Secondary structure of myristoylated recoverin determined by three-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 Ca(2+)-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 Ca(2+)-free, myristoylated recoverin (201 residues, 23 kDa). Complete polypeptide backbone 1H, 15N, and 13C 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 Ca(2+)-free myristoylated protein. The N-terminal helix of EF-2 is flexible in the myristoylated Ca(2+)-free protein, whereas it has a well-defined structure in the unmyristoylated Ca(2+)-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 Ca(2+)-free recoverin is significantly longer than that seen in the unmyristoylated Ca(2+)-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 Ca(2+)-free state.
Ames
J B
JB
Department of Neurobiology, Stanford University School of Medicine, California 94305.
Tanaka
T
T
Stryer
L
L
Ikura
M
M
D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000648:ultrastructure; D005136:Eye Proteins; D051597:Hippocalcin; D006860:Hydrogen Bonding; D008074:Lipoproteins; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009226:Myristates / Q000737:chemistry; D009419:Nerve Tissue Proteins; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D051603:Recoverin
D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D008074:Lipoproteins; D009226:Myristates; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D051603:Recoverin; D051597:Hippocalcin; D002118:Calcium