Calcium-myristoyl protein switch.

Abstract

Recoverin, a recently discovered member of the EF-hand superfamily of Ca2+-bindig proteins, serves as a Ca2+ sensor in vision. The amino terminus of the protein from retinal rod cells contains a covalently attached myristoyl or related N-acyl group. We report here studies of unmyrtylated and myristoylated recombinant recoverin designed to delineate the biological role of this hydrophobic unit. Ca2+ induces the binding of both the unmyristoylated and myristoylated proteins to phenyl-agarose, a hydrophobic support. MATERIALS AND METHODSRecoverin. Recombinant myristoylated and unmyristoylated recoverins were expressed in the overproducing E. coli strains pTrec2/pBB131/DH5aF' and pTrec2/DH5a, respectively, and were purified by a modification of a previously described procedure (5, 15). Cells from 20 liters of a bacterial culture were resuspended in 200 ml of 100 mM KCl/1 mM dithiothreitol/1 mM MgCl2/50 mM potassium Hepes, pH 7.5 (buffer A) supplied with 1 mM EGTA and disrupted by sonication. Streptomycin sulfate was added with stirring over a 10-min period to a final concentration of 0.1% from a 5% stock prepared in buffer A, and the cell debris was removed by centrifugation at 40C for 30 min at 18,000 x g. The cleared lysate was filtered through filter paper (Whatman 1), brought to 2 mM CaCl2, and applied to an 80-ml phenyl-Sepharose CL-4B (Pharmacia) column previously equilibrated with buffer A containing 2 mM CaC12. The column was washed with 400 ml of the same buffer, and the fraction containing recoverin was eluted with 100-150 ml of buffer A containing 2 mM EGTA. The eluate was diluted with 3 volumes of cold water, applied to a HiLoad 26/10 Q-Sepharose HP column (Pharmacia), and chromatographed with a gradient of 0-200 mM KCl in 500 ml of 20 mM Tris-HCl, pH 8.0/1 mM dithiothreitol/1 mM MgCl2/0.1 mM EGTA. Purified recoverin was concentrated to 10-20 mg/ml using Centriprep 10 centrifugal concentrators (Amicon), flash-frozen in liquid nitrogen, and stored at -70'C.To produce tritium-labeled myristoylated recoverin, the overproducing strain pTrec2/pBB131/DHSFa' containing the yeast N-myristoyltransferase expression vector (22) 11569The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Kv channel-interacting proteins as neuronal and non-neuronal calcium sensors

“…Notably, the N-terminus of NCS proteins may play a critical role in post-translational modification and protein function: Proteins in classes A – D and the class E protein KChIP1 possess an N-terminal myristoylation sequence, and myristoylation of these proteins allows their membrane association [24,25]. Moreover, recoverin (class C) and all class B members of the NCS family exhibit a so-called Ca 2+ /myristoyl switch, meaning that the N-terminal myristoyl moiety, which is sequestered in a hydrophobic groove in the Ca 2+ -free state, swings out when the protein binds Ca 2+ [26,27]. In recoverin, which is a calcium sensor par excellence , this Ca 2+ -dependent conformational change allows the interaction with its target proteins (rhodopsin kinase and rhodopsin), and, at the same time, the association of the protein complex with the photoreceptor outer segment disc membrane (Figure 1A) to control light sensitivity in a Ca 2+ -dependent manner [28].…”

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“…Recently, the phospholipid/membrane binding domain of the plasma membrane Ca 2+ pump was shown to bind calmodulin (21). Several members of the EFhand superfamily, including calmodulin, are known to bind membranes or hydrophobic supports in the presence of calcium (19,63). Therefore, it seems possible that in vivo, this region represents a phospholipid/membrane binding site rather than a calmodulin-binding site.…”

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“…This mechanism is an induced change in the orientation of myristic acid from the hydrophobic interior of a protein to the hydrophobic interior of the plasma membrane, and it has been well described for recoverin, a myristylated cellular protein found in retinal rod cells (2,24,38,41). NMR structures of myristylated recoverin have shown that the myristyl group is buried in the center of a nonfunctional helix-loop-helix, the EF hand-like motif (38).…”