Zihe Rao

Mycobacterial-derived lipid-linked galactan (LLG) is an essential component of the Mycobacterial tuberculosis (Mtb) cell wall core arabinogalactan (AG), a well-established virulence factor and target of anti-tubercular drugs. Cross-membrane translocation of LLG is energetically unfavorable and is therefore catalyzed by WzmWzt, a prominent member of the polysaccharide ABC transporter sub-family. Here we present cryo-EM structures of WzmWzt_Mtb to uncover critical steps of Mtb LLG translocation. Notably, densities suggestive of the mycobacterial-derived LLG substrates were observed in the structure of WzmWzt_Mtb, indicating that the substrate may bind through two Wzm and two Wzt subunits. This proposed LLG binding is fulfilled by a fully upward extension of the Wzt gate helix, which serves as a critical step to prime Wzt for dimerization. The ATP-bound structure revealed a remarkable downward movement of the gate helix, together with the Wzt dimerization. The third, DDM/ATP-bound structure captured WztWzm_Mtb in an intermediate state with two DDM molecules in the Wzm central cavity. Using two conditional mycobacterial mutants for functional analyses, we have identified residues on the Wzm N-terminal interface helix (IF_N) and on the Wzt helical domain are essential for LLG recognition and translocation in vivo. These data provide a clear structural basis for the development of Mtb cell wall targeted therapeutics for global tuberculosis treatment.

MFSD6 is a newly identified receptor that mediates the invasion of respiratory cells by enterovirus D68 (EV-D68), a non-polio enterovirus that causes severe respiratory disease and poliomyelitis-like illness in children. Here, we report near-atomic-resolution cryo-electron microscopy (cryo-EM) structures of historical and contemporary AFM-associated EV-D68 strains, together with their complexes bound to the third extracellular loop of MFSD6 (MFSD6-L3). These structures uncover a previously unrecognized “binary gating switch” mechanism of virus-receptor engagement that differs from the reported model. In this mechanism, the N200-V208 segment of MFSD6, carrying a glycosylated Asn207, inserts into one capsomer, whereas the sialyl-Gal-terminated glycan of MFSD6 engages an adjacent capsomer. Neu5Ac binding induces conformational rearrangements that expel the pocket factor, destabilize the virion, and prime infection. Functional analyses further define the contributions of the receptor-contacting residues and glycans to viral attachment and entry. Together, our findings refine the molecular basis of EV-D68 recognition of MFSD6 and reveal a glycan-mediated dual-lock mechanism that may enhance receptor specificity, prevent premature uncoating, and ensure productive infection only upon engagement of the correct host receptor. These results provide broader insight into enterovirus tropism and establish a framework for structure-guided antiviral design.

Integrated virtual and biological screening guided the design and synthesis of 50 novel pyrimido[1,2- a ]imidazole derivatives, leading to the discovery of potent Pks13-TE inhibitor 34 (IC ₅₀ = 0.23 μM).

Type IIA topoisomerases modulate DNA topology by coordinating the cleavage of gate-segment DNA and the passage of transport-segment DNA—a mechanism conserved across species and essential for diverse cellular processes. While gate-segment interactions have been extensively studied, direct structural evidence of transport-segment capture has remained elusive, limiting our understanding of the full catalytic cycle. Here, we present a cryo–electron microscopy structure of the T4 bacteriophage topoisomerase II with a transport-segment DNA bound directly to its central domain. The structure reveals conformational rearrangements in the central domain that accommodate the transport-segment DNA, suggesting an alternative sequence of events in which the enzyme sliding along the loosely bound religated gate segment may precede transport-segment passage through the coiled-coil gate. Supported by mutational and biochemical assays, our findings provide previously unidentified mechanistic insights and open potential avenues for the development of next-generation type IIA topoisomerase inhibitors.

Influenza virus ribonucleoprotein complexes (RNPs), composed of the polymerase complex (FluPol), nucleoprotein (NP), and RNA, are essential for replication and transcription. We report atomic-resolution cryo-EM structures of mini-vRNPs in two states: FluPol located inside (State-In) or at the outer rim (State-Out) of the NP–RNA ring. In both states, the 5′ and 3′ termini of vRNA are bound to FluPol as previously reported. One NP (NP-0) contacts PA/PB1 of FluPol and binds the distal double-stranded vRNA promoter, with its D72–K90 loop inserting into the RNA fork; separated strands occupy NP-0 RNA-binding grooves. Grooves from other NPs form a continuous RNA-protective path, consistent with negative-strand RNA virus mechanisms. In State-In, interfaces for FluPol dimerization or Pol II interaction are blocked, but fully exposed in State-Out. These structures reveal detailed FluPol–NP–RNA coupling and suggest a conformational shift in RNPs during the viral life cycle.

The pre-dimerization of endosome-localized RNA sensor Toll-like receptor 3 (TLR3) is required for its innate recognition, yet how TLR3 pre-dimers are formed and precisely primed for innate activation remains unclear. Here, we demonstrate that endosome-localized self RNA Rmrp directly binds to TLR3 and induces TLR3 dimerization in the early endosome but does not interact with endosome-localized TLR7, TLR8, TLR9 or cytoplasmic RNA sensor RIG-I under homeostatic conditions. Cryo-EM structure of Rmrp–TLR3 complex reveals a novel lapped conformation of TLR3 dimer engaged by Rmrp, which is distinct from the activation mechanism by dsRNA and the specific structural feature at the 3’-end of Rmrp is critical for its functional interaction with TLR3. Furthermore, K42 residue of TLR3 is essential for binding to Rmrp and subsequent dimerization. Rmrp dissociates from TLR3 following endosomal acidification, generating a matured TLR3 dimer which is primed for innate recognition and activation. Myeloid-cell deficiency of Rmrp reduces TLR3 dimerization and attenuates TLR3-mediated antiviral responses against influenza A both in vitro and in vivo. These findings elucidate the structural mode of self RNA Rmrp-primed TLR3 dimerization and ready for efficient innate recognition on endosomal membrane, extending our knowledge of how membrane-associated TLRs pre-dimerize and suggesting a new function of subcellular localized self RNAs in empowering innate activation.