Ca2+-dependent conformational changes in bovine GCAP-2.
GCAP-2, a mammalian photoreceptor-specific protein, is a Ca2+-dependent regulator of the retinal membrane guanylyl cyclases (Ret-GCs). Sensing the fall in intracellular free Ca2+ after photo-excitation, GCAP-2 stimulates the activity of Ret-GC leading to cGMP production. Like other members of the recoverin superfamily, GCAP-2 is a small N-myristoylated protein containing four EF-hand consensus motifs. In this study, we demonstrate that like recoverin and neurocalcin, GCAP-2 alters its conformation in response to Ca2+-binding as measured by a Ca2+-dependent change in its far UV CD spectrum. Differences in the conformation of the Ca2+-bound and Ca2+-free forms of GCAP-2 were also observed by examining their relative susceptibility to V8 protease. In contrast to recoverin, we do not observe proteolytic cleavage of the myristoylated N-terminus of Ca2+-bound GCAP-2. NMR spectra also show that, in contrast to recoverin, the chemical environment of the N-terminus of GCAP-2 is not dramatically altered by Ca2+ binding. Despite the similarity of GCAP-2 and recoverin, the structural consequences of Ca2+-binding for these two proteins are significantly dissimilar.
Hughes
R E
RE
Howard Hughes Medical Institute, University of Washington, Seattle 98195, USA.
Brzovic
P S
PS
Dizhoor
A M
AM
Klevit
R E
RE
Hurley
J B
JB
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D005136:Eye Proteins; D051604:Guanylate Cyclase-Activating Proteins; D051597:Hippocalcin; D008074:Lipoproteins; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009226:Myristates / Q000378:metabolism; D009419:Nerve Tissue Proteins; D010786:Photoreceptor Cells / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D051603:Recoverin; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D012997:Solvents
D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D051604:Guanylate Cyclase-Activating Proteins; D008074:Lipoproteins; D009226:Myristates; D009419:Nerve Tissue Proteins; D012997:Solvents; D051603:Recoverin; D051597:Hippocalcin; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D002118:Calcium