Tumor necrosis factor (TNF) is a key regulator of inflammatory responses and has been implicated in many pathological conditions. We used structure-based design to engineer variant TNF proteins that rapidly form heterotrimers with native TNF to give complexes that neither bind to nor stimulate signaling through TNF receptors. Thus, TNF is inactivated by sequestration. Dominant-negative TNFs represent a possible approach to anti-inflammatory biotherapeutics, and experiments in animal models show that the strategy can attenuate TNF-mediated pathology. Similar rational design could be used to engineer inhibitors of additional TNF superfamily cytokines as well as other multimeric ligands.

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 ofE. 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. cofl 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 cytoxicity and membrane translocation in mammalian cells and lethality for E. col 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 a-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.Bacterial proteinaceous toxins that insert into and traverse membranes offer interesting opportunities to study the interactions of proteins with lipid bilayers. Diphtheria toxin (DT, 535 residues) belongs to a class of toxins that enzymically modify substrates within mammalian cells (1). Many of these toxins are initially taken into cells by receptor-mediated endocytosis, and subsequently, an enzymically active polypeptide moiety of the toxin is released into the cytosol from endosomes or another membrane-bound compartment. For DT, a proteolytic fragment (fragment A, or DTA) is transferred to the cytosol and catalyzes the ADP-ribosylation of elongation factor 2 and thereby inhibits protein synthesis and causes death of the cell (2). How the enzymically active moiety crosses a membrane is not well-understood for any toxin but, for DT and certain other toxins (and for many animal viruses, as well), acidic intravesicular pH is known to trigger the process. A variety of evidence indicates that the membrane translocation event for DT occurs when the toxin is exposed to a pH near 5.0 in the endosomal compartment (3-6). In vitro, treatment of DT with buffer of pH near 5.0 induces a conformational change that causes the toxin to insert into artificial lipid bilayers (7-10), as manifested, for example, by the formation of ion-conductive m...

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