Abstract
Recoverin is a Ca(2+)-regulated signal transduction modulator expressed in the vertebrate retina that has been implicated in visual adaptation. An intriguing feature of recoverin is a cluster of charged residues at its C terminus, the functional significance of which is largely unclear. To elucidate the impact of this segment on recoverin structure and function, we have investigated a mutant lacking the C-terminal 12 amino acids. Whereas in myristoylated recoverin the truncation causes an overall decrease in Ca(2+) sensitivity, results for the non-myristoylated mutant indicate that the truncation primarily affects the high affinity EF-hand 3. The three-dimensional structure of the mutant has been determined by x-ray crystallography. In addition to significant changes in average coordinates compared with wild-type recoverin, the structure provides strong indication of increased conformational flexibility, particularly in the C-terminal domain. Based on these observations, we propose a novel role of the C-terminal segment of recoverin as an internal modulator of Ca(2+) sensitivity.
Highlights
Motif [1, 2]
Homogeneous preparations of myristoylated and non-myristoylated recoverin variants were used to determine some of their key biochemical properties
While the residues following Glu189 could not be resolved in the NMR structures of myristoylated recoverin [12, 15, 16], residues 190 –196 form an ␣-helix in the crystal structure of the non-myristoylated protein [21, 22]
Summary
In addition to significant changes in average coordinates compared with wild-type recoverin, the structure provides strong indication of increased conformational flexibility, in the C-terminal domain Based on these observations, we propose a novel role of the C-terminal segment of recoverin as an internal modulator of Ca2؉ sensitivity. Ca2ϩ-binding proteins work as intracellular Ca2ϩ sensors and regulate their targets with high specificity and high spatial and temporal resolution To achieve these remarkable tasks, Ca2ϩ is recognized by specific amino acid sequence motifs, for example the C2 domain and the EF-hand. We have investigated key biochemical properties of the mutant, such as Ca2ϩ binding, association with rod outer segment (ROS) membranes and inhibition of rhodopsin kinase Based on these data, we propose a novel function of the C terminus of recoverin as an internal modulator of EF-hand function
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