Structural dynamics of receptor recognition and pH-induced dissociation of full-length Clostridioides difficile Toxin B.

Clostridioides difficile secretes Toxin B (TcdB) as one of its major virulence factors, which binds to intestinal epithelial and subepithelial receptors, including frizzled proteins and chondroitin sulfate proteoglycan 4 (CSPG4). Here, we present cryo-EM structures of full-length TcdB in complex with the CSPG4 domain 1 fragment (D1401-560) at cytosolic pH and the cysteine-rich domain of frizzled-2 (CRD2) at both cytosolic and acidic pHs. CSPG4 specifically binds to the autoprocessing and delivery domains of TcdB via networks of salt bridges, hydrophobic and aromatic/proline interactions, which are disrupted upon acidification eventually leading to CSPG4 drastically dissociating from TcdB. In contrast, FZD2 moderately dissociates from TcdB under acidic pH, most likely due to its partial unfolding. These results reveal structural dynamics of TcdB during its preentry step upon endosomal acidification, which provide a basis for developing therapeutics against C. difficile infections.

Efficient cell delivery mediated by lipid-specific endosomal escape of supercharged branched peptides.

Various densely charged polycationic species, whether of biological or synthetic origin, can penetrate human cells, albeit with variable efficiencies. The molecular underpinnings involved in such transport remain unclear. Herein, we assemble 1, 2 or 3 copies of the HIV peptide TAT on a synthetic scaffold to generate branched cell-permeable prototypes with increasing charge density. We establish that increasing TAT copies dramatically increases the cell penetration efficiency of the peptides while simultaneously enabling the efficient cytosolic delivery of macromolecular cargos. Cellular entry involves the leaky fusion of late endosomal membranes enriched with the anionic lipid BMP. Derivatives with multiple TAT branches induce the leakage of BMP-containing lipid bilayers, liposomal flocculation, fusion and an increase in lamellarity. In contrast, while the monomeric counterpart 1TAT binds to the same extent and causes liposomal flocculation, 1TAT does not cause leakage, induce fusion or a significant increase in lamellarity. Overall, these results indicate that an increase in charge density of these branched structures leads to the emergence of lipid specific membrane-disrupting and cell-penetrating activities.

KETCH1 imports HYL1 to nucleus for miRNA biogenesis in Arabidopsis.

MicroRNA (miRNA) is processed from primary transcripts with hairpin structures (pri-miRNAs) by microprocessors in the nucleus. How cytoplasmic-borne microprocessor components are transported into the nucleus to fulfill their functions remains poorly understood. Here, we report KETCH1 (karyopherin enabling the transport of the cytoplasmic HYL1) as a partner of hyponastic leaves 1 (HYL1) protein, a core component of microprocessor in Arabidopsis and functional counterpart of DGCR8/Pasha in animals. Null mutation of ketch1 is embryonic-lethal, whereas knockdown mutation of ketch1 caused morphological defects, reminiscent of mutants in the miRNA pathway. ketch1 knockdown mutation also substantially reduced miRNA accumulation, but did not alter nuclear-cytoplasmic shuttling of miRNAs. Rather, the mutation significantly reduced nuclear portion of HYL1 protein and correspondingly compromised the pri-miRNA processing in the nucleus. We propose that KETCH1 transports HYL1 from the cytoplasm to the nucleus to constitute functional microprocessor in Arabidopsis This study provides insight into the largely unknown nuclear-cytoplasmic trafficking process of miRNA biogenesis components through eukaryotes.