An approach to characterizing single‐subunit mutations in multimeric prepores and pores of anthrax protective antigen

Abstract

Heptameric pores formed by the protective antigen (PA) moiety of anthrax toxin translocate the intracellular effector moieties of the toxin across the endosomal membrane to the cytosol of mammalian cells. We devised a protocol to characterize the effects of individual mutations in a single subunit of heptameric PA prepores (pore precursors) or pores. We prepared monomeric PA containing a test mutation plus an innocuous Cys-replacement mutation at a second residue (Lys563, located on the external surface of the prepore). The introduced Cys was biotinylated, and the protein was allowed to cooligomerize with a 20-fold excess of wild-type PA. Finally, biotinylated prepores were freed from wild-type prepores by avidin affinity chromatography. For the proof of principle, we examined single-subunit mutations of Asp425 and Phe427, two residues where Ala replacements have been shown to cause strong inhibitory effects. The single-subunit D425A mutation inhibited pore formation by >10 4 and abrogated activity of PA almost completely in our standard cytotoxicity assay. The single-subunit F427A mutation caused $100-fold inhibition in the cytotoxicity assay, and this effect was shown to result from a combination of strong inhibition of translocation and smaller effects on pore formation and ligand affinity. Our results show definitively that replacing a single residue in one subunit of the heptameric PA prepore can inhibit the transport activity of the oligomer almost completely-and by different mechanisms, depending on the specific residue mutated.

Effects of Introducing a Single Charged Residue into the Phenylalanine Clamp of Multimeric Anthrax Protective Antigen

“…N ␣ -(3-maleimidylpropionyl)biocytin was from Invitrogen, and UltraLink immobilized monomeric avidin was obtained from Pierce. LF N , LF N -DTA (20,27,31), and [WT] 6 [F427A] 1 (25) were prepared as described. [ 3 H[rsq]Leucine was from PerkinElmer Life Sciences.…”

“…LF N ) also depends on Phe 427 (23,25). Binding of LF N to wild-type pore results in an almost complete blockage of current, but binding to F427A pores, for example, causes only partial blockage (23,25).…”

“…In an earlier report, we described a protocol for preparing heteroheptamers with an inhibitory mutation in only a single subunit (25). Both F427A and F425A were known to have dominant negative inhibitory effects on toxicity in PA homo-oligomers (26 -30), and isolating heteroheptamers with the inhibitory mutation in only one subunit enabled us to characterize the effects of these mutations in greater detail.…”

Ultrasensitive detection of protein translocated through toxin pores in droplet-interface bilayers

“…Recombinant WT PA and PA mutants were grown in ECPM1 (31) medium and overexpressed in the periplasm of Escherichia coli BL21 (DE3) before purification by anion-exchange chromatography (32). The heteroheptameric PA 63 ½F427D 1 ½WT 6 mutant PA protein contained two mutations, K563C and F427D, and was generated as previously described (9). WT PA 63 and homoheptameric PA 63 R178A/K214E prepore were formed by limited trypsin digestion and purified by anionexchange chromatography, as previously described (23,33,34).…”

“…In anthrax toxin, for example, cargo molecules block current through the pores formed by the protective antigen (PA) moiety, and after an appropriate electrical potential or a proton gradient is applied, the current is restored to the pore's open-state level, suggesting translocation of the cargo (5)(6)(7)(8). A number of control experiments support this interpretation (5,(7)(8)(9). Detecting and assaying cargo protein in the trans chamber, opposite to that to which the protein was added, has proven challenging, however, mainly because the quantities of the cargo believed to be translocated are so small.…”