Pub Date : 2025-11-05DOI: 10.1016/j.jmb.2025.169533
Ilias Zarguit , Marc K.M. Cajili , Bert van Erp , Samuel Schwab , Nico van der Vis , Marianne Julienne Bakker , John van Noort , Remus T. Dame
DNA-wrapping histone proteins play a central role in chromatin organization, gene expression and regulation in most eukaryotes and archaea. While the structure and function of eukaryotic histones are well-characterized, archaeal histones and their complexes with DNA require further scrutiny. Distinct from their eukaryotic counterparts, previously characterized canonical archaeal histones assemble on DNA into an ‘endless’ superhelical nucleoprotein complex called a hypernucleosome. In this study, we explored whether hypernucleosome formation is a conserved feature of canonical archaeal histones. Moreover, to further elucidate how hypernucleosomes are regulated, we also explored how changes in the physico-chemical conditions, particularly the presence of Mg2+, influence the hypernucleosome. Using a combination of Tethered Particle Motion (TPM) and single-molecule force spectroscopy, we established that T. kodakarensis histones assemble into hypernucleosomes on DNA, similar to the M. fervidus histones HMfA and HMfB, the only canonical histones structurally characterized in previous studies. However, the effects of Mg2+ ions are distinct despite the histones’ high sequence- and structural similarity. We propose a model in which Mg2+ ions exert a generic effect on hypernucleosome compactness and stability due to electrostatic DNA shielding, with additional differential effects depending on histone identity.
{"title":"Modulation of Archaeal Hypernucleosome Structure and Stability by Mg2+","authors":"Ilias Zarguit , Marc K.M. Cajili , Bert van Erp , Samuel Schwab , Nico van der Vis , Marianne Julienne Bakker , John van Noort , Remus T. Dame","doi":"10.1016/j.jmb.2025.169533","DOIUrl":"10.1016/j.jmb.2025.169533","url":null,"abstract":"<div><div>DNA-wrapping histone proteins play a central role in chromatin organization, gene expression and regulation in most eukaryotes and archaea. While the structure and function of eukaryotic histones are well-characterized, archaeal histones and their complexes with DNA require further scrutiny. Distinct from their eukaryotic counterparts, previously characterized canonical archaeal histones assemble on DNA into an ‘endless’ superhelical nucleoprotein complex called a hypernucleosome. In this study, we explored whether hypernucleosome formation is a conserved feature of canonical archaeal histones. Moreover, to further elucidate how hypernucleosomes are regulated, we also explored how changes in the physico-chemical conditions, particularly the presence of Mg<sup>2+</sup>, influence the hypernucleosome. Using a combination of Tethered Particle Motion (TPM) and single-molecule force spectroscopy, we established that <em>T. kodakarensis</em> histones assemble into hypernucleosomes on DNA, similar to the <em>M. fervidus</em> histones HMfA and HMfB, the only canonical histones structurally characterized in previous studies. However, the effects of Mg<sup>2+</sup> ions are distinct despite the histones’ high sequence- and structural similarity. We propose a model in which Mg<sup>2+</sup> ions exert a generic effect on hypernucleosome compactness and stability due to electrostatic DNA shielding, with additional differential effects depending on histone identity.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 24","pages":"Article 169533"},"PeriodicalIF":4.5,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145470483","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}
Pub Date : 2025-11-04DOI: 10.1016/j.jmb.2025.169510
Julia Kowal , Dongchun Ni , Scott M. Jackson , Ioannis Manolaridis , Henning Stahlberg , Kaspar P. Locher
{"title":"Corrigendum to “Structural Basis of Drug Recognition by the Multidrug Transporter ABCG2”. [J. Mol. Biol. 433 (2021) 166980]","authors":"Julia Kowal , Dongchun Ni , Scott M. Jackson , Ioannis Manolaridis , Henning Stahlberg , Kaspar P. Locher","doi":"10.1016/j.jmb.2025.169510","DOIUrl":"10.1016/j.jmb.2025.169510","url":null,"abstract":"","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 24","pages":"Article 169510"},"PeriodicalIF":4.5,"publicationDate":"2025-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145450465","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}
Pub Date : 2025-11-01Epub Date: 2025-07-25DOI: 10.1016/j.jmb.2025.169357
Yunpeng He, Dongyue Hou, Yuzong Chen, Xian Zeng
Biomolecular interaction kinetics underpin essential cellular mechanisms, yet quantitative databases remain scarce for RNA-protein interactions (RPIs)-a critical regulatory axis in post-transcriptional control, synthetic biology, and therapeutic development. We previously established KDBI (Kinetic Data of Bio-molecular Interactions database) to catalog quantitative kinetics data across diverse biomolecular interaction types. Here, we present KDBI-RP, a dedicated extension focused on RPI kinetics, addressing the growing demand for RNA-centric kinetic research. KDBI-RP systematically integrates binding data for RNA-protein interactions, including kinetic rate constants-association (kon, 3657 entries) and dissociation (koff, 3761 entries)-supplemented by equilibrium dissociation constants (Kd, 175,932 entries). The database offers well-curated information on kinetic constants, assay conditions, literature sources, and comprehensive sequence, structural, and functional annotations for proteins, RNAs, and their complexes. KDBI-RP is freely accessible at http://www.kdbirp.aiddlab.com. We anticipate that KDBI-RP will serve as a valuable resource for the RNA biology and RNA-based medicine research communities.
{"title":"KDBI-RP: Kinetic Data of RNA-Protein Interactions Database.","authors":"Yunpeng He, Dongyue Hou, Yuzong Chen, Xian Zeng","doi":"10.1016/j.jmb.2025.169357","DOIUrl":"10.1016/j.jmb.2025.169357","url":null,"abstract":"<p><p>Biomolecular interaction kinetics underpin essential cellular mechanisms, yet quantitative databases remain scarce for RNA-protein interactions (RPIs)-a critical regulatory axis in post-transcriptional control, synthetic biology, and therapeutic development. We previously established KDBI (Kinetic Data of Bio-molecular Interactions database) to catalog quantitative kinetics data across diverse biomolecular interaction types. Here, we present KDBI-RP, a dedicated extension focused on RPI kinetics, addressing the growing demand for RNA-centric kinetic research. KDBI-RP systematically integrates binding data for RNA-protein interactions, including kinetic rate constants-association (k<sub>on</sub>, 3657 entries) and dissociation (k<sub>off</sub>, 3761 entries)-supplemented by equilibrium dissociation constants (K<sub>d</sub>, 175,932 entries). The database offers well-curated information on kinetic constants, assay conditions, literature sources, and comprehensive sequence, structural, and functional annotations for proteins, RNAs, and their complexes. KDBI-RP is freely accessible at http://www.kdbirp.aiddlab.com. We anticipate that KDBI-RP will serve as a valuable resource for the RNA biology and RNA-based medicine research communities.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169357"},"PeriodicalIF":4.5,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144726322","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}
Ureaplasma is one of the smallest pathogenic bacteria, generating approximately 95% of its adenosine triphosphate (ATP) solely through urease. Studies on Ureaplasma parvum, a species of Ureaplasma, have confirmed that adding urease inhibitors inhibits bacterial growth. The Km and Vmax of the urease-mediated reaction were estimated to be 4.3 ± 0.2 mM and 3,333.3 ± 38.0 μmol NH3/min/mg protein, respectively. The cryo-electron microscopy (cryo-EM) structure of Ureaplasma parvum urease (UPU) at a resolution of 2.03 Å reveals a trimer of heterotrimers comprising three proteins: UreA, UreB, and UreC. The active site is well conserved among the known ureases. However, the Vmax of UPU was higher than that of most known ureases, including those ureases derived from Sporosarcina pasteurii (SPU) and Klebsiella aerogenes (KAU) with identical oligomeric state. All-atom molecular dynamics simulations showed that the flap and UreB are more open in UPU than SPU and KAU. His-tagged wild-type recombinant UPU (WT-rUPU) revealed estimated Km and Vmax values of 4.1 ± 0.3 mM and 769.2 ± 7.4 µmol NH3/min/mg protein, respectively. Amino acid substitutions of recombinant UPUs within the flap region to SPU. Amongst the flap region variants, the Vmax of K331N variant was 48-fold lower than that of WT-rUPU. ICP-MS analysis reveals that one molecule of UPU, WT-rUPU, and K331N-rUPU contains 3.7, 0.8, and 0.1 Ni2+ atoms, respectively, suggesting that a wide-open flap of urease may contribute to delivering nickel into the enzyme, resulting in a high Vmax. Ureaplasma evolved highly efficient UPU through a few amino acid substitutions in the disorganized loop of the mobile flap region.
{"title":"Structural Analysis and Molecular Dynamics Simulations of Urease From Ureaplasma parvum.","authors":"Heng Ning Wu, Junso Fujita, Yukiko Nakura, Masao Inoue, Koichiro Suzuki, Toru Ekimoto, Bingjie Yin, Yohta Fukuda, Kazuo Harada, Tsuyoshi Inoue, Mitsunori Ikeguchi, Keiichi Namba, Itaru Yanagihara","doi":"10.1016/j.jmb.2025.169368","DOIUrl":"10.1016/j.jmb.2025.169368","url":null,"abstract":"<p><p>Ureaplasma is one of the smallest pathogenic bacteria, generating approximately 95% of its adenosine triphosphate (ATP) solely through urease. Studies on Ureaplasma parvum, a species of Ureaplasma, have confirmed that adding urease inhibitors inhibits bacterial growth. The K<sub>m</sub> and V<sub>max</sub> of the urease-mediated reaction were estimated to be 4.3 ± 0.2 mM and 3,333.3 ± 38.0 μmol NH<sub>3</sub>/min/mg protein, respectively. The cryo-electron microscopy (cryo-EM) structure of Ureaplasma parvum urease (UPU) at a resolution of 2.03 Å reveals a trimer of heterotrimers comprising three proteins: UreA, UreB, and UreC. The active site is well conserved among the known ureases. However, the V<sub>max</sub> of UPU was higher than that of most known ureases, including those ureases derived from Sporosarcina pasteurii (SPU) and Klebsiella aerogenes (KAU) with identical oligomeric state. All-atom molecular dynamics simulations showed that the flap and UreB are more open in UPU than SPU and KAU. His-tagged wild-type recombinant UPU (WT-rUPU) revealed estimated K<sub>m</sub> and V<sub>max</sub> values of 4.1 ± 0.3 mM and 769.2 ± 7.4 µmol NH<sub>3</sub>/min/mg protein, respectively. Amino acid substitutions of recombinant UPUs within the flap region to SPU. Amongst the flap region variants, the V<sub>max</sub> of K331N variant was 48-fold lower than that of WT-rUPU. ICP-MS analysis reveals that one molecule of UPU, WT-rUPU, and K331N-rUPU contains 3.7, 0.8, and 0.1 Ni<sup>2+</sup> atoms, respectively, suggesting that a wide-open flap of urease may contribute to delivering nickel into the enzyme, resulting in a high V<sub>max</sub>. Ureaplasma evolved highly efficient UPU through a few amino acid substitutions in the disorganized loop of the mobile flap region.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169368"},"PeriodicalIF":4.5,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144768237","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}
Pub Date : 2025-11-01DOI: 10.1016/j.jmb.2025.169530
Yang Yu , Jinxing Ou , Nathalie Dostatni
Since its first adaptation in fruit fly embryos with the fluorescent tagging of RNA, live imaging of transcription during development revealed the unanticipated importance of the temporal dynamics in the decoding of positional information. At the molecular scale, these approaches revealed the bursty dynamics of transcription of many developmental genes. They allowed the precise dissection of these bursty transcription cycles at single loci in vivo and showed that burst frequency and duration are differentially regulated by distinct developmental clues. Live transcriptional imaging also uncovered enhancer-promoter interactions not only through direct looping but also via shared transcriptional hubs, challenging classical contact-based models. In addition, these RNA-labelling systems provided also new tools to visualize and mechanistically dissect in vivo transcriptional memory, dosage compensation, chromosome pairing and transvection. Finally, these approaches were also more recently adapted to the nematode, the flour beetle, the zebrafish, or the mouse, paving the way for a better understanding of cross-species developmental mechanisms.
{"title":"Lighting-up Transcription During Development","authors":"Yang Yu , Jinxing Ou , Nathalie Dostatni","doi":"10.1016/j.jmb.2025.169530","DOIUrl":"10.1016/j.jmb.2025.169530","url":null,"abstract":"<div><div>Since its first adaptation in fruit fly embryos with the fluorescent tagging of RNA, live imaging of transcription during development revealed the unanticipated importance of the temporal dynamics in the decoding of positional information. At the molecular scale, these approaches revealed the bursty dynamics of transcription of many developmental genes. They allowed the precise dissection of these bursty transcription cycles at single loci <em>in vivo</em> and showed that burst frequency and duration are differentially regulated by distinct developmental clues. Live transcriptional imaging also uncovered enhancer-promoter interactions not only through direct looping but also via shared transcriptional hubs, challenging classical contact-based models. In addition, these RNA-labelling systems provided also new tools to visualize and mechanistically dissect <em>in vivo</em> transcriptional memory, dosage compensation, chromosome pairing and transvection. Finally, these approaches were also more recently adapted to the nematode, the flour beetle, the zebrafish, or the mouse, paving the way for a better understanding of cross-species developmental mechanisms.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"438 1","pages":"Article 169530"},"PeriodicalIF":4.5,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145436866","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}
Pub Date : 2025-11-01DOI: 10.1016/j.jmb.2025.169528
Stephen R. Archuleta, Ryan C. Miller, Julia A. Mirita, James A. Goodrich, Jennifer F. Kugel
Transcription by RNA polymerase II (Pol II) requires general transcription factors that bind with Pol II at the promoters of protein-coding genes to form preinitiation complexes (PICs). Among these is TFIIE, which recruits TFIIH to the PIC and stimulates the kinase and translocase activities of TFIIH, thereby regulating the fate of formed PICs. In this study, we used a purified reconstituted human Pol II transcription system and single molecule total internal reflection fluorescence microscopy to monitor TFIIE binding dynamics in PICs under different conditions in real time. We observed dynamic interactions of the two subunits of TFIIE (TFIIEα and TFIIEβ) with PICs, consistent with rapid on/off binding as a heterodimer. TFIIH exclusion increased the rates of association and dissociation for both subunits, and enabled TFIIEα to associate with PICs more often than TFIIEβ; hence, TFIIEα and TFIIEβ behave asynchronously in the absence of TFIIH. Additionally, two disease-related TFIIEβ point mutations destabilized TFIIEβ and altered its kinetic behaviors within PICs. Our results contribute to an emerging model that PICs are not static assemblies and highlight important connections between the structural arrangement and kinetic behaviors of GTFs in PICs.
{"title":"Single-molecule Microscopy Reveals That TFIIE Subunits Dynamically Interact With Preinitiation Complexes in a Manner Impacted by TFIIH","authors":"Stephen R. Archuleta, Ryan C. Miller, Julia A. Mirita, James A. Goodrich, Jennifer F. Kugel","doi":"10.1016/j.jmb.2025.169528","DOIUrl":"10.1016/j.jmb.2025.169528","url":null,"abstract":"<div><div>Transcription by RNA polymerase II (Pol II) requires general transcription factors that bind with Pol II at the promoters of protein-coding genes to form preinitiation complexes (PICs). Among these is TFIIE, which recruits TFIIH to the PIC and stimulates the kinase and translocase activities of TFIIH, thereby regulating the fate of formed PICs. In this study, we used a purified reconstituted human Pol II transcription system and single molecule total internal reflection fluorescence microscopy to monitor TFIIE binding dynamics in PICs under different conditions in real time. We observed dynamic interactions of the two subunits of TFIIE (TFIIEα and TFIIEβ) with PICs, consistent with rapid on/off binding as a heterodimer. TFIIH exclusion increased the rates of association and dissociation for both subunits, and enabled TFIIEα to associate with PICs more often than TFIIEβ; hence, TFIIEα and TFIIEβ behave asynchronously in the absence of TFIIH. Additionally, two disease-related TFIIEβ point mutations destabilized TFIIEβ and altered its kinetic behaviors within PICs. Our results contribute to an emerging model that PICs are not static assemblies and highlight important connections between the structural arrangement and kinetic behaviors of GTFs in PICs.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 24","pages":"Article 169528"},"PeriodicalIF":4.5,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145436862","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}
J-domain proteins (JDPs) are co-chaperones of Hsp70/DnaK, conferring a broad range of functions to the Hsp70/DnaK chaperone system. The shortest characterized JDP is AtcJ, which is part of an essential four-protein system dedicated to the cold adaptation of the bacterium Shewanella oneidensis. AtcJ connects DnaK to the Atc system that comprises also the proteins AtcA, AtcB, and AtcC. Previous studies have shown that AtcB interacts with RNA polymerase, and that its overproduction leads to toxicity, likely due to the inhibition of transcription. In this study, we explore the interplay between DnaK, the Atc proteins and RNA polymerase in order to elucidate the mechanism of their coordinated function. First, we identified the molecular determinants underlying the interaction between RNA polymerase and AtcB, mapping mutations on the two proteins that abolish binding. Using these mutants, we demonstrated that binding between AtcB and RNA polymerase is crucial for cold growth. Then, to investigate the potential role of the other Atc proteins in coordination with DnaK and AtcB, we leveraged the toxicity phenotype associated with AtcB overproduction. We discovered that the AtcJ–AtcC complex recruits DnaK, thereby suppressing AtcB toxicity by limiting the accumulation of inactive RNA polymerase. Our study demonstrates the coupling between DnaK and RNA polymerase, mediated by the Atc protein system, revealing how this network recruits the chaperone to modulate RNA polymerase activity. While the exact mechanism is still unclear, our study suggests the notion that this system represents a new DnaK-dependent bacterial transcriptional regulatory pathway involved in cold adaptation.
{"title":"Functional Coupling Between DnaK and Bacterial RNA Polymerase Through a Dedicated J-domain Protein System","authors":"Safa Boussouar , Amine Ali Chaouche , Yann Denis , Olivier Genest , Sébastien Dementin","doi":"10.1016/j.jmb.2025.169529","DOIUrl":"10.1016/j.jmb.2025.169529","url":null,"abstract":"<div><div>J-domain proteins (JDPs) are co-chaperones of Hsp70/DnaK, conferring a broad range of functions to the Hsp70/DnaK chaperone system. The shortest characterized JDP is AtcJ, which is part of an essential four-protein system dedicated to the cold adaptation of the bacterium <em>Shewanella oneidensis</em>. AtcJ connects DnaK to the Atc system that comprises also the proteins AtcA, AtcB, and AtcC. Previous studies have shown that AtcB interacts with RNA polymerase, and that its overproduction leads to toxicity, likely due to the inhibition of transcription. In this study, we explore the interplay between DnaK, the Atc proteins and RNA polymerase in order to elucidate the mechanism of their coordinated function. First, we identified the molecular determinants underlying the interaction between RNA polymerase and AtcB, mapping mutations on the two proteins that abolish binding. Using these mutants, we demonstrated that binding between AtcB and RNA polymerase is crucial for cold growth. Then, to investigate the potential role of the other Atc proteins in coordination with DnaK and AtcB, we leveraged the toxicity phenotype associated with AtcB overproduction. We discovered that the AtcJ–AtcC complex recruits DnaK, thereby suppressing AtcB toxicity by limiting the accumulation of inactive RNA polymerase. Our study demonstrates the coupling between DnaK and RNA polymerase, mediated by the Atc protein system, revealing how this network recruits the chaperone to modulate RNA polymerase activity. While the exact mechanism is still unclear, our study suggests the notion that this system represents a new DnaK-dependent bacterial transcriptional regulatory pathway involved in cold adaptation.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 24","pages":"Article 169529"},"PeriodicalIF":4.5,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145436864","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}
Pub Date : 2025-11-01Epub Date: 2025-08-07DOI: 10.1016/j.jmb.2025.169372
Lingshen Meng, Shangxiang Ye, Kai Pei, Chun Tang
Coronaviruses, including SARS-CoV-2, pose a significant threat to global health. A critical step in viral maturation involves the proteolytic processing of viral polyproteins into functional nonstructural proteins (NSPs), with NSP4 being specifically cleaved by the main protease, NSP5, to release mature components. Through an integrative approach combining X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations, we reveal that the C-terminal domain of NSP4 (NSP4-CTD) undergoes dynamic interconversion among multiple conformational states on distinct timescales. These states are characterized by variations in the position and secondary structure of the NSP4-CTD's C-terminal tail region, encompassing an undocked conformation, a docked extended conformation, and a docked helical conformation. We demonstrate that the formation of this C-terminal helix is influenced by both local sequence and overall structural context, playing a crucial role in positioning NSP4 relative to NSP5 and, consequently, modulating the efficiency of the autoprocessing event. While current antiviral therapeutic development has predominantly focused on targeting the mature NSP5 protease, our findings highlight the dynamic NSP4 C-terminal tail as a novel and promising target for antiviral intervention.
{"title":"Structural Dynamics of SARS-CoV-2 NSP4 C-terminal Domain and Implications for Viral Processing.","authors":"Lingshen Meng, Shangxiang Ye, Kai Pei, Chun Tang","doi":"10.1016/j.jmb.2025.169372","DOIUrl":"10.1016/j.jmb.2025.169372","url":null,"abstract":"<p><p>Coronaviruses, including SARS-CoV-2, pose a significant threat to global health. A critical step in viral maturation involves the proteolytic processing of viral polyproteins into functional nonstructural proteins (NSPs), with NSP4 being specifically cleaved by the main protease, NSP5, to release mature components. Through an integrative approach combining X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations, we reveal that the C-terminal domain of NSP4 (NSP4-CTD) undergoes dynamic interconversion among multiple conformational states on distinct timescales. These states are characterized by variations in the position and secondary structure of the NSP4-CTD's C-terminal tail region, encompassing an undocked conformation, a docked extended conformation, and a docked helical conformation. We demonstrate that the formation of this C-terminal helix is influenced by both local sequence and overall structural context, playing a crucial role in positioning NSP4 relative to NSP5 and, consequently, modulating the efficiency of the autoprocessing event. While current antiviral therapeutic development has predominantly focused on targeting the mature NSP5 protease, our findings highlight the dynamic NSP4 C-terminal tail as a novel and promising target for antiviral intervention.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169372"},"PeriodicalIF":4.5,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144787990","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}
Pub Date : 2025-11-01DOI: 10.1016/j.jmb.2025.169513
Agata D. Misiaszek, Jeffrey A. Chao
Once an mRNA is exported to the cytoplasm, a transcript’s fate is determined by the combinatorial interaction of a myriad of RNA-binding proteins that will determine its localization, translation and, eventual, degradation. To understand how these processes are regulated temporally and spatially in cells, a host of single-molecule fluorescent microscopy methodologies have been developed to interrogate different steps in the gene expression pathway. Here, we have highlighted the recent contributions of single-molecule measurements towards understanding the cytoplasmic lives of mRNAs and provide an outlook for future challenges and developments in the field.
{"title":"Imaging the Cytoplasmic Lives of Single mRNAs","authors":"Agata D. Misiaszek, Jeffrey A. Chao","doi":"10.1016/j.jmb.2025.169513","DOIUrl":"10.1016/j.jmb.2025.169513","url":null,"abstract":"<div><div>Once an mRNA is exported to the cytoplasm, a transcript’s fate is determined by the combinatorial interaction of a myriad of RNA-binding proteins that will determine its localization, translation and, eventual, degradation. To understand how these processes are regulated temporally and spatially in cells, a host of single-molecule fluorescent microscopy methodologies have been developed to interrogate different steps in the gene expression pathway. Here, we have highlighted the recent contributions of single-molecule measurements towards understanding the cytoplasmic lives of mRNAs and provide an outlook for future challenges and developments in the field.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"438 1","pages":"Article 169513"},"PeriodicalIF":4.5,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145436934","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}
Pub Date : 2025-11-01DOI: 10.1016/j.jmb.2025.169531
Yangang Pan , Jingyu Zhan , Zhaokun Wang , Yining Jiang , Di Xia , Simon Scheuring
Bcs1 is a AAA-ATPase that transports the Rieske iron-sulfur protein (ISP) across the inner mitochondrial membrane. Bcs1 is a particular molecular machine in many regards: In contrast to canonical, hexameric, soluble AAA-ATPases (i) Bcs1 is heptameric, (ii) Bcs1 is a transmembrane protein with each subunit featuring a transmembrane helix, (iii) Bcs1 transports ISP in its folded state, and (iv) Bcs1 works using a concerted mechanism and not a hand-over-hand or stochastic mechanism. How Bcs1 binds and transports folded ISP is unknown. Here we used high-speed atomic force microscopy (HS-AFM) single-molecule analysis and report that Bcs1 subunits are conformationally fully coupled: When Bcs1 is exposed to a mixture of AMP-PNP and ADP where the probability to be in the AMP-PNP or the ADP conformation is equal, all Bcs1 ring complexes are either in AMP-PNP or ADP state, and none forms a hetero-conformer ring. When Bcs1 is exposed to a mixture of AMP-PNP and ATP, traces of AMP-PNP inhibit Bcs1 action showing that all subunits in the ring must be compatible to hydrolyze ATP and undergo a conformational change for function. Furthermore, ISP binds exclusively to the matrix cavity of apo-conformation AAA-domains. Finally, ISP-Bcs1 binding is long enough to outlast the apo-conformation lifetime in ATP-turnover conditions, assuring high transport efficiency. Our single-molecule structural and kinetic data reveals that Bcs1 works according to a unique mechanism so far unknown for any AAA-ATPase.
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