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Elucidating Anthrax Lethal Factor Interactions with MAP Kinase Kinase Substrates
Author(s) -
Goldberg Allison,
Ngongoni Rejoice,
Turk Benjamin
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.894.4
Subject(s) - bacillus anthracis , anthrax toxin , kinase , microbiology and biotechnology , cleavage (geology) , mutant , chemistry , allosteric regulation , mapk/erk pathway , biology , protein kinase a , biochemistry , enzyme , bacteria , fusion protein , recombinant dna , genetics , gene , paleontology , fracture (geology)
The metalloprotease anthrax lethal factor (LF) is the enzymatic component of a toxin produced by Bacillus anthracis and is thought to mediate bacterial dissemination and pathology associated with systemic anthrax disease. LF acts in the cytoplasm of host cells, cleaving MAP kinase kinases (MKKs). Combined inhibition of MAPK pathways abolishes host defense mechanisms and leads to vascular collapse and toxic shock in infected animals and humans. LF selectivity for MKKs is due, in part, to the recognition of specific sequences at the site of cleavage. However, LF also appears to contain an exosite distal from the cleavage site that is required for efficient cleavage of MKKs. Because of the transient, low affinity nature of the LF‐MKK complex, there is little structural insight into LF‐MKK exosite interactions. Through in silico modeling of predicted protein interaction surfaces, we have identified an allosteric site spanning two non‐catalytic domains of LF that is required for efficient cleavage of specific MKKs in vitro and in cultured macrophage cells. Relative binding experiments and kinetic analysis of LF exosite mutants suggests that this region does not substantially contribute to substrate affinity or Km, but primarily affects the maximal rate of catalysis. Interestingly, mutation of specific residues within this site has differential effects on the cleavage of MKK1/MKK2 vs. MKK3/MKK6, which may have applications in the use of engineered LF variants to treat BRAF‐driven cancers.