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.
Senin
Ivan I
II
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992, Moscow, Russian Federation.
Vaganova
Svetlana A
SA
Weiergräber
Oliver H
OH
Ergorov
Nikolay S
NS
Philippov
Pavel P
PP
Koch
Karl Wilhelm
KW
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002417:Cattle; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D051597:Hippocalcin; D066298:In Vitro Techniques; D007700:Kinetics; D008074:Lipoproteins; D008958:Models, Molecular; D009154:Mutation; D019814:Myristic Acid / Q000378:metabolism*; D009419:Nerve Tissue Proteins; D011487:Protein Conformation; D015202:Protein Engineering; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051603:Recoverin; D012374:Rod Cell Outer Segment / Q000378:metabolism; D014785:Vision, Ocular
D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D019814:Myristic Acid; D051603:Recoverin; D051597:Hippocalcin; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D002118:Calcium