Binding of centrins and yeast calmodulin to synthetic peptides corresponding to binding sites in the spindle pole body components Kar1p and Spc110p.
Centrins contain four potential Ca2+ binding sites, known as EF-hands, and have essential functions in centrosome duplication and filament contraction. Here we report that centrins from yeast, green algae, and humans bound with high affinity to a peptide of the yeast centrosomal component Kar1p. Interestingly, centrin binding was regulated by physiological relevant changes in [Ca2+], and this Ca2+ dependence was influenced by acidic amino acids within the Kar1p peptide, which also prevented efficient binding of the related yeast calmodulin. However, a hybrid protein with the third and fourth EF-hands from the yeast centrin Cdc31p and the amino-terminal half from yeast calmodulin behaved more like Cdc31p, indicating that the carboxyl-terminal half of Cdc31p influences binding specificity. Besides Kar1p, centrins bound to a yeast calmodulin binding site, explaining the dosage-dependent suppression of a calmodulin mutant by CDC31. Consistent with an essential role of Ca2+ for centrin functions, mutations in the first or the fourth EF-hands of Cdc31p, impairing the Ca2+-induced conformational change of Cdc31p, resulted in nonfunctional proteins in vivo. Our results suggest that centrins are involved in Ca2+ signaling, likely by influencing the properties of target proteins in response to changes in [Ca2+].
Max-Planck-Institut für Biochemie, Genzentrum, Am Klopferspitz 18a, D-82152 Martinsried, Germany.
B M
BM
Geier
H
H
Wiech
E
E
Schiebel
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins; D002868:Chromosomal Proteins, Non-Histone; D003285:Contractile Proteins / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D008941:Spindle Apparatus / Q000737:chemistry / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D002868:Chromosomal Proteins, Non-Histone; D003285:Contractile Proteins; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins; C074559:KAR1 protein, S cerevisiae; D009687:Nuclear Proteins; C073277:SPC110 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C056608:caltractin; D002118:Calcium