Structure and Calcium-Binding Properties of Frq1, a Novel Calcium Sensor in the Yeast Saccharomyces cereVisiae † James B. Ames,* ,‡ Kristin B. Hendricks, §,# Thomas Strahl, § Inken G. Huttner, § Nobuko Hamasaki, ‡ and Jeremy Thorner § Center for AdVanced Research in Biotechnology, UniVersity of Maryland Biotechnology Institute, RockVille, Maryland 20850, and Department of Molecular and Cell Biology, DiVision of Biochemistry and Molecular Biology, UniVersity of California, Berkeley, California 94720-3202 ReceiVed June 6, 2000; ReVised Manuscript ReceiVed August 3, 2000 ABSTRACT: The FRQ1 gene is essential for growth of budding yeast and encodes a 190-residue, N-myristoylated (myr) calcium-binding protein. Frq1 belongs to the recoverin/frequenin branch of the EF-hand superfamily and regulates a yeast phosphatidylinositol 4-kinase isoform. Conformational changes in Frq1 due to N-myristoylation and Ca 2+ binding were assessed by nuclear magnetic resonance (NMR), fluorescence, and equilibrium Ca 2+ -binding measurements. For this purpose, Frq1 and myr-Frq1 were expressed in and purified from Escherichia coli. At saturation, Frq1 bound three Ca 2+ ions at independent sites, which correspond to the second, third, and fourth EF-hand motifs in the protein. Affinity of the second site (K d ) 10 µM) was much weaker than that of the third and fourth sites (K d ) 0.4 µM). Myr- Frq1 bound Ca 2+ with a K d app of 3 µM and a positive Hill coefficient (n ) 1.25), suggesting that the N-myristoyl group confers some degree of cooperativity in Ca 2+ binding, as seen previously in recoverin. Both the NMR and fluorescence spectra of Frq1 exhibited very large Ca 2+ -dependent differences, indicating major conformational changes induced upon Ca 2+ binding. Nearly complete sequence-specific NMR assignments were obtained for the entire carboxy-terminal domain (residues K100-I190). Assignments were made for 20% of the residues in the amino-terminal domain; unassigned residues exhibited very broad NMR signals, most likely due to Frq1 dimerization. NMR chemical shifts and nuclear Overhauser effect (NOE) patterns of Ca 2+ -bound Frq1 were very similar to those of Ca 2+ -bound recoverin, suggesting that the overall structure of Frq1 resembles that of recoverin. A model of the three-dimensional structure of Ca 2+ -bound Frq1 is presented based on the NMR data and homology to recoverin. N-myristoylation of Frq1 had little or no effect on its NMR and fluorescence spectra, suggesting that the myristoyl moiety does not significantly alter Frq1 structure. Correspondingly, the NMR chemical shifts for the myristoyl group in both Ca 2+ -free and Ca 2+ -bound myr-Frq1 were nearly identical to those of free myristate in solution, indicating that the fatty acyl chain is solvent-exposed and not sequestered within the hydrophobic core of the protein, unlike the myristoyl group in Ca 2+ -free recoverin. Subcellular fractionation experiments showed that both the N-myristoyl group and Ca 2+ -binding contribute to the ability of Frq1 to associate with membranes. Calcium ion (Ca 2+ ) regulates many important physiological processes in the budding yeast, Saccharomyces cereVisiae (reviewed in ref 1), as it does in other eukaryotic cell types (reviewed in ref 2). Moreover, the yeast genome encodes channels (3) and pumps (4) homologous to those involved in regulated Ca 2+ entry and maintenance of Ca 2+ homeostasis in mammalian cells. In all eukaryotic cells, the effects of changes in intracellular Ca 2+ levels are mediated primarily by small Ca 2+ -binding proteins that belong to the EF-hand superfamily (5). In S. cereVisiae, five genes specify small, EF-hand-containing Ca 2+ -binding proteins: CMD1, cal- modulin (6); CDC31, centrin/caltractin (7); CNB1, cal- cineurin regulatory subunit (8); MLC1, myosin light chain (9); and, FRQ1, frequenin (10). The FRQ1 gene product is particularly interesting because it is a representative of a class of calcium-sensing proteins that is found in metazoans, in cells of the central nervous system, and in other excitable cell types (11, 12). Yeast Frq1 is necessary for cell growth and viability because it regulates the activity and localization of Pik1, a phosphatidylinositol 4-kinase isoform (10), an enzyme that has been shown to be essential for secretion in yeast cells (13, 14). Frequenin, the closest homologue of Frq1 in multicellular organisms, may play a similar role in neuronal and neuroendocrine cells (15, 16). The amino acid sequence of yeast Frq1 (Figure 1) demonstrates its homology to the recoverin branch of the EF-hand superfamily of Ca 2+ -binding proteins (17, 18). In addition to recoverin, this subgroup of the EF-hand super- family includes neuronal Ca 2+ sensors such as neurocalcin, † This work was supported by NIH Grants EY12347 to J.B.A. and GM21841 to J.T. Kristin B. Hendricks was supported by USPHS Traineeship GM07232, and Inken G. Huttner was supported by a traveling research fellowship from the Daimler-Benz Foundation. * To whom correspondence should be addressed. Dr. James B. Ames, Center for Advanced Research in Biotechnology, 9600 Gudelsky Drive, Rockville, MD 20850. Phone: (301) 738-6120. Fax: (301) 738-6255. E-mail: james@carb.nist.gov. ‡ University of Maryland Biotechnology Institute. § University of California. # Present address: Department of Cell Signaling, DNAX Research Institute, Palo Alto, CA 94304. 12149 Biochemistry 2000, 39, 12149-12161 10.1021/bi0012890 CCC: $19.00 © 2000 American Chemical Society Published on Web 09/13/2000