Calcium-binding activity of nucleobindin mediated by an EF hand moiety.
In a previous paper (Biochem. Biophys. Res. Commun. 187, 375-380, 1992), we reported cloning of a gene for nucleobindin (Nuc) from mouse and human. Nuc was identified as a secreted protein with DNA-binding properties. We noticed that Nuc includes two units of sequence similar to the EF hand motif, a calcium-binding motif, between a region rich in basic amino acids, which is presumably a DNA-binding site, and a leucine zipper which is a dimerization motif. In the present study, we examined the calcium-binding activity of Nuc. Mouse and human recombinant Nuc (rNuc), as well as two mutant rNuc proteins, were synthesized in Escherichia coli. Mouse and human rNuc and one mutant rNuc, in which the region from the second EF hand motif to the C-terminus had been deleted, all had calcium-binding activity. By contrast, another mutant rNuc in which both EF hand motifs had been deleted lacked this activity. Analysis by circular dichroism spectrometry indicated that addition of Ca2+ ions induces a structural change in Nuc, presumably an increase in the amount of alpha helix. However, addition of Ca2+ ions did not seem to cause any change in DNA-binding activity.
Miura
K
K
Institute for Comprehensive Medical Science, Fujita Health University, Aichi, Japan.
Kurosawa
Y
Y
Kanai
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D002147:Calmodulin / Q000737:chemistry; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006133:Growth Substances / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D010320:Parvalbumins / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014336:Troponin / Q000737:chemistry; D019209:Troponin C
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D004268:DNA-Binding Proteins; D006133:Growth Substances; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D010320:Parvalbumins; D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D002118:Calcium