Recoverin, a member of the neuronal calcium sensor branch of the EF-hand superfamily, serves as a calcium sensor that regulates rhodopsin kinase (RK) activity in retinal rod cells. We report here the NMR structure of Ca 2؉ -bound recoverin bound to a functional N-terminal fragment of rhodopsin kinase (residues 1-25, called RK25). The overall main-chain structure of recoverin in the complex is similar to structures of Ca 2؉ -bound recoverin in the absence of target (<1.8 Å root-mean-square deviation). The first eight residues of recoverin at the N terminus are solvent-exposed, enabling the N-terminal myristoyl group to interact with target membranes, and Ca 2؉ is bound at the second and third EF-hands of the protein. RK25 in the complex forms an amphipathic helix (residues 4 -16). The hydrophobic face of the RK25 helix (Val-9, Val-10, Ala-11, Ala-14, and Phe-15) interacts with an exposed hydrophobic groove on the surface of recoverin lined by side-chain atoms of Trp-31, Phe-35, Phe-49, Ile-52, Tyr-53, Phe-56, Phe-57, Tyr-86, and Leu-90. Residues of recoverin that contact RK25 are highly conserved, suggesting a similar target binding site structure in all neuronal calcium sensor proteins. Site-specific mutagenesis and deletion analysis confirm that the hydrophobic residues at the interface are necessary and sufficient for binding. The recoverin-RK25 complex exhibits Ca 2؉ -induced binding to rhodopsin immobilized on concanavalin-A resin. We propose that Ca 2؉ -bound recoverin is bound between rhodopsin and RK in a ternary complex on rod outer segment disk membranes, thereby blocking RK interaction with rhodopsin at high Ca 2؉ .Calcium ion (Ca 2ϩ ) in retinal rod cells plays a critical role in regulating the phototransduction cascade in vision (1-3). Recoverin, a 23 kDa Ca 2ϩ -binding protein and member of the EF-hand superfamily, serves as a Ca 2ϩ sensor in retinal rods (4, 5). Recoverin prolongs the lifetime of photoexcited rhodopsin by inhibiting rhodopsin kinase only at high Ca 2ϩ levels (5-8).Hence, recoverin makes the desensitization of rhodopsin responsive to Ca 2ϩ, and the shortened lifetime of photoexcited rhodopsin at low Ca 2ϩ levels may promote visual recovery and contribute to the adaptation to background light. More recently, recoverin was shown to have a different role in synaptic termini and was found localized in the rod inner segment (9). Upon light activation, 98% of recoverin is detected in the rod inner segment, whereas in the dark more than 10% of recoverin returns to the outer segment (10), consistent with the conventional role of recoverin in the inhibition of RK.Recoverin contains four EF-hand Ca 2ϩ binding motifs and a myristoyl or related fatty acyl group covalently attached at the N terminus (11). The cooperative binding of two Ca 2ϩ to the second and third EF-hands (EF-2 and EF-3) induces the binding of myristoylated, but not unmyristoylated recoverin to rod outer segment disc membranes (12, 13). The three-dimensional structures of myristoylated recoverin with 0, 1, and 2 Ca 2ϩ bound ha...

DREAM (calsenilin/KChIP3) is an EF-hand calcium-binding protein that binds to specific DNA sequences and regulates Ca2+-induced transcription of prodynorphin and c-fos genes. Here, we present the atomic-resolution structure of Ca2+-bound DREAM in solution determined by nuclear magnetic resonance (NMR) spectroscopy. Pulsed-field gradient NMR diffusion experiments and 15N NMR relaxation analysis indicate that Ca2+-bound DREAM forms a stable dimer in solution. The structure of the first 77 residues from the N-terminus could not be determined by our NMR analysis. The C-terminal DREAM structure (residues 78-256) contains four EF-hand motifs arranged in a tandem linear array, similar to that seen in KChIP1, recoverin, and other structures of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily. Mg2+ is bound at the second EF-hand, whereas Ca2+ is bound functionally at the third and fourth sites. The first and second EF-hands form an exposed hydrophobic groove on the protein surface lined by side-chain atoms of L96, F100, F114, I117, Y118, F121, F122, Y151, L155, L158, and L159 that are highly conserved in all NCS proteins. An exposed leucine near the C-terminus (L251) is suggested to form intermolecular contacts with leucine residues in the hydrophobic groove (L155, L158, and L159). Positively charged side chains of Arg and Lys (Lys87, Lys90, Lys91, Arg98, Lys101, Arg160, and Lys166) are clustered on one side of the protein surface and may mediate electrostatic contacts with DNA targets. We propose that Ca2+-induced dimerization of DREAM may partially block the putative DNA-binding site, which may suggest as to how Ca2+ abolishes DREAM binding to DNA to activate the transcription of prodynorphin and other downstream genes in pain control.

Yeast frequenin (Frq1), a small N-myristoylated EF-hand protein, activates phosphatidylinositol 4-kinase Pik1. The NMR structure of Ca 2؉ -bound Frq1 complexed to an N-terminal Pik1 fragment (residues 121-174) was determined. The Frq1 main chain is similar to that in free Frq1 and related proteins in the same branch of the calmodulin superfamily. The myristoyl group and first eight residues of Frq1 are solvent-exposed, and Ca 2؉ binds the second, third, and fourth EF-hands, which associate to create a groove with two pockets. The Pik1 peptide forms two helices ( In animal cells and yeast (1, 2), phosphoinositides mediate selective recruitment of proteins to membranes (3-6) and serve as precursors for intracellular second messengers (7-9). Phosphoinositide biosynthesis begins with phosphorylation of the myo-inositol headgroup of phosphatidylinositol (PtdIns) 3 at the D-4 position by PtdIns 4-kinase (ATP:1-phosphatidyl-1D-myo-inositol 4-phosphotransferase, EC 2.7.1.67) (10 -12). The first PtdIns 4-kinase to be purified (13), and the corresponding gene cloned (14), was Pik1 from the yeast Saccharomyces cerevisiae. PIK1 is an essential gene required for vesicular trafficking in the late secretory pathway (15, 16), for nuclear functions (17), and possibly cytokinesis (18). Pik1-like isoforms are conserved in metazoans (10,11,19).Yeast frequenin (Frq1), a 22-kDa Ca 2ϩ -binding protein, copurifies with Pik1 and is essential for its optimal activity (20). The site where Frq1 docks on Pik1 was localized to a region (residues 121-174) that lies far upstream of the catalytic domain (residues 792-1066) (21). Mammalian frequenin also interacts with Pik1 (22), and frequenin may regulate PtdIns 4-kinase activity in animal cells (23-25). Ca 2ϩ -dependent activation of PtdIns 4-kinase by frequenin may be especially important in neurons because modulation of phosphoinositide synthesis by intracellular Ca 2ϩ controls exocytosis (26) and is involved in synaptic plasticity (27).Frq1 and other frequenins belong to the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, which includes recoverin and neurocalcin (28 -31). These proteins are small (Յ25 kDa) and characterized by a consensus signal for N-terminal myristoylation and four EF-hand Ca 2ϩ -binding sites (Fig. 1). We have shown previously that, at saturation, Frq1 binds only three Ca 2ϩ (32). Frq1, which is itself essential for the viability of yeast cells (20), associates with membranes in a manner that depends on both the N-myristoyl group and conformational changes induced upon Ca 2ϩ binding, suggesting that Frq1, like other NCS proteins, may possess a Ca 2ϩ -myristoyl switch (32). Indeed, prior work indicated that N-myristoylation of Frq1 is important (but not essential) for stimulating both the catalytic activity (20) and the membrane recruitment of Pik1 (17).Three-dimensional structures for Frq1 and other NCS proteins have been determined by x-ray crystallography (23,(33)(34)(35)(36)(37) and NMR spectroscopy (32, 38 -40). The structure of * This work was su...

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