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Related Experiment Videos

Anthrax protective antigen: prepore-to-pore conversion.

C J Miller1, J L Elliott, R J Collier

  • 1Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.

Biochemistry
|August 11, 1999
PubMed
Summary
This summary is machine-generated.

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Anthrax protective antigen (PA) forms a pore precursor that converts to an SDS-resistant form under acidic conditions, involving conformational changes crucial for toxin translocation.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Anthrax protective antigen (PA) is essential for anthrax toxin entry into host cells.
  • PA(63) forms a heptameric prepore, a key intermediate in membrane pore formation.
  • Understanding PA(63) oligomerization and conformational changes is vital for developing countermeasures.

Purpose of the Study:

  • To investigate the structural and conformational changes of PA(63) prepore to pore conversion.
  • To elucidate the role of domain 2, loop 2 (D2L2) in PA(63) pore formation and function.
  • To characterize the properties of the SDS-resistant pore-like form of PA(63).

Main Methods:

  • Proteolytic cleavage assays (chymotrypsin) to assess conformational changes.
  • Fluorescence spectroscopy using pyrene-labeled PA(63) to monitor loop 2 conformation.

Related Experiment Videos

  • SDS-PAGE and beta-octylglucoside treatment to differentiate prepore and pore forms.
  • Analysis of D2L2 deletion mutants to assess functional roles.

    • Main Results:

    • PA(63) prepore converts to an SDS-resistant pore-like form under acidic conditions (pH ≤ 7) or with beta-octylglucoside.
    • Conformational changes in D2L2, including chymotrypsin site occlusion and pyrene excimer formation, indicate structural rearrangement.
    • The pore-like form binds toxin A and receptors but cannot form pores or mediate translocation.
    • A D2L2 deletion mutant forms SDS-resistant heptamers but is inactive in pore formation and translocation.
    • Domain 2 undergoes a significant rearrangement, independent of D2L2, conferring SDS resistance.

    Conclusions:

    • The D2L2 loops likely form a transmembrane beta-barrel, facilitating pore formation.
    • A major conformational rearrangement in domain 2, separate from D2L2, stabilizes the SDS-resistant form.
    • These findings provide insights into the mechanism of anthrax toxin translocation across membranes.