Structural basis for selective modification of Rho and Ras GTPases by Clostridioides difficile toxin B

Abstract

Diverse TcdB variants use a flexible structural platform to tune their specificity toward different GTPase substrates.

Structure of the glucosyltransferase domain of TcdA in complex with RhoA provides insights into substrate recognition

“…1 B,C, Fig. S1 A,B), and the overall architecture of the GTD TcdA –RhoA complex is very similar to the GTD TcdB –Cdc42/R-Ras complex (PDB code: 7S0Y, 7S0Z) 20 . The overall structure of RhoA-bound GTD TcdA ·UDP-glucose is highly similar to the previously reported GTD TcdA ·UDP-glucose (PDB code: 3SRZ) 25 and GTD TcdA ·U2F (a non-hydrolysable UDP-glucose homolog, PDB code: 5UQL) 32 complexes, with a root mean square deviation (RMSD) of ~ 0.525/0.504 Å over 408/407 residues, respectively (Fig.…”

“…To overcome this obstacle, we have developed a strategy to “freeze” the transient interactions between the GTD and its substrates by protein engineering. As reported in one of our recent studies, we designed a fusion protein where Cdc42 or R-Ras was covalently linked to the N-terminus of the GTD of TcdB via a flexible peptide linker 20 . The peptide linker does not restrict interactions between GTD TcdB and its substrates, while the covalent linking increases their local concentrations and thus strengthens the protein–protein interactions 29 , 30 .…”

“…Besides Rho proteins, GTD TcdA could target other GTPases, such as H/N/K-Ras, as minor substrates 26 – 28 . In a recent study, we demonstrated the structural mechanism by which GTD TcdB recognizes Rho and R-Ras 20 . Here we report the co-crystal structure of GTD TcdA from strain VPI10463 in complex with human RhoA in the presence of UDP-glucose, GDP, Mn 2+ and Mg 2+ .…”

“…These toxins then transport the GTD and the CPD into the cytosol 10 – 13 , where the CPD cleaves off and release the GTD in the presence of cytosolic inositol hexakisphosphate (InsP6) 14 , 15 . Once in the cytosol, the GTD inactivates the Rho/Ras-family of small guanosine triphosphatases (GTPases) via glucosylation, leading to disruption of the actin cytoskeleton in target cells and damage of the barrier function of epithelium in the intestine 16 – 20 .…”

Structure and conformational dynamics of Clostridioides difficile toxin A

“…Activated by cytosolic inositol hexakisphosphate (InsP6), the CPD cleaves off the GTD and releases it into the cytosol ( Egerer et al, 2007 ; Reineke et al, 2007 ). The GTD then glucosylates and inactivates the Rho and/or Ras families of small guanosine triphosphatases (GTPases) in host cells, resulting in depolymerization of the actin cytoskeleton, cell rounding, and ultimately cell death ( Just et al, 1995a , 1995b ; Chen et al, 2015 ; Liu et al, 2021 ).…”