Journal of Molecular Biology最新文献

{"title":"The specificity of RNA packaging in isometric RNA plant viruses is principally determined by replication.","authors":"Keith Saunders, Sachin N Shah, Hadrien Peyret, Yulia Meshcheriakova, Jake Richardson, Sandra Eltschkner, David M Lawson, George P Lomonossoff","doi":"10.1016/j.jmb.2025.169352","DOIUrl":"https://doi.org/10.1016/j.jmb.2025.169352","url":null,"abstract":"<p><p>A potato virus X (PVX)-based transient expression system (pEff) that produces replicating RNA has been used to examine the specificity of RNA packaging in the isometric viruses, turnip crinkle virus (TCV) and satellite tobacco necrosis virus-1 (STNV-1). Expression of the coat proteins from the subgenomic RNA derived from the replicating PVX genome results in the efficient production of virus-like particles (VLPs), indistinguishable in structure from native virus particles, and encapsidation of both the subgenomic RNA and truncated versions of the replicating genomic RNA. Non-specific encapsidation of host RNA (which is not replicating) could not be detected in this system, implying that replication is the major determinant of packaging in isometric as well as filamentous positive-strand RNA plant viruses. We further utilised the system to investigate the role of putative packaging signals previously identified within the coat protein open reading frames of both TCV and STNV-1. The results show that eliminating the hairpin structures previously identified as packaging signals has no detectable effect on the specificity of RNA packaging. Replacement of the 213 nucleotide sequence within the TCV coat protein coding region, believed to be important for genomic packaging, with an equivalent sequence codon-optimised for Plasmodium falciparum resulted in less efficient capsid formation and RNA packaging, but did not alter packaging specificity; addition of copies of the wild-type sequence did not complement the defects. We propose that replication is the major determinant of genome packaging specificity in plant RNA viruses, while packaging signals may play a role in packaging efficiency.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169352"},"PeriodicalIF":4.7,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144705989","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":"Rad23B delays Ataxin-3 liquid-to-solid phase transition through heterotypic buffering.","authors":"Archana Prasad, Sandhini Saha, Manisha Kumari, Krishna Singh Bisht, Tushar Kanti Maiti","doi":"10.1016/j.jmb.2025.169351","DOIUrl":"https://doi.org/10.1016/j.jmb.2025.169351","url":null,"abstract":"<p><p>The abnormal expansion of polyglutamine (polyQ) length in Ataxin-3 is associated with Machado-Joseph disease, forming nuclear inclusions in neurons. A truncated variant of Ataxin-3 with two ubiquitin-interacting motifs (UIMs) has recently been shown to undergo liquid-liquid phase separation (LLPS). However, the molecular mechanisms underlying Ataxin-3 aggregation through amyloid formation and phase separation remain unclear. Here we investigated the LLPS properties of Ataxin-3 with three UIMs, the most abundant isoform found inthe brain. Ataxin-3 Q25 forms phase-separated droplets which undergo aging, producing amyloid-like fibrillar structures. PolyQ expanded Q54 forms short-lived droplets that exhibit rapid maturation. Ataxin-3 C-terminal fragment forms unique straight, unbranched amyloid fibrils which display negligible Thioflavin-T binding. Rad23B, the main constituent driving the formation of proteasomal condensates, is a known Ataxin-3 interactor. We found that heterotypic interactions with Rad23B inhibit Ataxin-3 droplet maturation but do not inhibit amyloid formation under dilute conditions, suggesting that Ataxin-3 aggregation via misfolding pathway is distinct from condensation pathway. Finally, we show that Ataxin-3 is incorporated into liquid-like stress granules under arsenite stress, shedding light on its roles in aggregation dynamics and stress responses.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169351"},"PeriodicalIF":4.7,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144673653","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":"LncRNA SChLAP1 promotes cancer cell proliferation and invasion via its distinct structural domains and conserved regions.","authors":"Mihyun Oh, Roshni Nagesh Kadam, Zahra Sadruddin Charania, Srinivas Somarowthu","doi":"10.1016/j.jmb.2025.169350","DOIUrl":"10.1016/j.jmb.2025.169350","url":null,"abstract":"<p><p>Long non-coding RNAs (lncRNAs) play key roles in a range of biological processes and disease progression. Despite their functional significance and therapeutic potential, lncRNAs' mechanisms of action remain understudied. One such lncRNA is the Second Chromosome Locus Associated with Prostate-1 (SChLAP1). SChLAP1 is overexpressed in malignant prostate cancer and is associated with unfavorable patient outcomes, such as metastasis and increased mortality. In this study, we demonstrated that SChLAP1 possesses distinct structural domains and conserved regions that may contribute to its function. We determined the secondary structure of SChLAP1 using chemical probing methods combined with mutational profiling (DMS-MaP and SHAPE-MaP). Our in vitro secondary structural model revealed that SChLAP1 consists of two distinct secondary-structural modules located at its 5' and 3' ends, both featuring regions with a high degree of structural organization. Our in vivo chemical probing identified structurally stable regions and areas that may undergo specific structural rearrangements in the cellular context. Overexpression of the modules led to a notable increase in cancer cell proliferation and invasion, proving their functional significance in the oncogenicity of SChLAP1. In conclusion, we discovered functionally important, independent modules with well-defined structures of SChLAP1. These results will serve as a guide to explore the detailed molecular mechanisms by which SChLAP1 promotes aggressive prostate cancer, ultimately contributing to the development of SChLAP1 as a novel therapeutic target.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169350"},"PeriodicalIF":4.7,"publicationDate":"2025-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144666753","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":"Search for Specific Inhibitors Targeting Type IA Topoisomerases.","authors":"Somaia Haque Chadni, Shomita Ferdous, Yuk-Ching Tse-Dinh","doi":"10.1016/j.jmb.2025.169349","DOIUrl":"10.1016/j.jmb.2025.169349","url":null,"abstract":"<p><p>The type IA topoisomerase subfamily includes bacterial topoisomerase I and topoisomerase III encoded by topA and topB genes, reverse gyrase found in thermophilic bacteria and archaea, as well as eukaryotic topoisomerase III. Type IA topoisomerases also act on RNA as substrate. Important functions in neurological development have been demonstrated for human TOP3B. Type IA topoisomerase present in all bacterial pathogens should be a novel target that can be utilized for the discovery of new antibacterial agents. Naturally produced bacterial toxins have been shown to inhibit cell growth by targeting topoisomerase I. Topoisomerase III in human and other eukaryotes could potentially also be targeted for treatment of cancer and viral or parasitic infections. Docking, machine-learning, enzyme or cell-based screening campaigns have identified compounds that can inhibit the catalytic activity of type IA topoisomerases, or poisons that can trap the covalent complex of the targeted type IA topoisomerase. Small molecule inhibitors identified thus far for bacterial topoisomerase I or human TOP3B have not been viable candidates as drug leads mostly due to lack of sufficient potency and selectivity. The barriers for obtaining better inhibitors include the lack of an X-ray or cryo-EM structure of topoisomerase-ligand complex and mutations in the topoisomerase gene that can confirm the topoisomerase as primary cellular target. Well-designed combination of virtual and experiment screening to explore large chemical space in future studies may improve the likelihood of success for identifying small molecule inhibitors of type IA topoisomerases that can form specific protein-ligand complexes amenable for structure determination.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169349"},"PeriodicalIF":4.7,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144666754","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":"S. cerevisiae Xrs2 binds DNA through its FHA domain.","authors":"Ajeak Vigneswaran, Marella D Canny, Stephan B Azatian, Michael P Latham","doi":"10.1016/j.jmb.2025.169348","DOIUrl":"https://doi.org/10.1016/j.jmb.2025.169348","url":null,"abstract":"<p><p>The MRE11-RAD50-NBS1/Xrs2 (MRN/X) complex is a conserved first responder to DNA double-strand breaks (DSBs). All three members of the complex have DNA binding properties that support the range of functions MRN/X performs in its role in DNA DSB repair. Previous structural and functional studies have localized DNA binding sites within MRE11 and RAD50, but no structural model exists for DNA association with NBS1/Xrs2. Here, we identify a DNA binding site within the N-terminal folded FHA-BRCT-BRCT domain of Saccharomyces cerevisiae Xrs2. Using NMR chemical shift perturbations and paramagnetic relaxation enhancements, we define a DNA binding interface on the FHA domain and generate integrative models of the DNA-bound complex via the program HADDOCK. DNA binding overlaps with the site involved in phosphorylated Sae2 peptide binding - an interaction analogous to that between Schizosaccharomyces pombe Nbs1 and phosphorylated Ctp1. Comparative binding assays and site-directed mutagenesis confirm a shared binding surface for DNA and pSae2 on the FHA domain of Xrs2 and highlight the need for functional assays and mutagenesis for validating HADDOCK models. Finally, NMR relaxation experiments reveal altered ps-ns timescale dynamics but unaltered µs-ms conformational exchange upon ligand binding. These findings define a direct DNA binding role for Xrs2 and provide a structural framework for understanding its dual recognition of DNA and phosphoprotein partners during DSB repair.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169348"},"PeriodicalIF":4.7,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144658090","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":"Transfer RNA Recognition Mechanism of Thermoplasma acidophilum Trm56, a SPOUT tRNA Methyltransferase that Possesses an Unusually Long C-terminal Region","authors":"Soichiro Hidetaka ,&nbsp;Shuhei Fukumoto ,&nbsp;Takahiro Hasegawa ,&nbsp;Takuya Kawamura ,&nbsp;Mami Ototake ,&nbsp;Shizuka Moriguchi ,&nbsp;Miyu Namba ,&nbsp;Chie Tomikawa ,&nbsp;Ryota Yamagami ,&nbsp;Akira Hirata ,&nbsp;Hiroyuki Hori","doi":"10.1016/j.jmb.2025.169328","DOIUrl":"10.1016/j.jmb.2025.169328","url":null,"abstract":"<div><div>Trm56 from <em>Thermoplasma acidophilum</em> is an exceptional SpoU-TrmD superfamily enzyme, which possesses an unusually long C-terminal region, and catalyzes methylation of 2′-OH of ribose of C56 in tRNA. <em>T. acidophilum</em> Trm56 methylates elongator tRNA^Met (tRNA^Mete) transcript effectively, however this enzyme methylates tRNA^Leu transcript slowly. This enzymatic feature is common in <em>T. acidophilum</em> Trm56 and <em>Pyrococcus horikoshii</em> Trm56, which possesses a short C-terminal region. Kinetic analysis revealed that Km value of <em>T. acidophilum</em> Trm56 for tRNA^Leu is larger than that for tRNA^Mete. Our analysis of native tRNAs revealed that the 2′-<em>O</em>-methylcytidine content at position 56 (Cm56) in tRNA^Leu is 1.9% while in tRNA^Mete it is 63.1%, showing that the <em>in vitro</em> enzymatic properties of <em>T. acidophilum</em> Trm56 are reflected in the Cm56 modification levels in living cells. Experiments with chimera tRNA transcript of tRNA^Leu and tRNA^Mete showed that <em>T. acidophilum</em> Trm56 recognizes the T-loop and the three-dimensional core of tRNA. Furthermore, experiments with mutant tRNA^Mete transcript revealed that the G53-C61 base pair and U54U55C56purine57purine58 sequence are essential for methylation. Crystal structures of apo- and 5′-methyl-5′-thioadenosine binding forms of the catalytic domain of <em>T. acidophilum</em> Trm56 revealed that the structure of the 5′-methyl-5′-thioadenosine binding pocket and overall structure of the catalytic domain of <em>T. acidophilum</em> Trm56 closely resemble those of <em>P. horikoshii</em> Trm56. Experiments with a chimera of <em>T. acidophilum</em> and <em>P. horikoshii</em> Trm56 proteins demonstrates that the catalytic domain of <em>T. acidophilum</em> Trm56 is responsible for the slow methylation of tRNA^Leu transcript.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 19","pages":"Article 169328"},"PeriodicalIF":4.7,"publicationDate":"2025-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144632172","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":"OmpA Specifically Modulates the Activity of Enzymes that Reside in the Crowded Bacterial Outer Membrane","authors":"Jonathan M. Machin,&nbsp;Neil A. Ranson,&nbsp;Sheena E. Radford","doi":"10.1016/j.jmb.2025.169346","DOIUrl":"10.1016/j.jmb.2025.169346","url":null,"abstract":"<div><div>The outer membrane (OM) of lipopolysaccharide (LPS) containing-diderm bacteria is crowded with outer membrane proteins (OMPs) that reside in a membrane that is relatively rich in protein and poor in lipid. As a consequence, extensive interactions between OMPs occur. Yet, how these interactions affect OMP function remains unexplored. Here, we examine the effect of OmpA on the activity of three different OMP enzymes, OmpLA (a phospholipase), PagP (a palmitoyltransferase) and OmpT (a protease). We show that OmpA-OmpT interactions enhance the activity of OmpT, and that this catalytic enhancement is mediated via their extracellular loops, an effect that is not observed with other common OMPs, including OmpF and OmpX. In contrast, OmpA specifically reduces the activity of PagP, while OmpLA activity shows no significant change. Possible interactions between the abundant <em>E. coli</em> OMPs (OmpA, OmpF/C, OmpT, OmpX, MipA) and all other <em>E. coli</em> OMPs were screened via Alphafold predictions, with the results suggesting that smaller OMPs are generally more promiscuous interactors, and identifying new interactions that may plausibly form in the OMP-rich islands in the OM. Together, the results identify a previously underappreciated role for specific OMP–OMP interactions in modulating protein function in the OM, and highlight how evolution may have exploited the high local concentrations of abundant OMPs in the OM to tune enzyme activity.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 19","pages":"Article 169346"},"PeriodicalIF":4.7,"publicationDate":"2025-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144648135","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":"Isotopologues of a Metabolic Precursor for Selective N-15 and C-13 Histidine Labeling.","authors":"Sven Brüschweiler, Matus Hlavac, Sarah Kratzwald, Julia Schörghuber, Katharina M Siess, Alisa Wimmer, Gerald Platzer, Robert Konrat, Roman J Lichtenecker","doi":"10.1016/j.jmb.2025.169347","DOIUrl":"10.1016/j.jmb.2025.169347","url":null,"abstract":"<p><p>Histidine is a versatile residue with distinct properties ensuring many proteins' structure and proper function. Its imidazole side-chain represents an ideal chemical entity to serve as a proton shuttle in enzyme mechanisms, control recognition interfaces either by contribution of its aromatic Pi system or in its cationic form, and acts as a coordinating ligand to metal cations. These functional capabilities are modulated by the local molecular environment, which influences pK_a values and tautomeric states. NMR spectroscopy has proven to be a reliable method for probing the distinct functions of histidine. Here, we describe the synthesis of isotopically labeled variants of a non-chiral precursor to introduce NMR active ¹³C and/or ¹⁵N nuclei into histidine side-chains. The compounds were employed to selectively label protein targets of significant interest in current drug discovery programs, such as the WD repeat containing protein 5 (WDR5), the Src homology 2 domain of the phospholipase C (PLCγ-SH2) and the product of the Kirsten rat sarcoma virus oncogene (KRAS).</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169347"},"PeriodicalIF":4.7,"publicationDate":"2025-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144648134","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":"Acid pH sensing by EnvZ-OmpR alters H-bonding networks to allosterically activate virulence gene expression.","authors":"Victoria Brady, Minoli Doshi, Andrew Wenzell, Linda Kenney, Ganesh S Anand","doi":"10.1016/j.jmb.2025.169345","DOIUrl":"https://doi.org/10.1016/j.jmb.2025.169345","url":null,"abstract":"<p><p>The molecular basis for how acidic pH environments trigger noncanonical phosphorylation-independent conformational changes in the EnvZ-OmpR two-component system for turning on virulence gene expression is not understood. Using amide hydrogen-deuterium exchange mass spectrometry (HDXMS), we compared kinetics of deuterium exchange of the cytosolic domain of EnvZ (EnvZc) with its cognate response regulator (RR) OmpR under conditions mimicking the intracellular environments encountered during host phagocytosis (pH range- 6.5-7.5). At pH 6.5 compared to pH 7.5, EnvZc showed lower deuterium exchange in its four-helical bundle subdomain, particularly in the region surrounding the conserved His 243, indicating acid stabilization. Similarly, pH-dependent changes were observed in OmpR, notably in peptides associated with its aromatic switch in the receiver and DNA binding domains. Interestingly, acidic pH elicited changes in the same allosteric loci associated with phosphorylation-dependent regulation in response to osmosensing. These findings suggest that the EnvZ-OmpR system adapts to acidic environments via a mechanism distinct from canonical phosphorylation but with different gene expression outcomes to favor expression of virulence factor genes. The study provides new insights into bacterial adaptation to host-induced stress and highlights potential targets for antimicrobial development.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169345"},"PeriodicalIF":4.7,"publicationDate":"2025-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144648133","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":"Allosteric Autoregulation of Ferroptosis Suppressor Protein 1 Activity by its N-myristoylated Tail.","authors":"Carlos Ventura, Xiaowei Bogetti, Ji Young Lee, Ivet Bahar","doi":"10.1016/j.jmb.2025.169344","DOIUrl":"10.1016/j.jmb.2025.169344","url":null,"abstract":"<p><p>Ferroptosis is a form of cell death characterized by iron-dependent accumulation of lipid peroxides. Ferroptosis suppressor protein 1 (FSP1) has been shown to work with glutathione peroxidase 4 (GPX4) to suppress ferroptosis through different antioxidant pathways. Many studies have been conducted on FSP1 to better understand its function and mechanism of action, which remained inconclusive in the absence of structural information on FSP1. Recent elucidation of FSP1 structures in different forms and advances in computational characterization of functional changes in its conformation provide us with the opportunity of dissecting FSP1 mechanism of action and gaining insights into critical sites and interactions that control its activity. We present the results from elastic network model analyses of cooperative changes in FSP1 structure, as well as those from molecular dynamics simulations of its interactions with the lipid bilayer and small molecules, toward assisting in future development of modulators of ferroptosis targeting FSP1. Our study reveals the critical role of N-terminal myristoylated tail in modulating the accessibility of the ligand-binding sites and in anchoring FSP1 to the membrane, giving insights into mechanisms of regulating FSP1 function.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169344"},"PeriodicalIF":4.7,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144625108","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}