biostudies-literatureChen BNational Institute of Allergy and Infectious DiseasesNIAID NIH HHSNIMH NIH HHSNational Institute of Mental HealthNational Institute of General Medical SciencesNIGMS NIH HHSe202201383https://www.ebi.ac.uk/biostudies/studies/S-EPMC8924006biostudies-literatureUnknown5(6)<i>Clostridioides difficile</i> toxin A and B (TcdA and TcdB) are two major virulence factors responsible for diseases associated with <i>C. difficile</i> infection (CDI). Here, we report the 3.18-Å resolution crystal structure of a TcdA fragment (residues L843-T2481), which advances our understanding of the complete structure of TcdA holotoxin. Our structural analysis, together with complementary single molecule FRET and limited proteolysis studies, reveal that TcdA adopts a dynamic structure and its CROPs domain can sample a spectrum of open and closed conformations in a pH-dependent manner. Furthermore, a small globular subdomain (SGS) and the CROPs protect the pore-forming region of TcdA in the closed state at neutral pH, which could contribute to modulating the pH-dependent pore formaLife science allianceStructure and conformational dynamics of &lt;i&gt;Clostridioides difficile&lt;/i&gt; toxin A.PMC8924006R01 AI158503P30 GM124165R01AI125704, R01AI158503, R21AI156092, and R21AI163178R01GM074830 and R01GM130144R01AI139087R01AI132387R21 AI163178R01 GM130144R01 AI132387R01 MH081923R21 AI156092R01 GM151334R01MH081923R01 AI139087R01 AI125704R01 GM074830Basak SChen PYu CJin RBowen MEPerry KDong MTian SHuang LZhang CChen BfalseStructure and conformational dynamics of &lt;i&gt;Clostridioides difficile&lt;/i&gt; toxin A.<i>Clostridioides difficile</i> toxin A and B (TcdA and TcdB) are two major virulence factors responsible for diseases associated with <i>C. difficile</i> infection (CDI). Here, we report the 3.18-Å resolution crystal structure of a TcdA fragment (residues L843-T2481), which advances our understanding of the complete structure of TcdA holotoxin. Our structural analysis, together with complementary single molecule FRET and limited proteolysis studies, reveal that TcdA adopts a dynamic structure and its CROPs domain can sample a spectrum of open and closed conformations in a pH-dependent manner. Furthermore, a small globular subdomain (SGS) and the CROPs protect the pore-forming region of TcdA in the closed state at neutral pH, which could contribute to modulating the pH-dependent pore forma2022-01-01T00:00:00Z2022 Jun2026-05-27T22:24:49.678Z2025-02-19T01:11:53.348ZS-EPMC89240063529253810.26508/lsa.202201383