Structure and mechanism of haem-dependent nitrogen–nitrogen bond formation in piperazate synthase
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TCR catch bonds nonlinearly control CD8 cooperation to shape T cell specificity
Naturally evolved T-cell receptors (TCRs) exhibit remarkably high specificity in discriminating non-self antigens from self-antigens under dynamic biomechanical modulation. In contrast, engineered high-affinity TCRs often lose this specificity, leading to cross-reactivity with self-antigens and off-target toxicity. The underlying mechanism for this difference remains unclear. Our study reveals that natural TCRs exploit mechanical force to form optimal catch bonds with their cognate antigens. This process relies on a mechanically flexible TCR–pMHC binding interface, which enables force-enhanced CD8 coreceptor binding to MHC-α1α2 domains through sequential conformational changes induced by force in both the MHC and CD8. Conversely, engineered high-affinity TCRs create rigid, tightly bound interfaces with cognate pMHCs of their parental TCRs. This rigidity prevents the force-induced conformational changes necessary for optimal catch-bond formation. Paradoxically, these high-affinity TCRs can form moderate catch bonds with non-stimulatory pMHCs of their parental TCRs, leading to off-target cross-reactivity and reduced specificity. We have also developed comprehensive force-dependent TCR–pMHC kinetics-function maps capable of distinguishing functional and non-functional TCR–pMHC pairs and identifying toxic, cross-reactive TCRs. These findings elucidate the mechano-chemical basis of the specificity of natural TCRs and highlight the critical role of CD8 in targeting cognate antigens. This work provides valuable insights for engineering TCRs with enhanced specificity and potency against non-self antigens, particularly for applications in cancer immunotherapy and infectious disease treatment, while minimizing the risk of self-antigen cross-reactivity.
Immediate and delayed micro shear bond strength evaluation of two glass ionomer cements to composite resin by using different bonding techniques—an in vitro study
Evaluating immediate and delayed micro shear bond strength (µSBS) between composite resin and glass ionomer cements using different adhesive systems and mechanical surface treatment.
Crystal structures of monomeric BsmI restriction endonuclease reveal coordinated sequential cleavage of two DNA strands
BsmI, a thermophilic Type IIS restriction endonuclease from Bacillus stearothermophilus, presents a unique structural composition, housing two distinct active sites within a single monomer. Recognition of the non-symmetrical 5’-GAATGC-3’ sequence enables precise cleavage of the top and bottom DNA strands. Synthetic biology interventions have led to the transformation of BsmI into Nb.BsmI, a nicking endonuclease. Here we introduce Nt*.BsmI, tailored for top-strand cleavage, which is inactive on standard double-stranded DNA, but active on bottom-strand nicked DNA, suggesting a sequential cleavage mechanism. Crystallographic structures of pre- and post-reactive complexes with cognate DNA show one major conformational change, a retractable loop possibly governing sequential active site accessibility. The x-ray structures reveal the position of the divalent metal ions in the active sites and the DNA:protein interactions, while the models predicted by Alphafold3 are incorrect. This comprehensive structural and functional study lays a foundation for rational enzyme redesign and potential applications in biotechnology.
Nitrogen transfer and cross-feeding between Azotobacter chroococcum and Paracoccus aminovorans promotes pyrene degradation
Nitrogen is a limiting nutrient for degraders function in hydrocarbon-contaminated environments. Biological nitrogen fixation by diazotrophs is a natural solution for supplying bioavailable nitrogen. Here, we determined whether the diazotroph Azotobacter chroococcum HN can provide nitrogen to the polycyclic aromatic hydrocarbon-degrading bacterium Paracoccus aminovorans HPD-2 and further explored the synergistic interactions that facilitate pyrene degradation in nitrogen-deprived environments. We found that A. chroococcum HN and P. aminovorans HPD-2 grew and degraded pyrene more quickly in co-culture than in monoculture. Surface-enhanced Raman spectroscopy combined with 15N stable isotope probing (SERS − 15N SIP) demonstrated that A. chroococcum HN provided nitrogen to P. aminovorans HPD-2. Metabolite analysis and feeding experiments confirmed that cross-feeding occurred between A. chroococcum HN and P. aminovorans HPD-2 during pyrene degradation. Transcriptomic and metabolomic analyses further revealed that co-culture significantly upregulated key pathways such as nitrogen fixation, aromatic compound degradation, protein export, and the TCA cycle in A. chroococcum HN and quorum sensing, aromatic compound degradation and ABC transporters in P. aminovorans HPD-2. Phenotypic and fluorescence in situ hybridization (FISH) assays demonstrated that A. chroococcum HN produced large amounts of biofilm and was located at the bottom of the biofilm in co-culture, whereas P. aminovorans HPD-2 attached to the surface layer and formed a bridge-like structure with A. chroococcum HN. This study demonstrates that distinct syntrophic interactions occur between A. chroococcum HN and P. aminovorans HPD-2 and provides support for their combined use in organic pollutant degradation in nitrogen-deprived environments.
Temperature-driven nitrogen mixotrophy shapes marine cyanobacteria Prochlorococcus and Synechococcus latitudinal distribution pattern
Temperature, photosynthetically active radiation, and nutrient availability are pivotal in determining the global distribution of marine cyanobacteria. This study demonstrates that the distinct latitudinal niches of Synechococcus and Prochlorococcus are modulated by their temperature-dependent, mixotrophic nitrogen utilization strategies. Comparative genomic analysis reveals that Prochlorococcus ecotypes differ in their nitrogen transporter repertoire, with low-light ecotypes harboring ~12 transporters, while high-light ecotypes possess ~5 transporters. Conversely, Synechococcus displays greater genomic flexibility, with 17 nitrogen transporters. Reanalysis of Tara Oceans metatranscriptomic data identifies ~15 °C as a key thermal threshold for cyanobacterial distribution. In warmer regions (15-30°C, 35˚S-40˚N), Prochlorococcus optimizes nitrogen transporter functions across their ecotypes, whereas Synechococcus, leveraging its broad nitrogen transporters, exhibits functional plasticity, utilizing inorganic nitrogen in warmer waters and organic nitrogen in cooler waters ranging from -2°C to 15 °C beyond 35˚S/40˚N. Our findings underscore the critical role of temperature-driven mixotrophic nitrogen utilization in shaping the biogeographical patterns of marine cyanobacteria.
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