One of the Ca2+ binding sites of recoverin exclusively controls interaction with rhodopsin kinase.

Recoverin is a neuronal calcium sensor protein that controls the activity of rhodopsin kinase in a Ca(2+)-dependent manner. Mutations in the EF-hand Ca2+ binding sites are valuable tools for investigating the functional properties of recoverin. In the recoverin mutant E121Q (Rec E121Q ) the high-affinity Ca2+ binding site is disabled. The non-myristoylated form of Rec E121Q binds one Ca2+ via its second Ca(2+)-binding site (EF-hand 2), whereas the myristoylated variant does not bind Ca2+ at all. Binding of Ca2+ to non-myristoylated Rec E121Q apparently triggers exposure of apolar side chains, allowing for association with hydrophobic matrices. Likewise, an interaction surface for the recoverin target rhodopsin kinase is constituted upon Ca2+ binding to the non-acylated mutant. Structural changes resulting from Ca(2+)-occupation of EF-hand 2 in myristoylated and non-myristoylated recoverin variants are discussed in terms of critical conditions required for biological activity.

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 Ca(2+)-myristoyl switch when Ca(2+) binds to its two functional EF-hand motifs (EF-hands 2 and 3), each present in one of recoverin's two domains. Impairment of Ca(2+)-binding in recoverin leads to a disturbance of the Ca(2+)-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 Ca(2+)-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 Ca(2+)-binding to EF-hand 4. For example, the myristoylated form of the latter mutant bound to membranes in a Ca(2+)-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 Ca(2+)-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.