Involvement of domain II in toxicity of anthrax lethal factor.
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.
Liang
Xudong
X
Laboratory of Cancer and Developmental Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan 49503, USA.
Young
John J
JJ
Boone
Sherrie A
SA
Waugh
David S
DS
Duesbery
Nicholas S
NS
D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D001408:Bacillus anthracis / Q000737:chemistry / Q000472:pathogenicity; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D066298:In Vitro Techniques; D008264:Macrophages / Q000187:drug effects; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011994:Recombinant Proteins; C030325:anthrax toxin; D020929:Mitogen-Activated Protein Kinase Kinases