GroEL as a molecular scaffold for structural analysis of the anthrax toxin pore

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Abstract
The protective antigen (PA) moiety of anthrax toxin exists as a stable prepore, converting into the pore form under low pH to translocate the enzymatic components across the host cell membrane. The PA pore rapidly aggregates in solution, and it is now shown that the chaperone GroEL can stabilize the PA pore, allowing single-particle EM analysis. This method could be useful for other membrane protein complexes. We analyzed the 440-kDa transmembrane pore formed by the protective antigen (PA) moiety of anthrax toxin in the presence of GroEL by negative-stain electron microscopy. GroEL binds both the heptameric PA prepore and the PA pore. The latter interaction retards aggregation of the pore, prolonging its insertion-competent state. Two populations of unaggregated pores were visible: GroEL-bound pores and unbound pores. This allowed two virtually identical structures to be reconstructed, at 25-Å and 28-Å resolution, respectively. The structures were mushroom-shaped objects with a 125-Å-diameter cap and a 100-Å-long stem, consistent with earlier biochemical data. Thus, GroEL provides a platform for obtaining initial glimpses of a membrane protein structure in the absence of lipids or detergents and can function as a scaffold for higher-resolution structural analysis of the PA pore.