Liang Xudong, Young John J, Boone Sherrie A, Waugh David S, Duesbery Nicholas S
Laboratory of Cancer and Developmental Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan 49503, USA.
J Biol Chem. 2004 Dec 10;279(50):52473-8. doi: 10.1074/jbc.M409105200. Epub 2004 Oct 1.
Anthrax lethal factor (LF) is a Zn2+ -metalloprotease that cleaves and inactivates mitogen-activated protein kinase kinases (MEKs). We have used site-directed mutagenesis to identify a cluster of residues in domain II of LF that lie outside the active site and are required for cellular proteolytic activity toward MEKs. Alanine substituted for Leu293, Lys294, Leu514, Asn516, or Arg491 caused a 10-50-fold reduction in LF toxicity. Further, whereas pairwise substitution of alanine for Leu514 and either Leu293, Lys294, or Arg491 completely abrogated LF toxicity, pairwise mutation of Leu514 and Asn516 resulted in toxicity comparable with N516A alone. The introduction of these mutations reduced LF-mediated cleavage of MEK2 in cell-based assays but altered neither the ability of LF to bind protective antigen nor its ability to translocate across a membrane. Interestingly, direct in vitro measurement of LF activity indicated that decreased toxicity was not always accompanied by reduced proteolytic activity. However, mutations in this region significantly reduced the ability of LF to competitively inhibit B-Raf phosphorylation of MEK. These results provide evidence that elements of domain II are involved in the association of LF into productive complex with MEKs.
炭疽致死因子（LF）是一种锌离子金属蛋白酶，可切割并使丝裂原活化蛋白激酶激酶（MEK）失活。我们利用定点诱变技术在LF的结构域II中鉴定出一组位于活性位点之外的残基，这些残基是LF对MEK进行细胞内蛋白水解活性所必需的。用丙氨酸取代Leu293、Lys294、Leu514、Asn516或Arg491会导致LF毒性降低10至50倍。此外，用丙氨酸分别取代Leu514和Leu293、Lys294或Arg491会完全消除LF毒性，而Leu514和Asn516的成对突变产生的毒性与单独的N516A相当。在基于细胞的实验中，这些突变的引入减少了LF介导的MEK2切割，但既未改变LF结合保护性抗原的能力，也未改变其跨膜转运的能力。有趣的是，LF活性的直接体外测量表明，毒性降低并不总是伴随着蛋白水解活性的降低。然而，该区域的突变显著降低了LF竞争性抑制MEK的B-Raf磷酸化的能力。这些结果证明结构域II的元件参与了LF与MEK形成有效复合物的过程。
