Unveiling the pH-Dependent Dynamics of the Prepore-to-Pore Transition of a Tc Toxin

Abstract

Tc toxins are 1.7 MDa protein complexes that are found in insect- and human-pathogenic bacteria. 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. After endocytosis, Tc penetrates the membrane of the host's cells and translocates a deadly enzyme into the cytosol. However, the kinetics of the prepore-to-pore transition are unknown. Here we present continuous wave (cw) EPR and Double Electron-Electron Resonance (DEER) kinetics, on two crucial steps of pore formation: shell opening and linker contraction of TcA. Both steps are triggered by basic pH and proceed slowly 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 new 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, must precede the prepore-to-pore transition to effectively activate Tc.