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

Blythe E. Janowiak, Alan Finkelstein, R. John Collier

Research output: Contribution to journalArticle

13 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)348-358
Number of pages11
JournalProtein Science
Volume18
Issue number2
DOIs
Publication statusPublished - Feb 1 2009

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Keywords

  • Anthrax toxin
  • Cytotoxicity
  • Phenylalanine clamp
  • Planar lipid bilayer
  • Pore formation
  • Translocation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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