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

Biochem J. 2011 Apr 15;435(2):441-50. doi: 10.1042/BJ20110013.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution / genetics
  • Amino Acid Substitution / physiology
  • Animals
  • Binding Sites / genetics
  • Cattle
  • G-Protein-Coupled Receptor Kinase 1 / chemistry*
  • G-Protein-Coupled Receptor Kinase 1 / genetics
  • G-Protein-Coupled Receptor Kinase 1 / metabolism*
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Interaction Domains and Motifs / genetics
  • Protein Interaction Domains and Motifs / physiology*
  • Protein Structure, Tertiary / genetics
  • Protein Structure, Tertiary / physiology
  • Recoverin / chemistry*
  • Recoverin / genetics
  • Recoverin / metabolism*
  • Sequence Homology, Amino Acid

Substances

  • RCV1 protein, Bos taurus
  • Recoverin
  • G-Protein-Coupled Receptor Kinase 1