Preparation of Clostridium perfringens binary iota-toxin pore complex for structural analysis using cryo-EM

Yamada, Tomohito; Tsuge, Hideaki

doi:10.1016/bs.mie.2021.01.032

Cited by 2 publications

(2 citation statements)

References 36 publications

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“…These dynamic features of D4II are likely important in binding the host cell LSR. In our previous study, we confirmed that the receptor-binding domain is also involved in determining oligomerization efficiency, using a D4II-deficient mutant Ib 36 .…”

Section: Discussionsupporting

confidence: 70%

Cryo-EM structures of the translocational binary toxin complex CDTa-bound CDTb-pore from Clostridioides difficile

et al. 2022

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Some bacteria express a binary toxin translocation system, consisting of an enzymatic subunit and translocation pore, that delivers enzymes into host cells through endocytosis. The most clinically important bacterium with such a system is Clostridioides difficile (formerly Clostridium). The CDTa and CDTb proteins from its system represent important therapeutic targets. CDTb has been proposed to be a di-heptamer, but its physiological heptameric structure has not yet been reported. Here, we report the cryo-EM structure of CDTa bound to the CDTb-pore, which reveals that CDTa binding induces partial unfolding and tilting of the first CDTa α-helix. In the CDTb-pore, an NSS-loop exists in ‘in’ and ‘out’ conformations, suggesting its involvement in substrate translocation. Finally, 3D variability analysis revealed CDTa movements from a folded to an unfolded state. These dynamic structural information provide insights into drug design against hypervirulent C. difficile strains.

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Section: Discussionsupporting

confidence: 70%

Cryo-EM structures of the translocational binary toxin complex CDTa-bound CDTb-pore from Clostridioides difficile

et al. 2022

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“…To prepare the heptameric CDTb-pore, we used a solubilization protocol similar to that used for preparing the iota toxin pore with lauryl maltose neopentyl glycol (LMNG), which mimics the buried liposome environment 31,37 . This protocol differed from the CDTb-pore preparation protocol 34 .…”

Section: Preparation Of the Cdtb Heptameric Porementioning

confidence: 99%

Cryo-EM structures of the translocational binary toxin complex CDTa-bound CDTb-pore from Clostridioides difficile

Kawamoto

Yamada

Yoshida

et al. 2021

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Besides two large cytotoxins (TcdA and TcdB), certain Clostridioides difficile strains also produce a binary toxin, called C. difficile toxin (CDT) composed of an enzymatic subunit involved in actin ADP-ribosylation (CDTa) and translocation pore (CDTb) that delivers CDTa into host cells through receptor-mediated endocytosis. CDTb is proposed to be a di-heptamer, but its physiological heptameric structure has not been reported to date. Here, we report the CDTa-bound CDTb-pore (heptamer) as a physiological complexes using cryo-EM. The high-resolution structure of the CDTa-bound CDTb-pore at 2.56-Å resolution revealed that CDTa binding to CDTb-pore induces partial unfolding and tilting of the first CDTa a-helix, and the translocation. In the CDTb-pore, the NSS-loop exists in “in” and “out” conformations, suggesting their involvement in substrate translocation through formation of weak, non-specific interactions. This structural information provides insights into drug design against hypervirulent C. difficile strains.

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Preparation of Clostridium perfringens binary iota-toxin pore complex for structural analysis using cryo-EM

Cited by 2 publications

References 36 publications

Cryo-EM structures of the translocational binary toxin complex CDTa-bound CDTb-pore from Clostridioides difficile

Cryo-EM structures of the translocational binary toxin complex CDTa-bound CDTb-pore from Clostridioides difficile

Cryo-EM structures of the translocational binary toxin complex CDTa-bound CDTb-pore from Clostridioides difficile

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