Abstract
The metalloproteinase anthrax lethal factor (LF) is secreted by Bacillus anthracis to promote disease virulence through disruption of host signaling pathways. LF is a highly specific protease, exclusively cleaving mitogen-activated protein kinase kinases (MKKs) and rodent NLRP1B (NACHT leucine-rich repeat and pyrin domain-containing protein 1B). How LF achieves such restricted substrate specificity is not understood. Previous studies have suggested the existence of an exosite interaction between LF and MKKs that promotes cleavage efficiency and specificity. Through a combination of in silico prediction and site-directed mutagenesis, we have mapped an exosite to a non-catalytic region of LF. Mutations within this site selectively impair proteolysis of full-length MKKs yet have no impact on cleavage of short peptide substrates. Although this region appears important for cleaving all LF protein substrates, we found that mutation of specific residues within the exosite differentially affects MKK and NLRP1B cleavage in vitro and in cultured cells. One residue in particular, Trp-271, is essential for cleavage of MKK3, MKK4, and MKK6 but dispensable for targeting of MEK1, MEK2, and NLRP1B. Analysis of chimeric substrates suggests that this residue interacts with the MKK catalytic domain. We found that LF-W271A blocked ERK phosphorylation and growth in a melanoma cell line, suggesting that it may provide a highly selective inhibitor of MEK1/2 for use as a cancer therapeutic. These findings provide insight into how a bacterial toxin functions to specifically impair host signaling pathways and suggest a general strategy for mapping protease exosite interactions.
Highlights
The metalloproteinase anthrax lethal factor (LF) is secreted by Bacillus anthracis to promote disease virulence through disruption of host signaling pathways
This region appears important for cleaving all LF protein substrates, we found that mutation of specific residues within the exosite differentially affects mitogen-activated protein kinase kinases (MKKs) and NLRP1B cleavage in vitro and in cultured cells
We found that the chimera in which the N terminus of MKK6 was fused to the kinase domain of MEK1 was cleavable by LF-W271A, albeit with slightly lower efficiency than WT LF
Summary
The metalloproteinase anthrax lethal factor (LF) is secreted by Bacillus anthracis to promote disease virulence through disruption of host signaling pathways. We found that LF-W271A blocked ERK phosphorylation and growth in a melanoma cell line, suggesting that it may provide a highly selective inhibitor of MEK1/2 for use as a cancer therapeutic These findings provide insight into how a bacterial toxin functions to impair host signaling pathways and suggest a general strategy for mapping protease exosite interactions. The LF catalytic domain is most closely related to MtfA, a putative intracellular aminopeptidase widespread in Gram-negative bacteria, and a group of secreted metalloproteinases found in some species of pathogenic Gram-positive bacteria, represented by Zmp from Clostridium difficile [25, 26] These proteases share with LF a conserved zinc-binding Glu residue and an additional active site Tyr residue important for catalysis. We identify and characterize in detail an LF exosite in domain II that is required for the efficient cleavage of MKK substrates
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