Mapping the Anthrax Protective Antigen Binding Site on the Lethal and Edema Factors

D Borden Lacy,Michael Mourez,Alexandre Fouassier,R John Collier

doi:10.1074/jbc.m109997200

Abstract

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.

Highlights

anthrax toxin (ATx) intoxication involves binding of protective antigen (PA) (83 kDa) to a specific mammalian cell-surface receptor and proteolytic activation by furin or a furin-like protease [7]
We showed by deletion mutagenesis that the first 36 residues of LFN are dispensible for binding to PA63 and translocation
Comparison to protein made from a prior construct (pET15b-LFN-(1–255) [17]) showed no change in activity as assessed by a cell surface binding and translocation assay

Summary

EXPERIMENTAL PROCEDURES

Reagents and Chemicals—Oligonucleotides were synthesized by Integrated DNA Technologies. The oligonucleotides introduced NdeI and BamHI sites to facilitate the ligation back into a pET15b vector. Mutants of this construct and their corresponding 35S-labeled proteins were made as described above for LFN. Each mutant was transformed into E. coli BL21(DE3) (Novagen) and purified using the protocol described previously for wildtype LFN-DTA [11]. Proteolysis was quenched with soybean trypsin inhibitor, and digestion profiles of wild-type and mutant LFN were compared by SDS-PAGE to assess protein stability. The cells were washed with PBS and incubated on ice with 35S-labeled EFN or LFN for 1 h. Inhibition of Protein Synthesis—The protein synthesis inhibition assay was used to measure the ability of PA to deliver mutants of LFN-DTA to the cytosol and was performed as described previously [18]. An equal volume of 0.1 M HCl was added, and the amount of tritiated protein was determined by scintillation counting

RESULTS

DISCUSSION

58 Ϯ 1 43 Ϯ 5