Hoffmann Claudia, Aktories Klaus, Schmidt Gudula
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Albert-Strasse 25, 79104 Freiburg, Germany.
J Biol Chem. 2007 Apr 6;282(14):10826-32. doi: 10.1074/jbc.M610451200. Epub 2007 Feb 12.
Cytotoxic necrotizing factors CNF1 and CNF2 are produced by pathogenic Escherichia coli strains. They constitutively activate small GTPases of the Rho family by deamidation of a glutamine, which is crucial for GTP hydrolysis. Recently, a novel CNF (CNF(Y)) encompassing 65% identity to CNF1 has been identified in Yersinia pseudotuberculosis. In contrast to the E. coli toxins, which activate several isoforms of Rho family GTPases, CNF(Y) is a strong and selective activator of RhoA in vivo. By constructing chimeras between CNF1 and CNF(Y), we show that this substrate specificity is based on differences in the catalytic domains, whereas the receptor binding and translocation domains have no influence. We further define a loop element (L8) on the surface of the catalytic domains as important for substrate recognition. A single amino acid exchange in L8 is sufficient to shift substrate specificity of CNF1. Moreover, it is shown that RhoA activation by CNF1 is transient, which may be the consequence of the broader substrate specificity of the E. coli toxin, leading to cross-talk between the activated GTPases.
细胞毒性坏死因子CNF1和CNF2由致病性大肠杆菌菌株产生。它们通过使谷氨酰胺脱酰胺来组成性激活Rho家族的小GTP酶，而谷氨酰胺对于GTP水解至关重要。最近，在假结核耶尔森菌中鉴定出一种与CNF1有65%同源性的新型CNF（CNF(Y)）。与激活Rho家族GTP酶的几种同工型的大肠杆菌毒素不同，CNF(Y)在体内是RhoA的强选择性激活剂。通过构建CNF1和CNF(Y)之间的嵌合体，我们表明这种底物特异性基于催化结构域的差异，而受体结合和易位结构域没有影响。我们进一步确定催化结构域表面的一个环元件（L8）对底物识别很重要。L8中的单个氨基酸交换足以改变CNF1的底物特异性。此外，研究表明CNF1对RhoA的激活是短暂的，这可能是大肠杆菌毒素更广泛的底物特异性导致激活的GTP酶之间相互作用的结果。
