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To investigate how diphtheria toxin (DT) undergoes pH-dependent membrane translocation in mammalian cells, we have isolated and characterized mutants of the toxin that are defective in acidic-pH-dependent killing of Escherichia coli. Cloned DT secreted to the periplasm of E. coli kills the bacteria under acidic conditions (near pH 5.0) by inserting into and permeabilizing the inner membrane (a mechanism independent of the toxin's ADP-ribosylation activity). Mutant forms of DT with reduced lethality for E. coli were selected by plating the bacteria under acidic conditions. CRM503, one of the full-length mutants selected by this protocol, also showed diminished cytotoxicity for mammalian cells. We traced the altered cytotoxicity of CRM503 to a Glu-349----Lys mutation (E349K), one of three point mutations, within the B fragment. The E349K mutation alone inhibited cytotoxicity and membrane translocation in mammalian cells and lethality for E. coli but did not affect enzymic activity or receptor binding. The recently determined crystallographic model of DT shows that Glu-349 resides within a short loop connecting two long hydrophobic alpha-helices of the translocation domain. Protonation of Glu-349 and two other nearby acidic residues, Asp-352 and Glu-362, may enable these helices to undergo membrane insertion and the intervening loop to be transferred to the opposite face of the bilayer. The E349K mutation introduces a positive charge at this site, which would be expected to inhibit membrane insertion and the insertion-dependent activities of DT. These results suggest that protonation of Glu-349 and nearby acidic residues may be important in triggering the translocation step of toxin action.

pH-dependent insertion of proteins into membranes: B-chain mutation of diphtheria toxin that inhibits membrane translocation, Glu-349----Lys.