Nuclear Magnetic Resonance Evidence for Ca2+-induced Extrusion of the Myristoyl Group of Recoverin

Abstract

Recoverin, a recently discovered 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 translocate to retinal disc membranes when the Ca2+ level is elevated. Two-dimensional 1H-13C shift correlation NMR spectra of recoverin containing a 13C-labeled myristoyl group were obtained to selectively probe the effect of Ca2+ on the environment of the attached myristoyl group. In the Ca(2+)-free state, each pair of methylene protons bonded to carbon atoms 2, 3, 11, and 12 of the myristoyl group gives rise to two peaks. The splittings, caused by nonequivalent methylene proton chemical shifts, indicate that the myristoyl group interacts intimately with the protein in the Ca(2+)-free state. By contrast, only one peak is seen for each pair of methylene protons in the Ca(2+)-bound state, indicating that the myristoyl group is located in an isotropic environment in this form. Furthermore, the 1H-13C shift correlation NMR spectrum of Ca(2+)-bound recoverin is very similar to that of myristic acid in solution. 1H-(13)C shift correlation NMR experiments were also performed with 13C-labeled recoverin to selectively probe the resonances of methyl groups in the hydrophobic core of the protein. The spectrum of Ca(2+)-bound myristoylated recoverin is different from that of Ca(2+)-free myristoylated recoverin but similar to that of Ca(2+)-bound unmyristoylated recoverin. Hence, the myristoyl group interacts little with the hydrophobic core of myristoylated recoverin in the Ca(2+)-bound state. Three-dimensional (13C/F1)-edited (13C/F3)-filtered heteronuclear multiple quantum correlation-nuclear Overhauser effect spectroscopy spectra of recoverin containing a 13C-labeled myristoyl group were obtained to selectively probe protein residues located within 5 A of the myristoyl group. The myristoyl group makes close contact with a number of aromatic residues in Ca(2+)-free recoverin, whereas the myristoyl group makes no observable contacts with the protein in the Ca(2+)-bound state. These NMR data demonstrate that the binding of Ca2+ to recoverin induces the extrusion of its myristoyl group into the solvent, which would enable it to interact with a lipid bilayer or a hydrophobic site of a target protein.