Wu Kai-Hui, Tai Phang C
Department of Biology, Georgia State University, Atlanta, Georgia 30303, USA.
J Biol Chem. 2004 Jan 9;279(2):901-9. doi: 10.1074/jbc.M308296200. Epub 2003 Oct 21.
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.
CvaB是ATP结合盒转运蛋白超家族的成员之一，是大肠杆菌中colicin V分泌系统的核心膜转运蛋白。CvaB N端结构域中的Cys32和His105被确定为colicin V分泌和半胱氨酸蛋白酶活性的关键残基。通过用N-乙基马来酰亚胺和蛋白酶抑制剂混合物抑制降解，纯化得到了一种稳定的野生型N端结构域（激活时显示半胱氨酸蛋白酶活性）。这种蛋白酶活性依赖于Ca2+且具有浓度依赖性，可被抑肽酶、N-乙基马来酰亚胺、EDTA和EGTA抑制。在低浓度下，Ca2+类似物Tb3+和La3+（但不是Fe3+）显著增强了蛋白水解活性，表明阳离子的大小对活性很重要。结合半胱氨酸蛋白酶家族成员序列的比较，这些结果表明Cys32和His105是CvaB N端结构域中钙依赖性半胱氨酸蛋白酶活性和colicin V分泌的关键残基。
