AMINO-TERMINAL MYRISTOYLATION INDUCES COOPERATIVE CALCIUM-BINDING TO RECOVERIN

Recoverin, a new member of the EF-hand protein superfamily, serves as a Ca2+ sensor in vision, A myristoyl or related N-acyl group covalently attached to the amino terminus of recoverin enables it to bind to disc membranes when the Ca2+ level is elevated. Ca2+-bound recoverin prolongs the lifetime of photoexcited rhodopsin, most likely by blocking its phosphorylation, We report here Ca2+ binding studies of myristoylated and unmyristoylated recombinant recoverin using flow dialysis, fluorescence, and NMR spectroscopy, Unmyristoylated recoverin exhibits heterogeneous and uncooperative binding of two Ca2+ with dissociation constants of 0.11 and 6.9 mu M. In contrast, two Ca2+ bind cooperatively to myristoylated recoverin with a Hill coefficient of 1.75 and an apparent dissociation constant of 17 mu M. Thus, the attached myristoyl group lowers the calcium affinity of the protein and induces cooperativity in Ca2+ binding, One-dimensional H-1 and two-dimensional N-15-H-1 shift correlation NMR spectra of myristoylated recoverin measured as a function of Ca2+ concentration show that a concerted conformational change occurs when two Ca2+ are bound. The Ca2+ binding and NMR data can be fit to a concerted allosteric model in which the two Ca2+ binding sites have different affinities in both the T and R states, The T and R conformational states are defined in terms of the Ca2+-myristoyl switch; in the T state, the myristoyl group is sequestered inside the protein, whereas in the R state, the myristoyl group is extruded, Ca2+ binds to the It state at least 10,000-fold more tightly than to T, In this model, the dissociation constants of the two sites in the It state of the myristoyl ated protein are 0.11 and 6.9 mu M, as in unmyristoylated recoverin, The ratio of the unliganded form of T to that of R is estimated to be 400 for myristoylated and <0.05 for unmyristoylated recoverin, Thus, the attached myristoyl group has two related roles: it shifts the T/R ratio of the unliganded protein more than 8000-fold, and serves as a membrane anchor for the fully Liganded protein.