Cys32 and His105 are the critical residues for the calcium-dependent cysteine proteolytic activity of CvaB, an ATP-binding cassette transporter.
CvaB, a member of the ATP-binding cassette transporter superfamily, is the central membrane transporter of the colicin V secretion system in Escherichia coli. Cys32 and His105 in the N-terminal domain of CvaB were identified as critical residues for both colicin V secretion and cysteine proteolytic activity. By inhibiting degradation with N-ethylmaleimide and a mixture of protease inhibitors, a stable wild-type N-terminal domain (which showed cysteine protease activity when activated) was purified. Such protease activity was Ca2+- and concentration-dependent and could be inhibited by antipain, N-ethylmaleimide, EDTA, and EGTA. At low concentrations, the Ca2+ analogs Tb3+ and La3+ (but not Fe3+) significantly enhanced proteolytic activity, suggesting that the size of the cations is important for activity. Together with comparisons of the sequences of members of the cysteine protease family, these results indicate that Cys32 and His105 are the critical residues in the CvaB N-terminal domain for the calcium-dependent cysteine protease activity and secretion of colicin V.
Department of Biology, Georgia State University, Atlanta, Georgia 30303, USA.
Kai-Hui
KH
Wu
Phang C
PC
Tai
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000737:chemistry; D002455:Cell Division; D003087:Colicins / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry*; D003546:Cysteine Endopeptidases / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000737:chemistry; D004533:Egtazic Acid / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005033:Ethylmaleimide / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D018528:ATP-Binding Cassette Transporters; D003087:Colicins; D003470:Culture Media; C102358:CvaB protein, E coli; D029968:Escherichia coli Proteins; D010455:Peptides; D011480:Protease Inhibitors; D006639:Histidine; D004533:Egtazic Acid; D004492:Edetic Acid; D002154:Calpain; D003546:Cysteine Endopeptidases; D003545:Cysteine; D005033:Ethylmaleimide; D002118:Calcium