25-30 August 2019
Henry Ford Building
Europe/Berlin timezone

An Integrative EPR and EM Approach to unveil the mechanics of a Tc toxin

Not scheduled
4h
Harnack House and Henry Ford Building

Harnack House and Henry Ford Building

Board: 247
Poster Posters

Speaker

Dr Svetlana Kucher (Ruhr University Bochum)

Description

Tc toxins are 1.7 MDa protein complexes that are found in insect- and human-pathogenic bacteria. After endocytosis, Tc penetrates the membrane of the host’s cells and translocates a deadly enzyme into the cytosol. The complex consists of three subunits: the 1.4 MDa TcA pentamer, which mediates target cell association, membrane insertion and toxin translocation, and two smaller subunits, TcB and TcC, which form a 250 kDa cocoon that encapsulates the toxic enzyme. TcA contains a ~40 residue long, stretched linker between the outer shell and the toxin translocation channel [1], which contracts upon the pH-triggered shell opening of TcA and drives membrane permeation [2]. However, the exact sequence and the kinetics of the prepore-to-pore transition are unknown. Here we present DEER and cw EPR kinetics, corroborated by ODNP data, on two crucial steps of pore formation: shell opening and linker contraction of TcA. Both steps are triggered by basic pH and proceed with half-lives in the 10 hours range, with no indications of enrichment of an open, non-contracted intermediate. Moreover, we found that mutations in the TcA subunit, which prevent membrane insertion, caused an acceleration of pore formation by at least three orders of magnitudes and modified the pH-dependency of the reaction such that pores are also formed at acidic pH values in vitro. A cryo-EM structure of the membrane-integration-deficient TcA variant shows a slight opening of the outer shell, which indicates that a structural rearrangement that might be caused by receptor binding in vivo, has to precede the prepore-to-pore transition to activate Tc.

  1. D. Meusch, C. Gatsogiannis and R. G. Efremov et al., Nature, 2014, 508, 61-65
  2. C. Gatsogiannis et al., Nat Struct Mol Biol, 2016, 10, 884-890

Primary authors

Dr Svetlana Kucher (Ruhr University Bochum) Dr Daniel Roderer (Max Planck Institute of Molecular Physiology) Dr Tufa Assafa (Ruhr University Bochum) Prof. Stefan Raunser (Max Planck Institute of Molecular Physiology) Prof. Enrica Bordignon (Ruhr University Bochum)

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