Calcium-binding by p26olf, an S100-like protein in the frog olfactory epithelium.
Frog p26olf is a novel S100-like Ca2+-binding protein found in olfactory cilia. It consists of two S100-like domains aligned sequentially, and has a total of four Ca2+-binding sites (known as EF-hands). In this study, to elucidate the mechanism of Ca2+-binding to each EF-hand (named EF-A, -B, -C and -D from the N-terminus of p26olf), we examined Ca2+-binding in wild-type p26olf and also in its mutants in which a glutamate at the -z coordinate position within each Ca2+-binding loop was substituted for a glutamine. Flow dialysis experiments showed that the wild-type binds nearly four Ca2+ per molecule maximally, while all the mutants bind approximately three Ca2+. Although EF-B and -D are p26olf-specific EF-hands and their role in Ca2+-binding is not known, the result unequivocally showed that they actually bind Ca2+. The overall Ca2+-binding affinity decreased in the three mutants. The decrease was very large in the mutants of EF-A and -B, which suggested that the Ca2+-affinities are high in EF-A and -B in the wild-type. Assuming the presence of four steps of Ca2+-binding, we determined the dissociation constant of each step in wild-type p26olf. To assign which step takes place at which EF-hand, we measured the antagonistic effect of K+ on each step, as the effect of K+ is thought to be a function of the number of the carboxyl groups in an EF-hand. Although the actual Ca2+-binding mechanism may not be so simple, this study together with the mutation study suggested a tentative Ca2+-binding model of p26olf: the order of Ca2+-binding to p26olf is EF-B, EF-A, EF-C and EF-D. Based on these results, we speculate that similar Ca2+-binding takes place in an S100 dimer.
Miwa
N
N
Department of Biology, Graduate School of Science, Osaka University, Japan.
Shinmyo
Y
Y
Kawamura
S
S
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D029845:Amphibian Proteins; D000818:Animals; D001001:Anura; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D066298:In Vitro Techniques; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009831:Olfactory Mucosa / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009418:S100 Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D029845:Amphibian Proteins; C115659:P26OLF protein, Rana catesbeiana; D011994:Recombinant Proteins; D009418:S100 Proteins; D011188:Potassium; D002118:Calcium