Recoverin, a member of the EF-hand superfamily, serves as a calcium sensor in retinal rod cells. A myristoyl or related fatty acyl group covalently attached to the N-terminus of recoverin facilitates the binding of recoverin to retinal disk membranes by a mechanism known as the Ca2+-myristoyl switch. Previous structural studies revealed that the myristoyl group of recoverin is sequestered inside the protein core in the absence of calcium. The cooperative binding of two calcium ions to the second and third EF-hands (EF-2 and EF-3) of recoverin leads to the extrusion of the fatty acid. Here we present nuclear magnetic resonance (NMR), fluorescence, and calcium-binding studies of a myristoylated recoverin mutant (myr-E85Q) designed to abolish high-affinity calcium binding to EF-2 and thereby trap the myristoylated protein with calcium bound solely to EF-3. Equilibrium calcium-binding studies confirm that only one Ca2+ binds to myr-E85Q under the conditions of this study with a dissociation constant of 100 microM. Fluorescence and NMR spectra of the Ca2+-free myr-E85Q are identical to those of Ca2+-free wild type, indicating that the E85Q mutation does not alter the stability and structure of the Ca2+-free protein. In contrast, the fluorescence and NMR spectra of half-saturated myr-E85Q (one bound Ca2+) look different from those of Ca2+-saturated wild type (two bound Ca2+), suggesting that half-saturated myr-E85Q may represent a structural intermediate. We report here the three-dimensional structure of Ca2+-bound myr-E85Q as determined by NMR spectroscopy. The N-terminal myristoyl group of Ca2+-bound myr-E85Q is sequestered within a hydrophobic cavity lined by many aromatic residues (F23, W31, Y53, F56, F83, and Y86) resembling that of Ca2+-free recoverin. The structure of Ca2+-bound myr-E85Q in the N-terminal region (residues 2-90) is similar to that of Ca2+-free recoverin, whereas the C-terminal region (residues 100-202) is more similar to that of Ca2+-bound wild type. Hence, the structure of Ca2+-bound myr-E85Q represents a hybrid between the structures of recoverin with zero and two Ca2+ bound. The binding of Ca2+ to EF-3 leads to local structural changes within the EF-hand that alter the domain interface and cause a 45 degrees swiveling of the N- and C-terminal domains, resulting in a partial unclamping of the myristoyl group. We propose that Ca2+-bound myr-E85Q may represent a stable intermediate state in the kinetic mechanism of the calcium-myristoyl switch.

The neuronal calcium sensor (NCS) proteins (e.g. recoverin, neurocalcins, and frequenin) are expressed at highest levels in excitable cells, and some of them regulate desensitization of G protein-coupled receptors. Here we present NMR analysis and genetic functional studies of an NCS homolog in fission yeast (Ncs1p binding was essential for both phenotypes, while myristoylation was less critical. Exogenous cAMP, a key regulator for sexual development, suppressed conjugation and sporulation of ncs1⌬, suggesting involvement of Ncs1p in the adenylate cyclase pathway turned on by the glucose-sensing G protein-coupled receptor Git3p. Starvation-independent sexual development of ncs1⌬ was also complemented by retinal recoverin, which controls Ca 2؉ -regulated desensitization of rhodopsin. In contrast, the Ca 2؉ intolerance of ncs1⌬ was not affected by cAMP or recoverin, suggesting that the two ncs1⌬ phenotypes are mechanistically independent. We propose that Schizosaccharomyces pombe Ncs1p negatively regulates sporulation perhaps by controlling Ca 2؉ -dependent desensitization of Git3p.Calcium ion (Ca 2ϩ ) regulates physiological processes in the fission yeast Schizosaccharomyces pombe (for reviews, see Refs.1-3) as it does in other eukaryotic cell types (for a review, see Ref. 4). The fission yeast genome encodes ion channels (5, 6) and pumps (7-9) homologous to those involved in regulated Ca 2ϩ entry and maintenance of Ca 2ϩ homeostasis in mammalian cells. In all eukaryotic cells, the effects of changes in intracellular Ca 2ϩ levels are mediated primarily by Ca 2ϩ -binding proteins that belong to the EF-hand superfamily (10). In S. pombe, a total of 23 genes specify EF-hand-containing Ca 2ϩ -binding proteins as exemplified by cam1, calmodulin (SPAC3A12.14) (11); fim1, fimbrin (SPBC1778.06c) (12); plc1, phospholipase C (SPAC22F8.11) (13) (17)) expressed mostly in the central nervous system and in other excitable cell types (18,19).The primary sequence of S. pombe Ncs1p demonstrates its homology to the NCS branch of the EF-hand superfamily of Ca 2ϩ -binding proteins ( Fig. 1) (20, 21). Recoverin, the most intensively studied NCS protein, serves as a Ca 2ϩ sensor in retinal rod and cone cells where it controls the desensitization of rhodopsin by inhibiting rhodopsin kinase only at high Ca 2ϩ levels (22-25). The NCS family also includes neuronal Ca 2ϩ sensors such as neurocalcin (26), hippocalcin (27), and frequenin (28) as well as the budding yeast homolog Frq1 (29). All members of the NCS family are myristoylated and possess four EF-hands, although the first EF-hand motif (EF-1) contains substitutions that prevent Ca 2ϩ binding at this site (Fig. 1). In recoverin, substitutions in EF-4 also prevent Ca 2ϩ binding, and hence only EF-2 and EF-3 are functional (30). Frq1, frequenin, and neurocalcin contain three occupied Ca 2ϩ -binding sites (EF-2, EF-3, and EF-4) in their atomic resolution structures (31-33). The S. pombe Ncs1p shares highest sequence identity with Frq1 and frequenin (60%) but is also highly similar in s...