Identification of a Substrate-selective Exosite within the Metalloproteinase Anthrax Lethal Factor

J Biol Chem. 2017 Jan 20;292(3):814-825. doi: 10.1074/jbc.M116.761734. Epub 2016 Dec 1.

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.

Keywords: NACHT leucine-rich repeat and pyrin domain containing protein (NLRP); anthrax toxin; bacterial pathogenesis; host-pathogen interaction; macrophage; melanoma; mitogen-activated protein kinase kinase; protein kinase; proteinase.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Antigens, Bacterial / chemistry
  • Antigens, Bacterial / genetics
  • Antigens, Bacterial / pharmacology*
  • Apoptosis Regulatory Proteins / genetics
  • Apoptosis Regulatory Proteins / metabolism*
  • Bacillus anthracis / chemistry*
  • Bacterial Toxins / chemistry
  • Bacterial Toxins / genetics
  • Bacterial Toxins / pharmacology*
  • Cell Line, Tumor
  • MAP Kinase Signaling System / drug effects*
  • MAP Kinase Signaling System / genetics
  • Mice
  • Mitogen-Activated Protein Kinase Kinases / genetics
  • Mitogen-Activated Protein Kinase Kinases / metabolism*
  • Mutation, Missense
  • Phosphorylation

Substances

  • Antigens, Bacterial
  • Apoptosis Regulatory Proteins
  • Bacterial Toxins
  • Nalp1b protein, mouse
  • anthrax toxin
  • Mitogen-Activated Protein Kinase Kinases

Associated data

  • PDB/1J7N
  • PDB/1JKY
  • PDB/1PWV
  • PDB/1ZXV
  • PDB/1PWP
  • PDB/1PWW
  • PDB/1PWQ
  • PDB/1PWU