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

Area of Science:

• Biochemistry
• Molecular Biology
• Toxicology

Background:

• Bacillus anthracis secretes anthrax lethal factor (LF), a metalloproteinase that disrupts host signaling pathways.
• LF's high substrate specificity for mitogen-activated protein kinase kinases (MKKs) and rodent NLRP1B is not fully understood.
• Previous research suggested an exosite interaction between LF and MKKs enhances cleavage efficiency and specificity.

Purpose of the Study:

• To identify and characterize the exosite responsible for LF's substrate specificity.
• To elucidate the molecular mechanisms underlying LF's selective cleavage of MKKs and NLRP1B.
• To explore the therapeutic potential of LF variants as targeted inhibitors.

Main Methods:

• In silico prediction and site-directed mutagenesis to map the LF exosite.
• In vitro and cell-based assays to assess proteolysis of full-length and peptide substrates.
• Analysis of chimeric substrates and evaluation of ERK phosphorylation inhibition.

Main Results:

• A non-catalytic exosite was identified in LF, crucial for cleaving full-length MKKs but not short peptides.
• Specific exosite residues differentially affect MKK and NLRP1B cleavage.
• Trp-271 was found essential for MKK3, MKK4, and MKK6 cleavage but not for MEK1, MEK2, or NLRP1B.
• LF-W271A variant blocked ERK phosphorylation and melanoma cell growth, indicating therapeutic potential.

Conclusions:

• The LF exosite plays a critical role in substrate recognition and cleavage specificity.
• Residue Trp-271 exhibits differential interactions with MKK and NLRP1B substrates.
• LF variants targeting specific signaling pathways, like MEK1/2, could serve as novel cancer therapeutics.
• This study provides a general strategy for mapping protease exosite interactions.