Borden Lacy
Last active: 3/24/2020

Mapping the anthrax protective antigen binding site on the lethal and edema factors.

Lacy DB, Mourez M, Fouassier A, Collier RJ
J Biol Chem. 2002 277 (4): 3006-10

PMID: 11714723 · DOI:10.1074/jbc.M109997200

Entry of anthrax edema factor (EF) and lethal factor (LF) into the cytosol of eukaryotic cells depends on their ability to translocate across the endosomal membrane in the presence of anthrax protective antigen (PA). Here we report attributes of the N-terminal domains of EF and LF (EF(N) and LF(N), respectively) that are critical for their initial interaction with PA. We found that deletion of the first 36 residues of LF(N) had no effect on its binding to PA or its ability to be translocated. To map the binding site for PA, we used the three-dimensional structure of LF and sequence similarity between EF and LF to select positions for mutagenesis. We identified seven sites in LF(N) (Asp-182, Asp-187, Leu-188, Tyr-223, His-229, Leu-235, and Tyr-236) where mutation to Ala produced significant binding defects, with H229A and Y236A almost completely eliminating binding. Homologous mutants of EF(N) displayed nearly identical defects. Cytotoxicity assays confirmed that the LF(N) mutations impact intoxication. The seven mutation-sensitive amino acids are clustered on the surface of LF and form a small convoluted patch with both hydrophobic and hydrophilic character. We propose that this patch constitutes the recognition site for PA.

MeSH Terms (19)

Adenylyl Cyclases Alanine Animals Antigens, Bacterial Bacterial Toxins Binding Sites Carrier Proteins Cell Membrane CHO Cells Cricetinae Cytosol Models, Molecular Mutagenesis, Site-Directed Mutation Plasmids Protein Binding Protein Conformation Protein Structure, Tertiary Protein Transport

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