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Amino-terminal Myristoylation Induces Cooperative Calcium Binding to Recoverin (*)

J. Ames,T. Porumb,Toshiyuki Tanaka,M. Ikura,L. Stryer

Published 1995 in Journal of Biological Chemistry

ABSTRACT

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 μM. In contrast, two Ca2+ bind cooperatively to myristoylated recoverin with a Hill coefficient of 1.75 and an apparent dissociation constant of 17 μM. Thus, the attached myristoyl group lowers the calcium affinity of the protein and induces cooperativity in Ca2+ binding. One-dimensional 1H and two-dimensional 15N-1H 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 R state at least 10,000-fold more tightly than to T. In this model, the dissociation constants of the two sites in the R state of the myristoylated protein are 0.11 and 6.9 μM, 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.

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