Monitoring the kinetics of the pH driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance

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Issue Date
2013-09-17Author
Naik, Subhashchandra
Brock, Susan R.
Akkaladevi, Narahari
Tally, Jon
Mcginn-Staub, Wesley
Zhang, Na
Gao, Philip
Gogol, E. P.
Pentelute, B. L.
Collier, R. John
Fisher, Mark T.
Publisher
ACS
Type
Article
Article Version
Scholarly/refereed, author accepted manuscript
Rights
Copyright © 2013 American Chemical Society
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Show full item recordAbstract
Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å beta barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor (EF), from the endosome into the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance (SPR) and bio-layer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from pH 7.5 to pH 5.0, mirroring acidification of the endosome. Once transitioned, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto EM grids, where the PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early or late endosomal pH conditions (5.5 to 5.0 respectively). Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions.
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Citation
Naik, S., Brock, S., Akkaladevi, N., Tally, J., Mcginn-Straub, W., Zhang, N., … Fisher, M. T. (2013). Monitoring the kinetics of the pH driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance. Biochemistry, 52(37), 6335–6347. http://doi.org/10.1021/bi400705n
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