Jb. Ames et al., SECONDARY STRUCTURE OF MYRISTOYLATED RECOVERIN DETERMINED BY 3-DIMENSIONAL HETERONUCLEAR NMR - IMPLICATIONS FOR THE CALCIUM MYRISTOYL SWITCH, Biochemistry, 33(35), 1994, pp. 10743-10753
Recoverin, a new member of the EF-hand superfamily, serves as a Ca2+ s
ensor in vision. A myristoyl or related N-acyl group is covalently att
ached 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 Ca2has recently been solved by X-ray crystallography [Flaherty, K. M., Zo
zulya, S., Stryer, L., & McKay, D. B. (1993) Cell 75, 709-716]. We rep
ort here multidimensional heteronuclear NMR studies on Ca2+-free, myri
stoylated recoverin (201 residues, 23 kDa). Complete polypeptide backb
one H-1, N-15, and C-13 resonance assignments and secondary structure
are presented. We find 11 helical segments and two pairs of antiparall
el beta-sheets, in accord with the four EF-hands seen in the crystal s
tructure. 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 myristoylate
d Ca2+-free recoverin is significantly longer than that seen in the un
myristoylated Ca2+-bound protein. We propose that this helix is stabil
ized by the attached myristoyl group and may play a role in sequesteri
ng the myristoyl group within the protein in the Ca2+-free state.