Web Release Date: May 8,
Structure, Topology, and Dynamics of Myristoylated Recoverin Bound to
Phospholipid Bilayers
and
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, Division of Molecular and Structural Biology, Ontario Cancer Institute, and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada, and Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850
Received November 27, 2002
Revised Manuscript Received March 19, 2003
Abstract:
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 Ca2+-myristoyl switch. Samples of 15N-labeled
Ca2+-bound myristoylated recoverin bind anisotropically to phospholipid membranes as judged by analysis
of 15N and 31P chemical shifts observed in solid-state NMR spectra. On the basis of a 2H 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 (1H-15N PISEMA) spectra of uniformly and selectively 15N-labeled recoverin show that the Ca2+-bound protein
is positioned on the membrane surface such that its long molecular axis is oriented ~45
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.
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