Structure, topology, and dynamics of myristoylated recoverin bound to phospholipid bilayers.
Recoverin, a member of the EF-hand protein superfamily, serves as a calcium sensor in retinal rod cells. A myristoyl group covalently attached to the N-terminus of recoverin facilitates its binding to retinal disk membranes by a mechanism known as the Ca(2+)-myristoyl switch. Samples of (15)N-labeled Ca(2+)-bound myristoylated recoverin bind anisotropically to phospholipid membranes as judged by analysis of (15)N and (31)P chemical shifts observed in solid-state NMR spectra. On the basis of a (2)H NMR order parameter analysis performed on recoverin containing a fully deuterated myristoyl group, the N-terminal myristoyl group appears to be located within the lipid bilayer. Two-dimensional solid-state NMR ((1)H-(15)N PISEMA) spectra of uniformly and selectively (15)N-labeled recoverin show that the Ca(2+)-bound protein is positioned on the membrane surface such that its long molecular axis is oriented approximately 45 degrees with respect to the membrane normal. The N-terminal region of recoverin points toward the membrane surface, with close contacts formed by basic residues K5, K11, K22, K37, R43, and K84. This orientation of the membrane-bound protein allows an exposed hydrophobic crevice, near the membrane surface, to serve as a potential binding site for the target protein, rhodopsin kinase. Close agreement between experimental and calculated solid-state NMR spectra of recoverin suggests that membrane-bound recoverin retains the same overall three-dimensional structure that it has in solution. These results demonstrate that membrane binding by recoverin is achieved primarily by insertion of the myristoyl group inside the bilayer with apparently little rearrangement of the protein structure.
Valentine
Kathleen G
KG
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Mesleh
Michael F
MF
Opella
Stanley J
SJ
Ikura
Mitsuhiko
M
Ames
James B
JB
D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005136:Eye Proteins; D051597:Hippocalcin; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008074:Lipoproteins; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015233:Models, Statistical; D009227:Myristic Acids / Q000378:metabolism; D009419:Nerve Tissue Proteins; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D051603:Recoverin
D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D008051:Lipid Bilayers; D008074:Lipoproteins; D009227:Myristic Acids; D009419:Nerve Tissue Proteins; D010743:Phospholipids; D011994:Recombinant Proteins; D051603:Recoverin; D051597:Hippocalcin; D002118:Calcium