Mapping the anthrax protective antigen binding site on the lethal and edema factors.
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.
Lacy
D Borden
DB
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mourez
Michael
M
Fouassier
Alexandre
A
Collier
R John
RJ
D000262:Adenylyl Cyclases / Q000737:chemistry*; D000409:Alanine / Q000737:chemistry; D000818:Animals; D000942:Antigens, Bacterial; D001427:Bacterial Toxins / Q000737:chemistry*; D001665:Binding Sites; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D002352:Carrier Proteins; C030325:anthrax toxin; D000262:Adenylyl Cyclases; D000409:Alanine