Journal of Molecular Biology最新文献

{"title":"Evolution of Temperature Dependence of TRPM2 Channel Gating and Inactivation in Vertebrates","authors":"Adam Bartok,&nbsp;Adam V. Toth,&nbsp;László Csanády","doi":"10.1016/j.jmb.2025.169507","DOIUrl":"10.1016/j.jmb.2025.169507","url":null,"abstract":"<div><div>TRPM2 is a Ca²⁺ permeable cation channel activated by cytosolic Ca²⁺ and ADP ribose (ADPR). In contrast to invertebrate orthologs, vertebrate TRPM2 channels inactivate due to evolutionary alterations in amino acid sequence around the selectivity filter. Human TRPM2 (hsTRPM2) serves as a deep-brain temperature sensor important for body temperature regulation, and its gating is exquisitely temperature dependent. To address whether TRPM2 is temperature sensitive also in ectotherms that lack body temperature regulation, here we investigated the functional properties of zebrafish (<em>Danio rerio</em>) TRPM2 (drTRPM2) across the temperature range of 15–37 °C. The rate of ion permeation through the open pore was weakly temperature sensitive (<em>Q</em>₁₀ ∼ 1.3) as expected for a diffusion-limited process. In the presence of saturating concentrations of ligands (ADPR and Ca²⁺) drTRPM2 open probability showed no temperature dependence. Moreover, for both ADPR and Ca²⁺, the apparent affinities for channel activation were unaffected by temperature, reporting a standard enthalpy of opening near zero for drTRPM2. Inactivation of drTRPM2 is an order of magnitude slower than that of hsTRPM2. To address whether in both orthologs the same mechanism underlies inactivation, we studied its temperature sensitivity. For both drTRPM2 and hsTRPM2 inactivation rate was modestly temperature dependent (<em>Q</em>₁₀ ∼ 2.7 and ∼4.4). A triple substitution which converts the post-filter sequence of drTRPM2 into the corresponding human sequence accelerated drTRPM2 inactivation by 10–20-fold. The data suggest that temperature dependence of hsTRPM2 gating evolved in the course of vertebrate evolution, whereas inactivation was temperature dependent already when it first appeared in early vertebrates.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 24","pages":"Article 169507"},"PeriodicalIF":4.5,"publicationDate":"2025-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145462866","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Exploring the Intrinsic Antimicrobial Activity of Klebsiella Phage KP27 Endopeptidase Against Pseudomonas aeruginosa: Insights into Membrane Interactions Using Experimental and computational Approaches","authors":"Barbara Maciejewska ,&nbsp;Grzegorz Guła ,&nbsp;Angelika Bołoz ,&nbsp;Aleksander Czogalla ,&nbsp;Mateusz Kogut ,&nbsp;Jacek Czub ,&nbsp;Manish Kumar ,&nbsp;Sabina Oleksy ,&nbsp;Piotr Hinc ,&nbsp;Régis Tournebize ,&nbsp;Łukasz Berlicki ,&nbsp;Karol Ciepluch ,&nbsp;Michał Arabski ,&nbsp;Zuzanna Drulis-Kawa ,&nbsp;Daria Augustyniak","doi":"10.1016/j.jmb.2025.169491","DOIUrl":"10.1016/j.jmb.2025.169491","url":null,"abstract":"<div><div>Gram-negative pathogens are surrounded by lipopolysaccharide-containing outer membrane that is crucial in protecting them against antimicrobials and host defence proteins. Some phage endolysins, which are peptidoglycan-degrading enzymes, can overcome this barrier and kill bacteria. However studies on their structure–function relationships remain scarce. The present study verified that the muralytic activity of <em>Klebsiella</em> myovirus endopeptidase EndoKP27 is not required for <em>Pseudomonas aeruginosa</em> killing. EndoKP27 is a 14 kDa protein with moderate thermostability and a cationic N-terminus. We show that the antibacterial activity of EndoKP27 is boosted in the presence of membrane-targeting agents such as polymyxin B, cathelicidin LL-37, or human serum complement, as well as innate antimicrobials from <em>Galleria mellonella</em> in response to <em>P. aeruginosa</em> and <em>A.baumannii</em> infection. Using potential zeta measurements and dynamic light scattering, we documented that EndoKP27 binds effectively to the pseudomonal surface, causing disorganization of the LPS layer. We established the structure–function relationship by demonstrating the permeabilizing activity of native and heat-inactivated EndoKP27 against liposomal vesicles resembling bacterial inner and outer membranes, and against <em>P. aeruginosa</em> strains with altered LPS structures and varying susceptibility to polymyxins. Computational molecular dynamics simulations revealed the interaction between EndoKP27 and its N-terminal part with bacterial membranes leading to channel formation. Finally, we showed that synthesized 30-amino-acid N-terminal peptide of EndoKP27 exhibits antibacterial activity against a range of <em>P. aeruginosa</em> strains. By integrating experimental findings with computational simulation, we propose the possible LPS-destabilizing and membranolytic mechanism underlying <em>P. aeruginosa</em> sensitization to exogenously applied endolysin without structural modifications. Our findings identify EndoKP27 as a promising antipseudomonal agent.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 24","pages":"Article 169491"},"PeriodicalIF":4.5,"publicationDate":"2025-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145462868","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"EGFR Activation and Conformational “Convergence”: An Emerging Allosteric Model From Cancer Mutations","authors":"Laura Orellana","doi":"10.1016/j.jmb.2025.169493","DOIUrl":"10.1016/j.jmb.2025.169493","url":null,"abstract":"<div><div>Since its isolation nearly 50 years ago, the Epidermal Growth Factor Receptor (EGFR) has become one of the most extensively studied oncogenes. However, scarce data on the full-length receptor, together with its flexibility, has left critical aspects of its activation mechanism unresolved. This has hampered the interpretation of mutations, which are highly asymmetric, tissue-specific, and linked to antagonistic drug sensitivities. While in lung cancer, oncogenic mutations target the kinase and respond to type-I tyrosine kinase inhibitors (TKIs), in brain glioblastoma (GBM), highly heterogeneous mutations focus on the ectodomain (ECD) but share preferential responses to inhibitors that target intermediate active/inactive features in the kinase. Here, we recapitulate current structural and mutational data to shed light on how its extra- and intracellular domains are coupled, with a special focus on an ECD transition state in GBM. This intermediate, first detected by antibodies and later captured in simulations, is characterized by the flexible rotation of the same extracellular fragment deleted in the most frequent GBM mutation, EGFRvIII. The convergence of missense and deletion ECD changes to flexibilize or remove the same piece, hallmarked by a cryptic epitope, offers an explanation for shared drug responses. Based on these observations, we discuss a putative allosteric mechanism, where the extracellular and intracellular sides are tightly coupled in preformed dimers, and the rigidity of the ECD is key to preventing kinase activation. While GBM mutations bypass the steric blockade, lung cancer mutants directly enhance catalytic activity, stabilizing antagonistic states and explaining their opposite drug sensitivities.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 24","pages":"Article 169493"},"PeriodicalIF":4.5,"publicationDate":"2025-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145367259","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DNA Topoisomerase VI: Structure, Function and Mechanism.","authors":"Adam M B Allen, Shannon J McKie, Christian G Noble, Anthony Maxwell","doi":"10.1016/j.jmb.2025.169492","DOIUrl":"10.1016/j.jmb.2025.169492","url":null,"abstract":"<p><p>DNA topoisomerase VI (topo VI) is a type IIB topoisomerase that was originally found in archaea but later found in plants and some other eukaryotes and certain bacteria; claims that it is present in plasmodial parasites have yet to be substantiated. In plants it appears to have an essential role in endoreduplication, but its role in other organisms is less clear. Although topo VI is evolutionarily related to type IIA topoisomerases, it shows a different domain organisation and lacks an exit gate (C gate). Crystal structures of topo VI consolidate these distinctions and show protein cavities and subunit interfaces that are consistent with a double-strand passage mechanism. Single-molecule and ensemble measurements of topo VI reactions show that the rate of DNA relaxation is much slower than with its IIA counterparts, but that it shows a preference for decatenation over relaxation reactions. Radicicol, a known inhibitor of human topo II, also inhibits some topo VI enzymes, and recent drug screens have identified further compounds. Topo VI is ripe for exploitation as a target for herbicides and potentially for antibacterials.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169492"},"PeriodicalIF":4.5,"publicationDate":"2025-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145367246","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biochemical and Structural Analyses of the Tardigrade DNA-Damage Suppressor Protein, Dsup","authors":"Tyler J. Woodward,&nbsp;M. Todd Washington","doi":"10.1016/j.jmb.2025.169490","DOIUrl":"10.1016/j.jmb.2025.169490","url":null,"abstract":"<div><div>Tardigrades are extremophiles that withstand harsh environments through unique molecular strategies. One such strategy involves Damage Suppressor (Dsup), a protein shown to protect cells from radiation-induced DNA damage. Little is known about the biochemical and structural characteristics of Dsup that lead to DNA protection. To gain insight into the mechanism of DNA protection by Dsup, we examined its fundamental biochemical and structural properties using mass photometry, biolayer interferometry, small-angle X-ray scattering, and microfluidic modulation spectroscopy. We found that Dsup is largely intrinsically disordered and binds DNA with high affinity via a multi-valent interface. This interaction induced conformational changes in both Dsup and the DNA, suggesting a potential structural mechanism of its DNA protection ability. We propose that Dsup alters DNA structure, possibly by partially unwinding it, to reduce its susceptibility to damage. These findings offer new insights into how a disordered protein such as Dsup functions as radioprotectants in extreme environments.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 24","pages":"Article 169490"},"PeriodicalIF":4.5,"publicationDate":"2025-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145336090","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Light-induced FTIR Spectroscopy of Visual Rhodopsin Microcrystals Grown in Lipidic Cubic Phase","authors":"Yosuke Mizuno ,&nbsp;Valérie Panneels ,&nbsp;Yuji Furutani ,&nbsp;Gebhard F.X. Schertler ,&nbsp;Hideki Kandori ,&nbsp;Kota Katayama","doi":"10.1016/j.jmb.2025.169487","DOIUrl":"10.1016/j.jmb.2025.169487","url":null,"abstract":"<div><div>Time-resolved X-ray crystallographic analysis of mammalian visual rhodopsin has allowed to visualize the <em>cis</em>-to-<em>trans</em> isomerization of the retinal chromophore, a pivotal event in the early stages of vision, in a temporal and atomic resolution. This achievement provides a foundation for visualizing the subsequent photoreaction dynamics of bovine rhodopsin, paving the way for a comprehensive understanding of the molecular mechanism underlying scotopic vision. However, a critical question remains: Do the structural changes induced by the photoreactions in crystalline environments faithfully mirror those occurring in native membrane environments?</div><div>To start addressing this essential question and improve the reliability of future time-resolved X-ray crystallographic analyses, we first applied low-temperature light-induced FTIR spectroscopy to bovine rhodopsin microcrystals formed using the lipidic cubic phase (LCP) method, in order to compare with conformational changes in native retina membrane (rod outer segment). By encapsulating the microcrystals in custom-made FTIR measurement windows, we successfully obtained FTIR difference spectra of photoreaction intermediates trapped at various temperatures. A detailed comparison with spectra obtained from rhodopsin from native retina membrane revealed both similarities and differences in photoreaction-induced structural changes between crystalline and membrane environments, including alterations in local hydrogen-bonding networks and structural rearrangements of the α-helical backbone. Notably, the formation of Meta-I and Meta-IIa intermediates, which are precursors of G-protein activation, was confirmed even in the crystalline environment, demonstrating that the core photoreaction dynamics proceed comparably in both environments.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 24","pages":"Article 169487"},"PeriodicalIF":4.5,"publicationDate":"2025-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145312056","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural Studies on the M. tuberculosis Nucleoid-associated-Protein, NapA, Indicates DNA Bridging Mechanism","authors":"Maria A. Schumacher,&nbsp;Rajiv R. Singh,&nbsp;Raul Salinas","doi":"10.1016/j.jmb.2025.169486","DOIUrl":"10.1016/j.jmb.2025.169486","url":null,"abstract":"<div><div>Nucleoid-associated proteins (NAPs) play central roles in bacterial chromosome organization and DNA processes. Interestingly, <em>Mycobacterium tuberculosis</em> (<em>Mtb</em>) lacks most common NAPs and only recently have NAPs been uncovered in this bacterium. One such protein, NapA, was revealed to be an essential <em>Mtb</em> NAP that can bridge DNA. NapA shows no sequence homology to any protein and hence its DNA-binding functions remain unclear. Here we describe structures of apo NapA and a DNA-bound complex of NapA. The NapA structures reveal a dimeric fold for the protein, which is supported by mass photometry analyses, with each subunit comprised of an extended α1 helix and C-terminal three-helix module. The α1 helices combine to form a helical-bundle dimer scaffold that forms dimer-of-dimers at elevated protein concentrations. Each NapA dimer projects two DNA interacting elements, that bind and link between DNA sites. Combined these studies provide mechanistic insight into the DNA binding and bridging capabilities of a unique NAP that appears broadly conserved among most Actinobacteria.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 24","pages":"Article 169486"},"PeriodicalIF":4.5,"publicationDate":"2025-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145312013","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"NoAC: an automatic builder for knowledge bases and query interfaces on genomes of non-model organisms","authors":"Tzu-Hsien Yang ,&nbsp;You-Yi Chen ,&nbsp;Chien-Chi Liao ,&nbsp;Hao-Chen Zheng ,&nbsp;Chun-Lin Hsieh ,&nbsp;Jia-Syuan Chen ,&nbsp;Wen-Chieh Tsai ,&nbsp;Yan-Yuan Tseng ,&nbsp;Wei-Sheng Wu","doi":"10.1016/j.jmb.2025.169488","DOIUrl":"10.1016/j.jmb.2025.169488","url":null,"abstract":"<div><div>The cost of sequencing a genome has become affordable for many research groups. However, with the growing number of sequenced genomes from non-model organisms, manually building functional genome annotation knowledge databases for each species is no longer feasible. To address this, we developed NoAC (Non-model Organism Atlas Constructor), a web tool that automatically constructs knowledge bases and query interfaces for non-model organism genomes without programming skills. In NoAC, users simply upload the gene or transcript information of a given non-model organism genome and select an appropriate reference model organism. NoAC then identifies orthologous genes, infers functional annotations, and sets up a searchable knowledge base. Functional annotations for the non-model organism such as gene ontology (GO) terms, protein domains, pathways, and physical/genetic interactors are predicted and transferred from the reference organism to the target genome. In an example non-model organism <em>Phalaenopsis equestris</em>, NoAC associates functional annotations for more than half of its 21,938 genes. Through case studies of the non-model organism <em>Phalaenopsis equestris</em>, we demonstrated that the knowledge base constructed by NoAC can reveal key functional aspects of <em>PeSEP2</em> and <em>PaMLS</em>, supporting the study of novel genes involved in flower development. Another case study on the gene <em>Wnt-1</em> in <em>Bicyclus anynana</em> further illustrates the applicability of NoAC in investigating insect segmentation and morphogen activity, highlighting its broader utility across diverse taxonomic genomes. In summary, NoAC allows general researchers to study non-model organisms with minimal <em>in silico</em> barriers. NoAC and its user tutorial are freely available at <span><span>https://github.com/cosbi-nckuee/NoAC/</span><svg><path></path></svg></span>.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 24","pages":"Article 169488"},"PeriodicalIF":4.5,"publicationDate":"2025-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145312078","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"NMR Studies of Biomolecular Systems","authors":"Lewis E. Kay,&nbsp;Remco Sprangers","doi":"10.1016/j.jmb.2025.169489","DOIUrl":"10.1016/j.jmb.2025.169489","url":null,"abstract":"","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 23","pages":"Article 169489"},"PeriodicalIF":4.5,"publicationDate":"2025-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145312062","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Slow Dynamics Orchestrate Communication Between Binding Sites in the Condensation Domain of a Non-ribosomal Peptide Synthetase","authors":"Megha N. Karanth ,&nbsp;Debajyoti De ,&nbsp;John P. Kirkpatrick ,&nbsp;Mark Jeeves ,&nbsp;Teresa Carlomagno","doi":"10.1016/j.jmb.2025.169484","DOIUrl":"10.1016/j.jmb.2025.169484","url":null,"abstract":"<div><div>Non-ribosomal peptide synthetases (NRPSs) are complex molecular machineries that synthesize non-proteinaceous peptides in microorganisms. These peptides (NRPs) usually present a wide range of biological activities and are highly regarded as potential anti-cancer and anti-infective agents. Because of their chemical complexity, derivatives of NRPs with tailored pharmacological properties are difficult to synthesize chemically, which has triggered efforts to understand the functional mechanisms of NRPS systems and develop protein engineering strategies aimed at enabling enzymatic synthesis of non-natural NRPs. A fundamental reaction step of NRPS systems is the formation of peptide bonds between amino-acid-like building blocks. This reaction is catalyzed by so-called condensation domains. The structures of several condensation domains and their complexes have been solved by crystallography and electron microscopy, but these structures have failed to provide the key to the design of artificial condensation domains. Here, we use NMR spectroscopy to reveal a complex network of dynamics in the condensation domain of the NRPS responsible for the synthesis of Tomaymycin and reveal how these motions mediate communication between the two substrate binding sites, providing a means to synchronize interactions for efficient catalysis. Our results underline the impact of dynamics, next to structure, on the function of enzymatic units and reinforce the need to consider conformational flexibility in the design of proteins with altered functions.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 23","pages":"Article 169484"},"PeriodicalIF":4.5,"publicationDate":"2025-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145285397","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}