Journal of Molecular Biology最新文献_第10页

Sequence Determinants of Allosteric Back-to-front Control of the Arf Nucleotide Switch Arf核苷酸开关变构前后控制的序列决定因素。

IF 4.5 2区生物学

Journal of Molecular Biology Pub Date : 2025-07-26 DOI: 10.1016/j.jmb.2025.169361

Noam Hantman , Tejaswi Koduru , Edgar V. Peters , Michel W. Jaworek , Scott A. McCallum , Richard E. Gillilan , Roland Winter , Jacqueline Cherfils , Catherine A. Royer

{"title":"Sequence Determinants of Allosteric Back-to-front Control of the Arf Nucleotide Switch","authors":"Noam Hantman , Tejaswi Koduru , Edgar V. Peters , Michel W. Jaworek , Scott A. McCallum , Richard E. Gillilan , Roland Winter , Jacqueline Cherfils , Catherine A. Royer","doi":"10.1016/j.jmb.2025.169361","DOIUrl":"10.1016/j.jmb.2025.169361","url":null,"abstract":"<div><div>Unlike AI-based protein structure prediction, the sequence determinants of protein dynamics, and thus function, remain elusive. The nucleotide switch in Arf GTPases involves a massive structural change, which we showed recently in Arf1 is facilitated by a dynamic molten globule ensemble. Here we investigate the unresolved sequence-dynamics paradigm by comparing Arf1 and Arf6 using a combination of high-pressure NMR and other biophysical methods. We show that, as for Arf1, the Arf6 nucleotide switch implicates a functional molten globule ensemble, suggesting that this mechanism is a general feature of Arf and Arf-like GTPases. Comparison of the local stability distributions identifies key sequence determinants for the differences in switching between Arf1 and Arf6. Remarkably, these determinants are distant from the nucleotide-binding site, revealing back-to-front allosteric coupling controlling the switch. Evolutionary covariance analysis suggests that this back-to-front allosteric coupling is a fundamental characteristic of the Arf and Arf-like family, and may extend to the entire small GTPase kingdom. Our study thus establishes notable sequence-dynamics relationships with implications for signaling and diseases.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 19","pages":"Article 169361"},"PeriodicalIF":4.5,"publicationDate":"2025-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144726324","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}

引用次数: 0

NMR Insights Into the Structural Dynamics of p47, A Key Adaptor Protein of p97 in Golgi Reassembly. 高尔基重组中p97的关键接头蛋白p47结构动力学的NMR研究。

IF 4.5 2区生物学

Journal of Molecular Biology Pub Date : 2025-07-26 DOI: 10.1016/j.jmb.2025.169362

Megan K Black, Rajivan Raseekan, Paige Kanters, Peter Kim, Rui Huang

{"title":"NMR Insights Into the Structural Dynamics of p47, A Key Adaptor Protein of p97 in Golgi Reassembly.","authors":"Megan K Black, Rajivan Raseekan, Paige Kanters, Peter Kim, Rui Huang","doi":"10.1016/j.jmb.2025.169362","DOIUrl":"10.1016/j.jmb.2025.169362","url":null,"abstract":"<p><p>The Golgi apparatus undergoes systematic disassembly and reassembly during the cell cycle, a process requiring membrane fusion mediated by the AAA+ ATPase p97/VCP and its adaptor p47. While the p97-p47 complex plays a pivotal role in post-mitotic Golgi reassembly, the exact molecular mechanism underlying its function has not been completely understood. In particular, the conformational flexibility and dynamic feature of p47 hinders its structural characterization by cryo-electron microscopy and X-ray crystallography. Here, using NMR spectroscopy, we characterize the conformational dynamics of p47 and investigate its intra- and intermolecular interactions. p47 consists of three folded domains connected by intrinsically disordered regions (IDRs). We show that p47 adopts a \"beads-on-a-string\" arrangement and identify several regions that undergo microsecond-to-millisecond timescale motions, which may have functional significance. Using paramagnetic relaxation enhancement (PRE) experiments, we capture transient inter-domain and domain-linker interactions, gaining insights into the structural organization of the domains and linkers in p47. Notably, we identify and characterize an intramolecular interaction between a SEP-interacting motif (SIM), located on the flexible linker, and the SEP domain, suggesting a conserved structural and functional feature among SEP-containing p97 adaptors. Additionally, we observed transient intermolecular interactions between p47 molecules, primarily mediated by the SEP domain. These findings provide a comprehensive view of the structural organization and dynamics of p47, shedding light on how its modular architecture and multivalent interactions may modulate p97 activity and assist Golgi membrane reassembly.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169362"},"PeriodicalIF":4.5,"publicationDate":"2025-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144726323","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}

引用次数: 0

Structural and Functional Characterization of Pseudomonas aeruginosa Virulence Factor AaaA, an Autotransporter with Arginine-Specific Aminopeptidase Activity 具有精氨酸特异性氨基肽酶活性的铜绿假单胞菌毒力因子AaaA的结构和功能特征。

IF 4.5 2区生物学

Journal of Molecular Biology Pub Date : 2025-07-25 DOI: 10.1016/j.jmb.2025.169358

Erandi Jayawardana Arachchige , Md Shafiqur Rahman , Katharina S. Singendonk, Kelly H. Kim

{"title":"Structural and Functional Characterization of Pseudomonas aeruginosa Virulence Factor AaaA, an Autotransporter with Arginine-Specific Aminopeptidase Activity","authors":"Erandi Jayawardana Arachchige , Md Shafiqur Rahman , Katharina S. Singendonk, Kelly H. Kim","doi":"10.1016/j.jmb.2025.169358","DOIUrl":"10.1016/j.jmb.2025.169358","url":null,"abstract":"<div><div>AaaA is a virulence-associated outer membrane protein found in the Gram-negative pathogen <em>Pseudomonas aeruginosa</em>. Classified as both an autotransporter and a member of the M28 family of aminopeptidases, AaaA has been shown to cleave N-terminal arginine residues from host-derived peptides. This activity has been demonstrated to enhance bacterial survival and suppress host immune responses by increasing local arginine availability. Here, we report the first successful purification and combined structural and biochemical characterization of full-length AaaA. We resolved its cryo-EM structure at 3.87 Å resolution, revealing the canonical three-domain architecture of autotransporters: a signal peptide, a passenger domain, and a translocator domain. Notably, the passenger domain adopts a compact globular fold characteristic of M28 aminopeptidases, which is less common than the extended or β-helical structures observed in the majority of autotransporters structurally characterized to date. The structure reveals a zinc-coordinated catalytic site and a negatively charged substrate binding pocket, consistent with specificity for positively charged N-terminal arginine residues. Mutagenesis of active site residues confirmed the molecular basis for arginine recognition. Functional assays demonstrated that AaaA exhibits zinc-dependent aminopeptidase activity across a broad pH (6–10) and temperature (20–60 °C) range. Together, these findings provide fundamental insights into the structure and function of AaaA and establish a framework for future efforts to develop targeted inhibitors that may attenuate <em>P. aeruginosa</em> virulence.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 19","pages":"Article 169358"},"PeriodicalIF":4.5,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144726325","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}

引用次数: 0

Synergistic Methods to Investigate Eukaryotic Translation and Its Quality Control. 真核生物翻译及其质量控制的协同研究方法。

IF 4.5 2区生物学

Journal of Molecular Biology Pub Date : 2025-07-24 DOI: 10.1016/j.jmb.2025.169355

Aubrey J Emmi, Adrian S McFarland, Morgan M Grimes, Izaiah J Cole, Michael R Lawson

引用次数: 0

A Disassembly Intermediate of a Non-enveloped Virus Indicates the Pathway of Genome Release. 非包膜病毒的分解中间体表明了基因组释放的途径。

IF 4.5 2区生物学

Journal of Molecular Biology Pub Date : 2025-07-24 DOI: 10.1016/j.jmb.2025.169354

Milan Kumar Lokshman, Kirti Suhag, Devbrat Kumar, Subhomoi Borkotoky, Manidipa Banerjee

{"title":"A Disassembly Intermediate of a Non-enveloped Virus Indicates the Pathway of Genome Release.","authors":"Milan Kumar Lokshman, Kirti Suhag, Devbrat Kumar, Subhomoi Borkotoky, Manidipa Banerjee","doi":"10.1016/j.jmb.2025.169354","DOIUrl":"10.1016/j.jmb.2025.169354","url":null,"abstract":"<p><p>Disassembly of non-enveloped viruses in vivo are typically triggered by cellular factors such as host receptor binding, low pH in the early or late endosomal compartments, protease action in lysosomes, and localized changes in ionic concentrations. These triggers induce alterations in metastable capsids, resulting in the exposure of flexible capsid components and opening of gaps for genome release. Structural analysis of intermediate states is required to understand alterations in protein-protein and RNA-protein contacts in the pathway of capsid destabilization. Obtaining structural details of intermediates requires recreation of the in vivo transition states in stable forms, stepwise, in vitro. Here, we generated an asymmetric reconstruction of an early intermediate state in the disassembly pathway of Flock House Virus, a T = 3 icosahedral insect virus that is a model system for similar-sized non-enveloped viruses. The early intermediate was generated through judicious application, in vitro, of in vivo conditions such as receptor-binding-related transition and endosomal pH. The early intermediate showed asymmetric expansion, as well as asymmetric dynamic movement of the pocket factor, disordering of flexible membrane penetrating peptides and opening of gaps at the 2-fold axis, indicating that disassembly-related structural alterations may be local and not transpire throughout the icosahedral capsid. Surprisingly, the genomic RNA underwent a dramatic conformational alteration which superseded the relatively more subtle changes in the protein component. Recreation of disassembly-related transition states in vitro may be essential for structure-targeted, broadly effective inactivation strategies for non-enveloped viruses.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169354"},"PeriodicalIF":4.5,"publicationDate":"2025-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144717205","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}

引用次数: 0

A Reconstituted Capsid-Nucleus Platform Uncovers Distinct Roles of UL25 and UL36 in Herpesvirus Capsid Docking. 重组衣壳-核平台揭示UL25和UL36在疱疹病毒衣壳对接中的独特作用

IF 4.5 2区生物学

Journal of Molecular Biology Pub Date : 2025-07-23 DOI: 10.1016/j.jmb.2025.169356

Efthymios Tsimtsirakis, José Ramón Villanueva Valencia, Jamie B Huffman, Fred Homa, Alex Evilevitch

{"title":"A Reconstituted Capsid-Nucleus Platform Uncovers Distinct Roles of UL25 and UL36 in Herpesvirus Capsid Docking.","authors":"Efthymios Tsimtsirakis, José Ramón Villanueva Valencia, Jamie B Huffman, Fred Homa, Alex Evilevitch","doi":"10.1016/j.jmb.2025.169356","DOIUrl":"10.1016/j.jmb.2025.169356","url":null,"abstract":"<p><p>Herpes simplex virus type 1 (HSV-1) delivers its genome into the host nucleus via docking of the viral capsid at the nuclear pore complex (NPC), a process mediated by the capsid vertex-specific complex (CVSC), composed of UL17, UL25, and UL36. While prior in vivo studies have implicated UL25 and UL36 in NPC interactions, their interpretation is complicated by indirect effects from viral trafficking, host responses, and capsid stability defects resulting from CVSC protein deletions. To isolate the nuclear docking step, we employed a reconstituted in vitro system using purified fluorescent HSV-1 capsids (wild-type, ΔUL25, ΔUL36) and isolated mammalian nuclei. Quantitative confocal imaging analysis revealed a marked reduction in docking upon UL36 deletion, while UL25 deletion similarly impaired binding, likely due to loss of UL36 incorporation and compromised capsid integrity. These findings suggest UL36 is a key mediator of NPC engagement, with UL25 supporting CVSC assembly and stability. This reductionist platform enables precise dissection of herpesvirus nuclear entry mechanisms.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169356"},"PeriodicalIF":4.5,"publicationDate":"2025-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144717206","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}

引用次数: 0

The Specificity of RNA Packaging in Isometric RNA Plant Viruses is Principally Determined by Replication. 在等长RNA植物病毒中，RNA包装的特异性主要由复制决定。

IF 4.5 2区生物学

Journal of Molecular Biology Pub Date : 2025-07-22 DOI: 10.1016/j.jmb.2025.169352

Keith Saunders, Sachin N Shah, Hadrien Peyret, Yulia Meshcheriakova, Jake Richardson, Sandra Eltschkner, David M Lawson, George P Lomonossoff

{"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":"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":4.5,"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}

引用次数: 0

Rad23B Delays Ataxin-3 Liquid-to-solid Phase Transition Through Heterotypic Buffering Rad23B通过异型缓冲延迟Ataxin-3液相到固相转变。

IF 4.5 2区生物学

Journal of Molecular Biology Pub Date : 2025-07-18 DOI: 10.1016/j.jmb.2025.169351

Archana Prasad, Sandhini Saha, Manisha Kumari, Krishna Singh Bisht, Tushar Kanti Maiti

{"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":"10.1016/j.jmb.2025.169351","url":null,"abstract":"<div><div>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 <em>via</em> 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.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 19","pages":"Article 169351"},"PeriodicalIF":4.5,"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}

引用次数: 0

LncRNA SChLAP1 Promotes Cancer Cell Proliferation and Invasion Via Its Distinct Structural Domains and Conserved Regions LncRNA SChLAP1通过其独特的结构域和保守区域促进癌细胞的增殖和侵袭。

IF 4.5 2区生物学

Journal of Molecular Biology Pub Date : 2025-07-17 DOI: 10.1016/j.jmb.2025.169350

Mihyun Oh , Roshni Nagesh Kadam , Zahra Sadruddin Charania , Srinivas Somarowthu

{"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":"<div><div>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 <em>in vitro</em> 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 <em>in vivo</em> 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.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 19","pages":"Article 169350"},"PeriodicalIF":4.5,"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}

引用次数: 0

Saccharomyces cerevisiae Xrs2 Binds DNA Through Its FHA Domain. 酿酒葡萄球菌Xrs2通过其FHA结构域与DNA结合。

IF 4.5 2区生物学

Journal of Molecular Biology Pub Date : 2025-07-16 DOI: 10.1016/j.jmb.2025.169348

Ajeak Vigneswaran, Marella D Canny, Stephan B Azatian, Michael P Latham

{"title":"Saccharomyces 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":"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":4.5,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12313241/pdf/","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0