Gram-negative bacteria pose a threat to global healthcare mainly because their outer membrane OM provides an intrinsic barrier to many antimicrobials Key to this barrier function is the asymmetric structure of the OM with phospholipids constituting the inner leaflet and lipopolysaccharides the outer leaflet Although the mechanism of phospholipid transport ...More |Related Solutions: Rock Imager^®

Gram-negative bacteria pose a threat to global healthcare mainly because their outer membrane (OM) provides an intrinsic barrier to many antimicrobials. Key to this barrier function is the asymmetric structure of the OM, with phospholipids constituting the inner leaflet and lipopolysaccharides the outer leaflet. Although the mechanism of phospholipid transport between the inner membrane (IM) and OM remains poorly understood, recent studies implicate TamB, YhdP, and YdbH as functionally redundant proteins mediating this process in Escherichia coli. Accordingly, collective loss of these three paralogs is lethal and any one of them is sufficient for growth. YdbH is anchored to the IM and its periplasmic repeating β-sheet groove domain interacts with the OM lipoprotein YnbE via β-strand augmentation to form an intermembrane bridge. Additionally, YnbE multimerizes, and the periplasmic protein YdbL is proposed to modulate YnbE multimerization to facilitate its stacking on the C-terminus of YdbH. Here, we demonstrate that excess YdbL specifically inhibits the function of the YdbH-YnbE complex since overexpression of ydbL causes lethality in the ΔyhdP ΔtamB double mutant but the presence of both ydbH and ynbE in trans abrogates this lethality. We resolve high-resolution structural data for YdbL and ascertain its interaction site with the YnbE C-terminal α-helix, with residues mediating this interface highly conserved and critical for YdbL function. Finally, we show that YdbL is protected from degradation by the protease DegP when complexed with YnbE. Overall, our data supports a model in which YdbL ensures proper YdbH-YnbE intermembrane bridge formation by directly interacting with YnbE. Less |Related Solutions: Rock Imager^®

Parasite-derived homologs of the cytokine macrophage migration inhibitory factor MIF function as virulent factors during parasitic infection Recent evidence suggests MIF-like products from multicellular helminthic species can be targeted to ameliorate parasite burden Here we identify a broad contingent of hypothetical MIF-like proteins from genomic helminth repositories and perform structure ...More |Related Solutions: Rock Maker^®

Parasite-derived homologs of the cytokine macrophage migration inhibitory factor (MIF) function as virulent factors during parasitic infection. Recent evidence suggests MIF-like products from multicellular helminthic species can be targeted to ameliorate parasite burden. Here, we identify a broad contingent of hypothetical MIF-like proteins from genomic helminth repositories and perform structure analysis to validate conserved homology. Further, we employ a diverse subset of MIF-specific assays to establish cross-species functionality of helminth MIF proteins, including native enzymatic activity, binding to cognate receptor CD74, direct interactions with human MIF, and signaling through chemokine receptors CXCR2 and CXCR4. We demonstrate that MIF-like proteins retain a preserved architecture but are capable of diverse physiological outcomes due to small changes in key components of the conserved MIF structure. This work simultaneously provides a mechanistic understanding of helminth virulence upon infection, while broadly examining the potential to neutralize MIF-like proteins for protection against various pathological species. Less |Related Solutions: Rock Maker^®

Interactions between plants and pathogens drive long-term co-evolution through cycles of effector diversification and immune recognition Effectors play central roles in this molecular interplay and zinc-binding folds have been identified in a subset of pathogen effectors yet the contribution of zinc coordination to effector stability and immune recognition remains unclear ...More |Related Solutions: NT8^®

Interactions between plants and pathogens drive long-term co-evolution through cycles of effector diversification and immune recognition. Effectors play central roles in this molecular interplay, and zinc-binding folds have been identified in a subset of pathogen effectors, yet the contribution of zinc coordination to effector stability and immune recognition remains unclear.

Here, we investigated the structure and recognition of the AvrSr33 effector from the wheat stem rust fungus (Puccinia graminis f. sp. tritici, Pgt). Structural and biochemical analyses show that AvrSr33 adopts a fold containing two zinc-binding sites.

Transient expression assays in Nicotiana benthamiana show that AvrSr33 directly interacts with Sr33, and comparison of recognised and non-recognised AvrSr33 variants identifies a polymorphic loop associated with recognition. This loop is positioned adjacent to one of the zinc-binding sites with its orientation constrained by zinc coordination. Reciprocal mutations of key surface residues within this region alter recognition, whereas mutation of the zinc-binding site abolishes recognition.

Our data suggest that zinc coordination in AvrSr33 provides a structurally constrained scaffold that supports the surface features associated with Sr33 recognition. These findings provide a mechanistic framework for understanding how zinc coordination contributes to effector recognition and may influence the evolutionary trajectories of pathogen effectors. Less |Related Solutions: NT8^®

Mycobacterium tuberculosis Mtb encodes a huge repertoire of toxin antitoxin TA systems many of which remain uncharacterized Here we report the crystal structures of the VapC toxin and VapBC TA complex at a resolution of and respectively We show that VapC adopts a unique open dimeric conformation and inhibits mycobacterial ...More |Related Solutions: Rock Imager^®

Mycobacterium tuberculosis (Mtb) encodes a huge repertoire of toxin–antitoxin (TA) systems, many of which remain uncharacterized. Here, we report the crystal structures of the VapC52 toxin and VapBC52 TA complex at a resolution of 2.6 and 3.2 Å, respectively. We show that VapC52 adopts a unique open dimeric conformation and inhibits mycobacterial growth by cleaving tRNA at the variable or anticodon loop region. Structure reveals that VapB52 adopts a distinct structural architecture and binds VapC52 with a 1:2 stoichiometry, respectively. Interestingly, binding of ssDNA activates VapB52 peptidase domain, resulting in auto-cleavage of VapB52 N-terminal domain which is critical for VapBC complex formation and neutralization. In addition to VapB52, co-expression of several other non-cognate VapB antitoxins abrogates the growth inhibition associated with VapC52 overexpression in Mycobacterium smegmatis (Msm) suggesting crosstalk among VapBC TA systems. Further, we demonstrate that the vapBC52 locus is dispensable for in vitro growth but essential for Mtb intracellular growth in macrophages and guinea pigs. Notably, VapC52 also cleaves mycobacteriophage D29 encoded tRNAs and confers resistance to phage infection in Msm. Taken together, we show that VapBC52 adopts a unique structural architecture, plays role in pathogenesis, and is possibly involved in mycobacterial antiphage defense mechanisms. Less |Related Solutions: Rock Imager^®

Pyrophosphate-dependent phosphofructokinases provide an alternative route through central carbon metabolism by using inorganic pyrophosphate rather than ATP to phosphorylate fructose- -phosphate These enzymes are absent from mammals but occur in several anaerobic and parasitic organisms where they may support metabolic flexibility and represent potential therapeutic vulnerabilities Here we report three ...More |Related Solutions: NT8^®

Pyrophosphate-dependent phosphofructokinases provide an alternative route through central carbon metabolism by using inorganic pyrophosphate rather than ATP to phosphorylate fructose-6-phosphate. These enzymes are absent from mammals but occur in several anaerobic and parasitic organisms, where they may support metabolic flexibility and represent potential therapeutic vulnerabilities. Here, we report three crystal structures of Trichomonas vaginalis PPi-dependent phosphofructokinase 1 (TvPPi-PFK), a cytosolic enzyme that contributes substantially to parasite glycolytic flux. The structures show that TvPPi-PFK adopts the conserved two-domain PFK fold, assembles as a tetramer, and preserves the canonical PPi/MgPPi²⁻-binding motifs required for pyrophosphate-dependent catalysis. Mass photometry confirms that TvPPi-PFK is predominantly tetrameric in solution and shows that nucleotide- and Mg²⁺/PPi-containing conditions further stabilize the tetrameric assembly. Unexpectedly, the ligand-bound structures reveal ATP, AMP, and sugar-phosphate ligands at a recurring dimer-interface site that is spatially distinct from the canonical PPi/MgPPi²⁻ catalytic donor-binding pocket. This non-canonical interface is more accessible in TvPPi-PFK than in available PPi-PFK structures, suggesting a previously unrecognized ligand-recognition feature. These findings expand the structural landscape of the PFK superfamily and provide a framework for testing how nucleotide and sugar-phosphate binding may influence TvPPi-PFK function, regulation, and future antitrichomonal drug-discovery efforts. Less |Related Solutions: NT8^®