Functional Restoration of the Ca2+-myristoyl Switch in a Recoverin Mutant

Recoverin is a neuronal calcium sensor protein that plays a crucial role in vertebrate phototransduction. It undergoes a Ca2+-myristoyl switch when Ca2+ binds to its two functional EF-hand motifs (EF-hands 2 and 3), each present in one of recoverinʹs two domains. Impairment of Ca2+-binding in recoverin leads to a disturbance of the Ca2+-myristoyl switch and loss of its regulatory properties, i.e. inhibiton of rhodopsin kinase. We have engineered recoverin mutants with either of the two functional EF-hands disabled, but with a functional Ca2+-binding site in EF-hand 4. While a defect in EF-hand 2 could not be rescued by the additional EF-hand 4, the impairment of EF-hand 3 was powerfully compensated by Ca2+-binding to EF-hand 4. For example, the myristoylated form of the latter mutant bound to membranes in a Ca2+-dependent way and was able to inhibit rhodopsin kinase in a way similar to that of the wild-type protein. Thus, for recoverin to undergo a Ca2+-myristoyl switch, it is necessary and sufficient to have either of the two EF-hands in the second domain in a functional state. On the basis of these results and inspection of published three-dimensional structures of recoverin, we propose a model highlighting the mutual interdependence of sterical configurations in EF-hands 3 and 4 of recoverin.