteins capable of recognition and regulation of effector enzymes in a Ca2+-dependent manner, thus taking part in a number of signaling events in neurons. In spite of a high degree of structural similarity, NCSs demonstrate high selective recognition abilities for their specific target proteins and display different Ca2+-sensitivities. Based on the sequence alignment, we assumed that a variable sequence in C-terminus of NCS proteins, the so-called “C-terminal segment”, is a structural element which confers the specific Ca2+-dependent functional properties on NCSs. To prove this assumption a photoreceptor Ca2+-binding protein recoverin was taken as a model of NCS to study the role of the C-terminal segment in the NCS functionality. A number of recoverin mutants lacking the C-terminal 6, 10, 12, 14, 15, 16 or 18 amino acids, or containing P190G, Q191A, K192A, and V193G substitutions were generated by site-directed mutagenesis. The loss of fourteen or less C-terminal amino acids in recoverin, as well as all the point substitutions introduced, do not cause any destabilization of the protein structure as revealed by temperature dependence of the tryptophan fluorescence of recoverin. The further truncation of C-terminus of recoverin leads to substantial decrease in thermal stability of the protein. Using mutants obtained, it was demonstrated that the E189PQKVKEK196 sequence in the C-terminal segment of recoverin is involved in the mechanism of its Ca2+-myristoyl switch underlying the ability of recoverin to bind to photoreceptor membranes. Moreover, using affinity chromatography (pull down assays) and surface plasmon resonance measurements, it was shown that the 13 C-terminal amino acids of recoverin are necessary for the interaction of recoverin with its intracellular target, rhodopsin kinase. The latter result suggests the presence of a novel rhodopsin kinase binding site in recoverin in addition to the well-known site in the recoverin hydrophobic pocket. Based on the data obtained, we propose a novel role of the C-terminal segment as a sequence critical for the specific Ca2+-dependent recognition of the target enzymes by members of the NCS family.