Oncostatin M receptor beta (OSMRβ) is an essential signal-transducing subunit in the IL-31/IL-31 receptor alpha (IL-31RA)/OSMRβ complex and is implicated in inflammatory and pruritic diseases. We previously developed a high-affinity monoclonal antibody (2O2) that targets the proximal extracellular domain (amino acids 619-734) of canine OSMRβ (cOSMRβ) and shows no cross-reactivity with human OSMRβ (hOSMRβ). Here, we elucidate its functional mechanism of action and determine the co-crystal structure of the 2O2 Fab fragment in complex with this domain at a resolution of 2.76 Å. Consistent with the epitope location far away from the cytokine-binding domain (CBD) of cOSMRβ, 2O2 does not compete with canine IL-31 (cIL-31) for cOSMRβ binding. However, the in vitro functional assay demonstrated that 2O2 inhibits cIL-31-induced phosphorylation of STAT5 in DH-82 cells. Mechanistically, this inhibition is achieved through antibody-induced internalization of cOSMRβ from the cell surface. Structural analysis reveals that 2O2 binds at an oblique angle relative to the membrane, creating steric hindrance that induces localized lipid bilayer invagination. The bivalent IgG exhibits ∼34-fold higher potency than its monovalent Fab, reflecting not only avidity but also mechanical consequences of dual receptor engagement: crosslinking two receptor complexes exerts a stretching force that amplifies membrane invagination and promotes efficient endocytic uptake. This mechanism enables clearance of the entire signaling complex, including the cytokine, from the cell surface-a functional advantage over conventional cytokine-blocking antibodies. These findings establish 2O2 as a novel canine therapeutic candidate with a distinct internalization-driven mechanism of action.

Cholesterol (CHL) serves as a building block for membrane biogenesis and a precursor to oxysterols, steroid hormones, bile acids, and vitamin D. The lysosome serves as a major sorting station for low-density lipoproteins (LDLs), which carry dietary CHL, and it is also the cellular site where the master growth regulator, the protein kinase mechanistic Target of Rapamycin Complex 1 (mTORC1), is activated. Recently, the lysosomal transmembrane protein GPR155 was reported to signals CHL sufficiency to mTORC1 through sequestration of the GTPase-activating protein towards the Rags 1 (GATOR1). Although the recently reported structures of GPR155 have revealed the CHL binding site, how the signal is transduced from the CHL binding site to the soluble parts of GPR155 and GATOR1 remains unknown. Here, with our three cryo-EM structures of GPR155 captured in different conformations in complex with CHL, complemented by long-time scale molecular dynamics simulations, the dynamic rearrangement of different domains was observed. CHL binding induces a widening of the crevice between the transporter and GPCR domains. The extending helix preceding transmembrane helix (TM) 16, which was unresolved in other structures, acts as a linkage lever that transmits the rotation of the GPCR domain to the soluble parts of GPR155 in response to CHL binding. This work not only answers the question of how CHL is sensed by GPR155, but also addresses a more profound question: how the signal perceived by the TMs regions is transduced to the LED and DEP domains.

Interleukin-31 (IL-31) signals through the IL-31 receptor alpha (IL-31RA) and oncostatin M receptor beta (OSMRβ) heterodimer, mediating pruritus, dermatitis, inflammatory responses, neuroimmune interactions, and certain cancers. Here, we present the crystal structure of canine IL-31 (cIL-31) in complex with a neutralizing caninized monoclonal antibody (2D10-2). This antibody competitively inhibited cIL-31 binding to canine OSMRβ (cOSMRβ) but not to canine IL-31RA (cIL-31RA). Moreover, it effectively blocked cIL-31-induced STAT5 phosphorylation in vitro and alleviated cIL-31-induced pruritus in beagle dogs. Structural analysis identified key antibody-binding residues in α-helical A, α-helical D, and the AB loop of cIL-31. Systematic mutagenesis based on the complex structure further defined the conformational epitopes of cIL-31 recognized by cOSMRβ. In summary, this study reports the IL-31 structure, revealing a four-α-helical bundle cytokine, and elucidates 2D10-2's neutralizing mechanism by targeting the cIL-31-cOSMRβ interaction. These findings advance our understanding of IL-31 and offer insights for developing IL-31-targeted therapeutics.

EfpA, the first major facilitator superfamily (MFS) protein identified inMycobacterium tuberculosis(Mtb), is an essential efflux pump implicated in resistance to multiple drugs. EfpA-inhibitors have been developed to kill drug-tolerant Mtb. However, the biological function of EfpA has not yet been elucidated. Here, we present the cryo-EM structures of EfpA complexed with lipids or the inhibitor BRD-8000.3 at resolutions of 2.9 Å and 3.4 Å, respectively. Unexpectedly, EfpA forms an antiparallel dimer. Functional studies reveal that EfpA is a lipid transporter and BRD-8000.3 inhibits its lipid transport activity. Intriguingly, the mutation V319F, known to confer resistance to BRD-8000.3, alters the expression level and oligomeric state of EfpA. Based on our results and the observation of other antiparallel dimers in the MFS family, we propose an antiparallel-function model of EfpA. Collectively, our work provides structural and functional insights into EfpA’s role in lipid transport and drug resistance, which would accelerate the development of antibiotics against this promising drug target.

Interleukin-31 (IL-31), belonging to the IL-6 cytokine family, is involved in skin inflammation and pruritus, as well as some tumors' progression. Here, we reported the expression and purification of recombinant human IL-31 (rhIL-31) using a prokaryotic system. This recombinant protein was expressed in the form of inclusion bodies, refolded and purified by size-exclusion chromatography. Circular dichroism analysis revealed that the secondary structure of rhIL-31 was mainly composed of alpha-helix, which is in consistence with the 3D model structure built by AlphaFold server. In vitro studies showed that rhIL-31 exhibited a good binding ability to the recombinant hIL-31 receptor alpha fused with human Fc fragment (rhIL-31RA-hFc) with EC50 value of 16.36 µg/mL in ELISA assay. Meanwhile, flow cytometry demonstrated that rhIL-31 was able to bind to hIL-31RA or hOSMRβ expressed on the cell surface, independently. Furthermore, rhIL-31 could induce the phosphorylation of STAT3 in A549 cells. In conclusion, the prepared rhIL-31 in this study possesses the binding ability to its receptors, and can activate the signal pathway of JAK/STAT. Thus, it can be applied in further studies, including investigation of hIL-31-related diseases, structural analysis, and development of therapeutic drugs, and monoclonal antibodies targeting hIL-31.

Interleukin-31 (IL-31) is a pro-inflammatory cytokine involved in skin inflammation and tumor progression. The IL-31 signaling cascade is initiated by its binding to two receptors, IL-31 receptor alpha (IL-31RA) and oncostatin M receptor subunit beta (OSMRβ). The previous study suggested that human IL-31 (hIL-31) directly interacts with IL-31RA and OSMRβ, independently, but the binding ability of hIL-31 to IL-31RA is stronger than to OSMRβ. In different to its human ortholog, feline IL-31 (fIL-31) has a higher binding affinity for feline OSMRβ. However, the binding pattern of canine IL-31 to its receptors remains to be elucidated. In this study, we purified the recombinant canine IL-31 (rcIL-31) protein and revealed its secondary structure to be mainly composed of alpha-helices. Moreover, in vitro studies show that rcIL-31 has the ability to induce the phosphorylation of signal transducer activator of transcription 3 (STAT3) and STAT5 in DH-82 cells. In the following, the binding efficacies of bioactive rcIL-31 for its individual receptor components have been measured using a flow cytometry assay. The result demonstrates that correctly refolded rcIL-31 binds independently with cIL-31RA and cOSMRβ which were expressed on the cell surface. Of note, rcIL-31 has a greater than tenfold higher affinity to OSMRβ than to IL-31RA. Additionally, we demonstrated that D1–D4, especially D4 of cOSMRβ, is crucial for its binding to cIL-31. Furthermore, this study proved that rcIL-31 has a high binding affinity to the soluble cOSMRβ with a KD value of 3.59 × 10–8 M. The results presented in the current study will have a significant implication in the development of drugs or antibodies against diseases induced by cIL-31 signaling.

Oncostatin M receptor beta (OSMRβ) mediates signaling of Oncostatin M (OSM) and interleukine-31 (IL-31), two key cytokines involved in many important biological processes including inflammation and cancer progression. More importantly, OSMRβ might be a potential biomarker and therapeutic target for some diseases, such as inflammatory bowel disease, pruritus and ovarian cancer. In this study, soluble recombinant canine OSMRβ (cOSMRβ) was experimentally expressed as a native antigen to develop an effective cOSMRβ-specific monoclonal antibody (mAb), 2O2, using hybridoma technology. It was demonstrated that 2O2 is able to detect OSMRβ expressed on cell surface using immunofluorescence assay (IFA) and flow cytometry (FACS). This mAb exhibits very high binding affinity to cOSMRβ with the KD and half-maximal effective concentration (EC50) values of 2.49 nM and 96.96 ng/ml, respectively. Meanwhile, it didn't show any cross-relativities with feline OSMRβ (fOSMRβ) and human OSMRβ (hOSMRβ). Moreover, we determined the binding epitope of 2O2, which localizes in the domain VI (DVI, amino acids 623-734) of cOSMRβ. In conclusion, this novel mAb, 2O2, can be used in immunoassays, including IFA, FACS and enzyme-linked immunosorbent assay (ELISA) to facilitate studies in dogs.

The isolation of human monoclonal antibodies with broadly neutralizing breadth can provide a promising countermeasure for influenza A viruses infection. Most broadly neutralizing antibodies against influenza A viruses bind to the conserved stem region or the receptor-binding cavity of hemagglutinin and the interaction is dominated by the heavy chain. The light chain, however, contributes few or no direct contacts to the antigen. Here we report an H3-clade neutralizing human monoclonal antibody, AF4H1K1, which recognizes the hemagglutinin glycoproteins of all group 2 influenza A viruses. This human monoclonal antibody has been obtained through the screening by pairing different heavy and light chains from an H7N9-infected patient based on the next-generation sequencing technology. Further structural studies revealed that light chains modulate the neutralizing spectrum by affecting the local conformation of heavy chains, instead of direct interaction with the antigen. These findings provide important clues to understand the molecular basis of light chains in antigen recognition and to explore the strategies in particular of the use of light chain modification to develop broadly protective monoclonal antibodies against influenza A viruses and other emerging viruses.

Yellow fever virus (YFV), a deadly human pathogen, is the prototype of the genus Flavivirus. Recently, YFV re-emerged in Africa and Brazil, leading to hundreds of deaths, with some cases imported to China. Prophylactic or therapeutic countermeasures are urgently needed. Previously, several human monoclonal antibodies against YFV were screened out by phage display. Here, we find that one of them, 5A, exhibits high neutralizing potency and good protection. Crystallographic analysis of the YFV envelope (E) protein in its pre- and post-fusion states shows conformations similar to those observed in other E proteins of flaviviruses. Furthermore, the structures of 5A in complex with the E protein in both states are resolved, revealing an invariant recognition site. Structural analysis and functional data suggest that 5A has high neutralization potency because it interferes with virus entry by preventing both virus attachment and fusion. These findings will be instrumental for immunogen or inhibitor design.

Well structured: As a new triose phosphate isomerase (TIM) barrel-fold prenyl transferase, PcrB catalyzes the production of heptaprenylglyceryl phosphate from heptaprenyl diphosphate and glycerol-1-phosphate. Crystal structures of PcrB from Bacillus subtilis and Staphylococcus aureus in complex with ligands were solved, and together with site-directed mutagenesis and bioinformatics analyses, clearly reveal the catalytic mechanism of the enzyme.

Xylan-1,4-β-xylosidase (β-xylosidase) hydrolyses xylo-oligomers at their non-reducing ends into individual xylose units. Recently, XylC, a β-xylosidase from Thermoanaerobacterium saccharolyticum JW/SL-YS485, was found to be structurally different from corresponding glycosyl hydrolases in the CAZy database (http://www.cazy.org/), and was subsequently classified as the first member of a novel family of glycoside hydrolases (GH120). In the present paper, we report three crystal structures of XylC in complex with Tris, xylobiose and xylose at 1.48-2.05 Å (1 Å=0.1 nm) resolution. XylC assembles into a tetramer, and each monomer comprises two distinct domains. The core domain is a right-handed parallel β-helix (residues 1-75 and 201-638) and the flanking region (residues 76-200) folds into a β-sandwich domain. The enzyme contains an open carbohydrate-binding cleft, allowing accommodation of longer xylo-oligosaccharides. On the basis of the crystal structures and in agreement with previous kinetic data, we propose that XylC cleaves the glycosidic bond by the retaining mechanism using two acidic residues Asp382 (nucleophile) and Glu405 (general acid/base). In addition to the active site, nine other xylose-binding sites were consistently observed in each of the four monomers, providing a possible reason for the high tolerance of product inhibition.