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Structure-Function Studies of Anthrax Protective Antigen Octamer

  • Author(s): Kintzer, Alexander F.
  • Advisor(s): Krantz, Bryan A
  • et al.
Abstract

Abstract

Structure-Function Studies of Anthrax Protective Antigen Octamer

by

Alexander Frederick Kintzer

Doctor of Philosophy in Chemistry

University of California, Berkeley

Professor Bryan Krantz, Chair

The assembly of bacterial toxins and virulence factors is critical to their function, but the regulation of holotoxin assembly during infection has not been studied. The protective antigen (PA) component of the toxin assembles into ring-shaped homooligomers that bind the two other enzyme components of the toxin, lethal factor (LF) and edema factor (EF), to form toxic complexes. To disrupt the host, these toxic complexes are endocytosed, such that the PA oligomer forms a membrane-spanning channel that LF and EF translocate through to enter the cytosol.

We found using single-channel electrophysiology that PA channels contain two populations of conductance states, which correspond with two different PA pre-channel oligomers observed by electron microscopy--the well-described heptamer and a novel octamer. Octameric complexes are likely integral to the PA assembly mechanism, as dimeric and tetrameric intermediates are the intial populated species during assembly. While complexes are functional translocases, a 3.2Å crystal structure suggests that the PA octamer prechannel may be more thermodynamically stable than the heptamer. Indeed, the octamer comprises 20-30% of the oligomers on cells, but predominates (70-80%) during assembly in physiological plasma.

To probe the mechanism of octamer stability, we measured the pH-dependence of channel formation using circular dichroism, mass spectrometry, and electron microscopy. We found that octamers form channels at lower pH than heptamers. This results in heptamer inactivation under physiological conditions, while LT complexes containing octameric PA maintain maximal cytotoxic activity. Thus the PA octamer is a physiological, stable, and active assembly state capable of forming lethal toxins that may withstand the hostile conditions encountered in the bloodstream. This assembly mechanism demonstrates a novel means to control cytotoxicity during anthrax infection.

Finally, we studied the mechanism of channel formation. We found from structural, thermodynamic, and kinetic studies that two barriers define the prechannel to channel transition--a pH-dependent and independent process. Further analysis revealed that the protonation of the membrane insertion loop, dissociation of domains 2 and 4, and interactions with phenylalanine-clamp comprise the rate-limiting, pH-dependent barrier.

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