title: Structure and calcium-binding properties of Frq1, a novel calcium sensor in the yeast Saccharomyces cerevisiae.
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 microM) was much weaker than that of the third and fourth sites (K(d) = 0.4 microM). Myr-Frq1 bound Ca(2+) with a K(d)app of 3 microM 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.
authors.affiliation: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850, USA. james@carb.nist.gov
authors.forename: J B
authors.initials: JB
authors.lastname: Ames
authors.forename: K B
authors.initials: KB
authors.lastname: Hendricks
authors.forename: T
authors.initials: T
authors.lastname: Strahl
authors.forename: I G
authors.initials: IG
authors.lastname: Huttner
authors.forename: N
authors.initials: N
authors.lastname: Hamasaki
authors.forename: J
authors.initials: J
authors.lastname: Thorner
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D002247:Carbon Isotopes; D020832:EF Hand Motifs; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019814:Myristic Acid / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D002247:Carbon Isotopes; C400903:FRQ1 protein, S cerevisiae; D005656:Fungal Proteins; D009419:Nerve Tissue Proteins; D029701:Saccharomyces cerevisiae Proteins; D019814:Myristic Acid; D002118:Calcium