Abstract
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.
Highlights
Anthrax toxin is derived from an exotoxin produced by the Gram-positive bacterium Bacillus anthracis
Pairwise mutation of Leu514 and either Leu293, Lys294, or Arg491 completely abrogated lethal factor (LF) toxicity (Fig. 2c), pairwise mutation of Leu514 and Asn516 instead resulted in toxicity comparable with N516A alone (EC50 ϭ 200 Ϯ 98 nM for L514A/N516A versus 164 Ϯ 13 nM for N516A; Fig. 2c). These results indicate that subtle perturbations of the surface composition of domain II caused by alanine substitution of these residues can have a substantial impact upon LF toxicity
In lieu of a direct assay of LF binding to mitogen-activated protein kinase kinases (MEKs), we previously demonstrated that LF could competetively inhibit B-Raf phosphorylation of MEK and that this inhibition was independent of its proteolytic activity [19]
Summary
Anthrax toxin is derived from an exotoxin produced by the Gram-positive bacterium Bacillus anthracis. Following binding to ANTXR, PA is cleaved by cell surface-associated furin, removing a 20-kDa fragment and leaving a 63-kDa fragment (PA63) attached to the ANTXR. This step is necessary to expose a binding site for EF. The crystal structure of LF has been solved to a resolution of 2.2 Å [22] (see Fig. 1) Acidic residues in domain III form specific contacts with the basic NH2 termini of MEKs. Domain IV has limited structural homology to thermolysin and contains the catalytic core. Elements of domains II, III, and IV together create a long catalytic groove into which the NH2 terminus of MEK fits, forming an active site complex
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