Phenylalanine-427 of anthrax protective antigen functions in both pore formation and protein translocation

Abstract

The protective antigen (PA) moiety of anthrax toxin forms a heptameric pore in endosomal membranes of mammalian cells and translocates the enzymatic moieties of the toxin to the cytosol of these cells. Phenylalanine-427 (F427), a solvent-exposed residue in the lumen of the pore, was identified earlier as being crucial for the transport function of PA. The seven F427 residues were shown in electrophysiological studies to form a clamp that catalyzes protein translocation through the pore. Here, we demonstrate by a variety of tests that certain F427 mutations also profoundly inhibit the conformational transition of the heptameric PA prepore to the pore and thereby block pore formation in membranes. Lysine, arginine, aspartic acid, or glycine at position 427 strongly inhibited this acidic pH-induced conformational transition, whereas histidine, serine, and threonine had virtually no effect on this step, but inhibited translocation instead. Thus, it is possible to inhibit pore formation or translocation selectively, depending on the choice of the side chain at position 427; and the net inhibition of the PA transport function by any given F427 mutation is the product of its effects on both steps. Mutations inhibiting either or both steps elicited a strong dominant-negative phenotype. These findings demonstrate the dual functions of F427 and underline its central role in transporting the enzymatic moieties of anthrax toxin across membranes.acidic pH ͉ dominant-negative ͉ endosome

Anthrax toxin protective antigen integrates poly-γ- d -glutamate and pH signals to sense the optimal environment for channel formation

“…An electron microscopy (EM) structure of the channel supports the hypothesis (3, 4) that D2 and D4 separate as the channel forms (8). A translocase active site (F427), the ϕ clamp (22), is also critical to channel formation (23). The molecular basis for the pH dependence of channel formation is unknown.…”

Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins

“…This hydrophobic belt forms a seal around translocating polypeptides and facilitates unidirectional transport through the pore (42). Mutations at position F427 in PA disrupt polypeptide translocation and in some cases also inhibit the prepore to pore transition (44). An interprotomer salt bridge lying directly above the φ-clamp (formed by K397 and D426) may help position F427 and neutralize charges near the constriction that interact strongly with a translocating protein (45).…”

Mycobacterium tuberculosis ESAT-6 Exhibits a Unique Membrane-interacting Activity That Is Not Found in Its Ortholog from Non-pathogenic Mycobacterium smegmatis

“…K ϩ Release Assay-The K ϩ release assay was performed as previously described (22,23). Briefly, 200 l of the K ϩ -doped liposomes were added into 5 ml of 10 mM buffers at various pH (NaAc, pH 4.5-5.0; BisTris, pH 5.5-6.5; HEPES, pH 7.0 -7.5; Tris-Cl, pH 8 -9) containing 150 mM NaCl.…”

“…Liposome Preparation-Liposomes were prepared as previously described (22,23). Briefly, 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (20 mg/ml in chloroform) was dried under nitrogen gas to form a lipid film, followed by vacuum overnight to remove residual solvent.…”