SECONDARY STRUCTURE OF MYRISTOYLATED RECOVERIN DETERMINED BY 3-DIMENSIONAL HETERONUCLEAR NMR - IMPLICATIONS FOR THE CALCIUM MYRISTOYL SWITCH

Recoverin, a new member of the EF-hand superfamily, serves as a Ca2+ sensor in vision. A myristoyl or related N-acyl group is covalently attached at its N-terminus and plays an essential role in Ca2+-dependent membrane targeting by a novel calcium-myristoyl switch mechanism. The structure of unmyristoylated recoverin containing a single bound Ca2+ has recently been solved by X-ray crystallography [Flaherty, K. M., Zozulya, S., Stryer, L., & McKay, D. B. (1993) Cell 75, 709-716]. We report here multidimensional heteronuclear NMR studies on Ca2+-free, myristoylated recoverin (201 residues, 23 kDa). Complete polypeptide backbone H-1, N-15, and C-13 resonance assignments and secondary structure are presented. We find 11 helical segments and two pairs of antiparallel beta-sheets, in accord with the four EF-hands seen in the crystal structure. The present NMR study also reveals some distinct structural features of the Ca2+-free myristoylated protein. The N-terminal helix of EF-2 is flexible in the myristoylated Ca2+-free protein, whereas it has a well-defined structure in the unmyristoylated Ca2+-bound form. This difference suggests that the binding of Ca2+ to EF-3 induces EF-2 to adopt a conformation favorable for the binding of a second Ca2+ to recoverin. Furthermore, the N-terminal helix (K5-E16) of myristoylated Ca2+-free recoverin is significantly longer than that seen in the unmyristoylated Ca2+-bound protein. We propose that this helix is stabilized by the attached myristoyl group and may play a role in sequestering the myristoyl group within the protein in the Ca2+-free state.