Pub Date : 2025-11-17DOI: 10.1016/j.jmb.2025.169549
Pei-Yu Hsieh , Ksenija Romane , Julia Kowal , Kaspar P. Locher , Hendrik W. van Veen
Multidrug and toxic compound extrusion (MATE) transport proteins contribute to multidrug resistance in human pathogens by extruding various cytotoxic compounds from the cellular interior. Despite their importance across all domains of life, the specificities and mechanisms of substrate transport of these proteins remain poorly understood due to limited structural and functional information. Here, we determined the cryo-electron microscopy structure of NorM from Vibrio cholerae (NorM-VC) in complex with the anthracycline antibiotic doxorubicin, using the NabFab approach. The structure reveals that the doxorubicin-binding pocket is located halfway through the membrane, within the C-lobe of the protein. Functional studies targeting the doxorubicin-interacting residues validated the binding pocket and enabled detailed analysis of the doxorubicin transport reaction. Our findings indicate doxorubicin binding within a multisite binding chamber engaged in a general transport mechanism for a variety of substrates.
{"title":"Doxorubicin Recognition and Transport by the MATE Multidrug Transporter NorM From Vibrio cholerae","authors":"Pei-Yu Hsieh , Ksenija Romane , Julia Kowal , Kaspar P. Locher , Hendrik W. van Veen","doi":"10.1016/j.jmb.2025.169549","DOIUrl":"10.1016/j.jmb.2025.169549","url":null,"abstract":"<div><div>Multidrug and toxic compound extrusion (MATE) transport proteins contribute to multidrug resistance in human pathogens by extruding various cytotoxic compounds from the cellular interior. Despite their importance across all domains of life, the specificities and mechanisms of substrate transport of these proteins remain poorly understood due to limited structural and functional information. Here, we determined the cryo-electron microscopy structure of NorM from <em>Vibrio cholerae</em> (NorM-VC) in complex with the anthracycline antibiotic doxorubicin, using the NabFab approach. The structure reveals that the doxorubicin-binding pocket is located halfway through the membrane, within the C-lobe of the protein. Functional studies targeting the doxorubicin-interacting residues validated the binding pocket and enabled detailed analysis of the doxorubicin transport reaction. Our findings indicate doxorubicin binding within a multisite binding chamber engaged in a general transport mechanism for a variety of substrates.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"438 2","pages":"Article 169549"},"PeriodicalIF":4.5,"publicationDate":"2025-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145556012","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 : 2025-11-15DOI: 10.1016/j.jmb.2025.169545
Justin C. Wheat , Robert A. Coleman , Ulrich Steidl
Hematopoiesis, or the process of blood production, has long served as a prototype for stem cell biology research owing to the relative ease of obtaining blood as well as functional testing in vitro and in vivo (through stem cell transplantation). One central question in the field over the last century is how the various forms and phenotypes of blood cells arise from a common pool of hematopoietic stem cells. Key to that exercise have been in depth studies into how the genes required for cell differentiation are expressed and regulated. Paramount to these efforts have been single cell analysis techniques, which have helped resolve heterogeneity at multiple levels of hematopoietic regulation. Recently, as major advances in the fields of quantitative microscopy and systems biology have revolutionized modern molecular biology, the hematopoietic research community has begun to employ these techniques, leading to significant advances in our understanding of blood production. In this review, we discuss the historical role imaging has played in developing heuristics in the field of hematopoiesis research and, importantly, discuss the exciting areas being explored with novel and innovative technologies. Finally, we close with a discussion of the main challenges that will face the field going forward.
{"title":"Gene Expression Imaging in Hematopoiesis","authors":"Justin C. Wheat , Robert A. Coleman , Ulrich Steidl","doi":"10.1016/j.jmb.2025.169545","DOIUrl":"10.1016/j.jmb.2025.169545","url":null,"abstract":"<div><div>Hematopoiesis, or the process of blood production, has long served as a prototype for stem cell biology research owing to the relative ease of obtaining blood as well as functional testing in vitro and in vivo (through stem cell transplantation). One central question in the field over the last century is how the various forms and phenotypes of blood cells arise from a common pool of hematopoietic stem cells. Key to that exercise have been in depth studies into how the genes required for cell differentiation are expressed and regulated. Paramount to these efforts have been single cell analysis techniques, which have helped resolve heterogeneity at multiple levels of hematopoietic regulation. Recently, as major advances in the fields of quantitative microscopy and systems biology have revolutionized modern molecular biology, the hematopoietic research community has begun to employ these techniques, leading to significant advances in our understanding of blood production. In this review, we discuss the historical role imaging has played in developing heuristics in the field of hematopoiesis research and, importantly, discuss the exciting areas being explored with novel and innovative technologies. Finally, we close with a discussion of the main challenges that will face the field going forward.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"438 1","pages":"Article 169545"},"PeriodicalIF":4.5,"publicationDate":"2025-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145534072","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 : 2025-11-14DOI: 10.1016/j.jmb.2025.169546
Qamar Shhade , Marianna E. Estrada , Rawad Hanna , Muhammad Makhzumy , Hagit Bar-Yosef , Guenter Kramer , Bernd Bukau , Ayala Shiber
Many newly synthesized proteins assemble co-translationally, providing a vital mechanism to prevent subunit misfolding in the crowded cytoplasm. Initial evidence indicates that the spatial organization of mRNAs aids this assembly, but it is unclear how these mRNAs are organized and how common this mechanism is. We used single-molecule Fluorescence in situ Hybridization in Saccharomyces cerevisiae to examine the spatial organization of mRNAs encoding subunits of various cytosolic complexes involved in critical cellular functions, such as fatty acid synthesis, glycolysis, translation and various amino acid biosynthesis. We found that mRNAs of the same protein complex often co-localize in specific cytoplasmic clusters. Additionally, we observed that the mRNAs encoding enzymes of biosynthetic pathways are organized in cytosolic clusters. Focusing on mRNAs encoding fatty acid synthase complex subunits, we discovered that non-coding cis elements significantly influence mRNA localization in an additive manner. Specifically, 5′ and 3′ UTRs, together with further upstream and downstream regions, facilitate co-localization. Inhibiting mRNA co-localization impaired growth when complex activity was essential, highlighting the importance of mRNA spatial organization for cellular survival. Transiently disrupting mRNA translation also affected clustering, indicating that both the nascent chains and mRNA sequence targeting cues are combinatorically contributing to spatial organization. Proteomics analysis demonstrates the impact of cis-elements on the abundance of the encoded subunits, as well as the entire pathway. In summary, we provide evidence that mRNA co-localization in cytoplasmic foci is coordinated by complementary mechanisms crucial for co-translational assembly, allowing efficient regulation of protein complex formation and entire pathways.
{"title":"Distinct Cis-acting Elements Combinatorically Mediate Co-localization of mRNAs Encoding for Co-translational Interactors in Cytoplasmic Clusters in S. cerevisiae","authors":"Qamar Shhade , Marianna E. Estrada , Rawad Hanna , Muhammad Makhzumy , Hagit Bar-Yosef , Guenter Kramer , Bernd Bukau , Ayala Shiber","doi":"10.1016/j.jmb.2025.169546","DOIUrl":"10.1016/j.jmb.2025.169546","url":null,"abstract":"<div><div>Many newly synthesized proteins assemble co-translationally, providing a vital mechanism to prevent subunit misfolding in the crowded cytoplasm. Initial evidence indicates that the spatial organization of mRNAs aids this assembly, but it is unclear how these mRNAs are organized and how common this mechanism is. We used single-molecule Fluorescence in situ Hybridization in <em>Saccharomyces cerevisiae</em> to examine the spatial organization of mRNAs encoding subunits of various cytosolic complexes involved in critical cellular functions, such as fatty acid synthesis, glycolysis, translation and various amino acid biosynthesis. We found that mRNAs of the same protein complex often co-localize in specific cytoplasmic clusters. Additionally, we observed that the mRNAs encoding enzymes of biosynthetic pathways are organized in cytosolic clusters. Focusing on mRNAs encoding fatty acid synthase complex subunits, we discovered that non-coding cis elements significantly influence mRNA localization in an additive manner. Specifically, 5′ and 3′ UTRs, together with further upstream and downstream regions, facilitate co-localization. Inhibiting mRNA co-localization impaired growth when complex activity was essential, highlighting the importance of mRNA spatial organization for cellular survival. Transiently disrupting mRNA translation also affected clustering, indicating that both the nascent chains and mRNA sequence targeting cues are combinatorically contributing to spatial organization. Proteomics analysis demonstrates the impact of <em>cis</em>-elements on the abundance of the encoded subunits, as well as the entire pathway. In summary, we provide evidence that mRNA co-localization in cytoplasmic foci is coordinated by complementary mechanisms crucial for co-translational assembly, allowing efficient regulation of protein complex formation and entire pathways.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"438 2","pages":"Article 169546"},"PeriodicalIF":4.5,"publicationDate":"2025-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145534035","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 : 2025-11-14DOI: 10.1016/j.jmb.2025.169547
Vladimir N Uversky
Extremophiles are organisms adapted not only to survive in harsh conditions but thrive in environments considered extreme or uninhabitable by most life forms. They serve as a living reflection of a popular motto "What doesn't kill you makes you stronger" and show that life on Earth is spread well outside the human-centric comfort zone. In fact, extremophiles, being found in deep crustal and oceanic depths, outer space, highly acidic and basic conditions, extreme temperatures (+122 °C to -20 °C), and in the presence of toxins and high radiation, demonstrate life's remarkable adaptability. Polyextremophiles, capable of enduring multiple extreme conditions, further highlight this adaptability, whereas super-extremophiles, exhibiting resilience in seemingly impossible environments, showcase the extraordinary capacity of life to endure and thrive. Even though they inhabit vastly different environments, extremophiles, polyextremophiles, and super-extremophiles are all Earth organisms. They are made of the same elements found on our planet and share basic biological similarities with other life forms. Their survival in extreme conditions is due to adaptations and changes in their existing cellular structures and metabolic processes, and not because they use entirely new fundamental components or chemical reactions. While extremophiles employ a multitude of factors, mechanisms, and survival strategies to withstand harsh environments, this study addresses a specific aspect of extremophile adaptation by concentrating solely on the functions of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs).
{"title":"Protein Intrinsic Disorder and Adaptation to Extreme Environments: Resilience of Chaos.","authors":"Vladimir N Uversky","doi":"10.1016/j.jmb.2025.169547","DOIUrl":"10.1016/j.jmb.2025.169547","url":null,"abstract":"<p><p>Extremophiles are organisms adapted not only to survive in harsh conditions but thrive in environments considered extreme or uninhabitable by most life forms. They serve as a living reflection of a popular motto \"What doesn't kill you makes you stronger\" and show that life on Earth is spread well outside the human-centric comfort zone. In fact, extremophiles, being found in deep crustal and oceanic depths, outer space, highly acidic and basic conditions, extreme temperatures (+122 °C to -20 °C), and in the presence of toxins and high radiation, demonstrate life's remarkable adaptability. Polyextremophiles, capable of enduring multiple extreme conditions, further highlight this adaptability, whereas super-extremophiles, exhibiting resilience in seemingly impossible environments, showcase the extraordinary capacity of life to endure and thrive. Even though they inhabit vastly different environments, extremophiles, polyextremophiles, and super-extremophiles are all Earth organisms. They are made of the same elements found on our planet and share basic biological similarities with other life forms. Their survival in extreme conditions is due to adaptations and changes in their existing cellular structures and metabolic processes, and not because they use entirely new fundamental components or chemical reactions. While extremophiles employ a multitude of factors, mechanisms, and survival strategies to withstand harsh environments, this study addresses a specific aspect of extremophile adaptation by concentrating solely on the functions of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs).</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169547"},"PeriodicalIF":4.5,"publicationDate":"2025-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145534015","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 : 2025-11-14DOI: 10.1016/j.jmb.2025.169548
Karolina Mikulska-Ruminska , Matthew Licht , Mehmed Z. Ertem , John Shanklin , Qun Liu , Ivet Bahar
The alkane monooxygenase AlkB and rubredoxin AlkG form an electron transfer complex that hydroxylates terminal alkanes to produce alcohols. The recent cryoEM study of Fontimonas thermophila AlkB–AlkG complex revealed its architecture, including a dodecane (D12) substrate at the active site. However, FtAlkBG molecular mechanism of action remains unknown. Here, we examined its dynamics and interactions by multiscale computations, including molecular dynamics simulations, elastic network models, and QM/MM of the oxygen activation mechanism at the AlkB catalytic site. D12 maintained stable interactions within the catalytic site during two MD runs, coordinated by hydrophobic residues L263–L264, I267, I133. A third extended run revealed that D12 could translocate to a membrane-exposed site near S49/F46 along a hydrophobic channel gated by I54. During this translocation, D12 was temporarily stabilized at intermediate sites IS1 (lined by I27/L30–G31/G50/L53–I54/P59/S124/A127–V128) and IS2 (I33–G34/L37/L45–F46/S49) before nearly exiting the protein, and diffused back to the active site, assisted by L30. Substrate binding and translocation across those intermediate sites affects the coupling between the iron centers in AlkBG, and interfacial interactions between AlkB–AlkG. The channel was further connected to the cytosol, near two surface-exposed arginines, potentially allowing for O2 passage. The allosteric effects between D12 putative entry site, catalytic site and AlkB–AlkG interface were analyzed by ENM-based methods which confirmed the cooperative perturbation responses and strongly correlated movements of residues belonging to those distal regions. Our study provides new mechanistic insights into key sites and their interactions that could be targeted for developing AlkB-variants with desirable alkane conversion functions.
{"title":"Binding and Translocation of Substrate Allosterically Promotes Functional Interactions Within the AlkB–AlkG Electron Transfer Complex","authors":"Karolina Mikulska-Ruminska , Matthew Licht , Mehmed Z. Ertem , John Shanklin , Qun Liu , Ivet Bahar","doi":"10.1016/j.jmb.2025.169548","DOIUrl":"10.1016/j.jmb.2025.169548","url":null,"abstract":"<div><div>The alkane monooxygenase AlkB and rubredoxin AlkG form an electron transfer complex that hydroxylates terminal alkanes to produce alcohols. The recent cryoEM study of <em>Fontimonas thermophila</em> AlkB–AlkG complex revealed its architecture, including a dodecane (D12) substrate at the active site. However, FtAlkBG molecular mechanism of action remains unknown. Here, we examined its dynamics and interactions by multiscale computations, including molecular dynamics simulations, elastic network models, and QM/MM of the oxygen activation mechanism at the AlkB catalytic site. D12 maintained stable interactions within the catalytic site during two MD runs, coordinated by hydrophobic residues L263–L264, I267, I133. A third extended run revealed that D12 could translocate to a membrane-exposed site near S49/F46 along a hydrophobic channel gated by I54. During this translocation, D12 was temporarily stabilized at intermediate sites <em>IS1</em> (lined by I27/L30–G31/G50/L53–I54/P59/S124/A127–V128) and <em>IS2</em> (I33–G34/L37/L45–F46/S49) before nearly exiting the protein, and diffused back to the active site, assisted by L30. Substrate binding and translocation across those intermediate sites affects the coupling between the iron centers in AlkBG, and interfacial interactions between AlkB–AlkG. The channel was further connected to the cytosol, near two surface-exposed arginines, potentially allowing for O<sub>2</sub> passage. The allosteric effects between D12 putative entry site, catalytic site and AlkB–AlkG interface were analyzed by ENM-based methods which confirmed the cooperative perturbation responses and strongly correlated movements of residues belonging to those distal regions. Our study provides new mechanistic insights into key sites and their interactions that could be targeted for developing AlkB-variants with desirable alkane conversion functions.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"438 2","pages":"Article 169548"},"PeriodicalIF":4.5,"publicationDate":"2025-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145534009","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 : 2025-11-13DOI: 10.1016/j.jmb.2025.169544
Osamu Tani , Tomomi Kubota , Tomoko Yamasaki , Takatsugu Hirokawa , Koji Furukawa , Kazuhiko Yamasaki
Dihydroorotate dehydrogenase (DHODH), containing a cofactor, flavin mononucleotide (FMN), catalyzes the oxidation of dihydroorotate to orotate. In Trypanosoma brucei, the causative parasite of African trypanosomiasis, the DHODH enzyme (TbDHODH) couples this reaction with the reduction of fumarate to succinate. These reactions occur successively in the same catalytic site through a ping-pong bi–bi mechanism. FMN is reduced in the first step and re-oxidized in the second one. Here, we investigated how the redox state of FMN influences the binding of substrates and products to TbDHODH by NMR analyses of a catalytically impaired mutant. The affinities for the substrates, dihydroorotate and fumarate, were redox-dependent: dihydroorotate preferred the oxidized state, whereas fumarate preferred the reduced state. One of the products, succinate, exhibited no detectable binding to either of the redox states, suggesting its efficient product release. In contrast, the product of the first-half reaction, orotate, showed stronger binding to the oxidized state, suggesting that product inhibition may occur unless orotate is rapidly consumed in the subsequent biosynthetic step. The structural basis of these findings was elucidated by crystallography of the complexes with the above ligands in both redox states, along with quantitative analyses of the interactions using quantum chemical calculations. Upon reduction, the FMN isoalloxazine ring gains two additional hydrogens and becomes more bent at its center. These changes perturb the interactions among the ligands, flavin, and surrounding residues (notably Lys44, Asn68, and Asn195), primarily in the electrostatic term, which quantitatively explains the difference in binding affinities.
{"title":"Structural Basis of Redox-Dependent Affinities of Dihydroorotate Dehydrogenase for Its Substrates and Products","authors":"Osamu Tani , Tomomi Kubota , Tomoko Yamasaki , Takatsugu Hirokawa , Koji Furukawa , Kazuhiko Yamasaki","doi":"10.1016/j.jmb.2025.169544","DOIUrl":"10.1016/j.jmb.2025.169544","url":null,"abstract":"<div><div>Dihydroorotate dehydrogenase (DHODH), containing a cofactor, flavin mononucleotide (FMN), catalyzes the oxidation of dihydroorotate to orotate. In <em>Trypanosoma brucei</em>, the causative parasite of African trypanosomiasis, the DHODH enzyme (TbDHODH) couples this reaction with the reduction of fumarate to succinate. These reactions occur successively in the same catalytic site through a ping-pong bi–bi mechanism. FMN is reduced in the first step and re-oxidized in the second one. Here, we investigated how the redox state of FMN influences the binding of substrates and products to TbDHODH by NMR analyses of a catalytically impaired mutant. The affinities for the substrates, dihydroorotate and fumarate, were redox-dependent: dihydroorotate preferred the oxidized state, whereas fumarate preferred the reduced state. One of the products, succinate, exhibited no detectable binding to either of the redox states, suggesting its efficient product release. In contrast, the product of the first-half reaction, orotate, showed stronger binding to the oxidized state, suggesting that product inhibition may occur unless orotate is rapidly consumed in the subsequent biosynthetic step. The structural basis of these findings was elucidated by crystallography of the complexes with the above ligands in both redox states, along with quantitative analyses of the interactions using quantum chemical calculations. Upon reduction, the FMN isoalloxazine ring gains two additional hydrogens and becomes more bent at its center. These changes perturb the interactions among the ligands, flavin, and surrounding residues (notably Lys44, Asn68, and Asn195), primarily in the electrostatic term, which quantitatively explains the difference in binding affinities.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"438 2","pages":"Article 169544"},"PeriodicalIF":4.5,"publicationDate":"2025-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145530301","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 : 2025-11-12DOI: 10.1016/j.jmb.2025.169543
Raymond Hartman , Ashleigh Blane , Fillmon Kubrom , Riyaadh Mayet , J. Carlos Penedo , Sylvia Fanucchi
TBR1 is a transcription factor critical for brain development that recognises a specific nucleotide sequence, the T-box binding element (TBE). Dysregulation or mutation of TBR1 is linked to a spectrum of neurodevelopmental disorders, including autism, speech delay, and intellectual disability, yet the molecular mechanism underlying its DNA recognition remains poorly defined. Here, we examine how the TBR1 T-box domain recognises DNA containing either a single TBE or a palindromic arrangement of two adjacent TBEs. Both sequences are derived from naturally occurring TBR1 binding motifs located within genomic regions annotated as candidate enhancers of autism-associated genes. The TBR1 T-box binds its cognate element in a sequence-specific, enthalpically driven, and entropically opposed manner. Single-molecule Förster resonance energy transfer (smFRET) reveals that the single-site sequence binds one monomer, whereas the palindromic sequence can recruit a second monomer and exhibits additional dynamic events unique to this architecture. These include short-range transitions of a monomer along the palindromic DNA before dissociation or before the arrival of a second monomer. Molecular docking and dynamics simulations support these observations, predicting fewer stabilising contacts per TBE and hence greater conformational flexibility for the palindromic complex. Together, our findings reveal how DNA architecture modulates affinity and kinetics within the T-box family: single TBEs promote stable monomeric binding, whereas palindromic arrangements enable dual occupancy and dynamic exchange. This study provides the first detailed mechanistic insight into DNA recognition by a T-box transcription factor, advancing understanding of its role in neurodevelopmental gene regulation.
{"title":"Molecular Insights Into the Binding Dynamics of Transcription Factor TBR1 to T-box DNA Sequences","authors":"Raymond Hartman , Ashleigh Blane , Fillmon Kubrom , Riyaadh Mayet , J. Carlos Penedo , Sylvia Fanucchi","doi":"10.1016/j.jmb.2025.169543","DOIUrl":"10.1016/j.jmb.2025.169543","url":null,"abstract":"<div><div>TBR1 is a transcription factor critical for brain development that recognises a specific nucleotide sequence, the T-box binding element (TBE). Dysregulation or mutation of TBR1 is linked to a spectrum of neurodevelopmental disorders, including autism, speech delay, and intellectual disability, yet the molecular mechanism underlying its DNA recognition remains poorly defined. Here, we examine how the TBR1 T-box domain recognises DNA containing either a single TBE or a palindromic arrangement of two adjacent TBEs. Both sequences are derived from naturally occurring TBR1 binding motifs located within genomic regions annotated as candidate enhancers of autism-associated genes. The TBR1 T-box binds its cognate element in a sequence-specific, enthalpically driven, and entropically opposed manner. Single-molecule Förster resonance energy transfer (smFRET) reveals that the single-site sequence binds one monomer, whereas the palindromic sequence can recruit a second monomer and exhibits additional dynamic events unique to this architecture. These include short-range transitions of a monomer along the palindromic DNA before dissociation or before the arrival of a second monomer. Molecular docking and dynamics simulations support these observations, predicting fewer stabilising contacts per TBE and hence greater conformational flexibility for the palindromic complex. Together, our findings reveal how DNA architecture modulates affinity and kinetics within the T-box family: single TBEs promote stable monomeric binding, whereas palindromic arrangements enable dual occupancy and dynamic exchange. This study provides the first detailed mechanistic insight into DNA recognition by a T-box transcription factor, advancing understanding of its role in neurodevelopmental gene regulation.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"438 2","pages":"Article 169543"},"PeriodicalIF":4.5,"publicationDate":"2025-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145522592","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 : 2025-11-12DOI: 10.1016/j.jmb.2025.169535
Mengqiu Wang , Zhiwei Zhang , Xinxin Zhang , Ruoyan Dai , Zhenghui Wang , Zeyao Chen , Lixin Lei , Zhenxing Li , Qianjin Guo
Recent advances in spatial transcriptomics (ST) have significantly enhanced our understanding of tissue structure and intercellular interactions. However, existing methods for spatial domain identification and cell type deconvolution still face challenges related to accuracy, robustness, and computational efficiency. To address these issues, we introduce SpatialFusion, an innovative deep learning model designed to improve both spatial domain identification and cell type deconvolution by integrating gene expression and spatial coordinates. The core innovation of SpatialFusion lies in its use of graph neural networks (GNN) and attention mechanisms to capture complex spatial relationships through multi-dimensional embeddings of spatial data. By employing a dual-encoding strategy (co-learning of spatial graphs and feature maps) and self-supervised contrastive learning, the model significantly enhances accuracy and robustness across datasets. Experimental results demonstrate that SpatialFusion outperforms existing methods in accuracy and resolution when applied to the human DLPFC dataset, particularly in capturing complex, layer-specific expression patterns. The model also shows strong robustness in cell type deconvolution, accurately mapping spatial cell type distributions despite noise and low cell density. In breast cancer tumor microenvironment analysis, SpatialFusion revealed spatial heterogeneity and identified potential therapeutic targets, COX6C and CCND1, providing valuable insights for precision medicine.
{"title":"SpatialFusion: A Unified Model for Integrating Spatial Transcriptomics to Unveil Cell-type Distribution, Interaction, and Functional Heterogeneity in Tissue Microenvironments","authors":"Mengqiu Wang , Zhiwei Zhang , Xinxin Zhang , Ruoyan Dai , Zhenghui Wang , Zeyao Chen , Lixin Lei , Zhenxing Li , Qianjin Guo","doi":"10.1016/j.jmb.2025.169535","DOIUrl":"10.1016/j.jmb.2025.169535","url":null,"abstract":"<div><div>Recent advances in spatial transcriptomics (ST) have significantly enhanced our understanding of tissue structure and intercellular interactions. However, existing methods for spatial domain identification and cell type deconvolution still face challenges related to accuracy, robustness, and computational efficiency. To address these issues, we introduce SpatialFusion, an innovative deep learning model designed to improve both spatial domain identification and cell type deconvolution by integrating gene expression and spatial coordinates. The core innovation of SpatialFusion lies in its use of graph neural networks (GNN) and attention mechanisms to capture complex spatial relationships through multi-dimensional embeddings of spatial data. By employing a dual-encoding strategy (co-learning of spatial graphs and feature maps) and self-supervised contrastive learning, the model significantly enhances accuracy and robustness across datasets. Experimental results demonstrate that SpatialFusion outperforms existing methods in accuracy and resolution when applied to the human DLPFC dataset, particularly in capturing complex, layer-specific expression patterns. The model also shows strong robustness in cell type deconvolution, accurately mapping spatial cell type distributions despite noise and low cell density. In breast cancer tumor microenvironment analysis, SpatialFusion revealed spatial heterogeneity and identified potential therapeutic targets, COX6C and CCND1, providing valuable insights for precision medicine.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"438 2","pages":"Article 169535"},"PeriodicalIF":4.5,"publicationDate":"2025-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145522560","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 : 2025-11-10DOI: 10.1016/j.jmb.2025.169537
Raechell, Wei-Ven Tee, Bingxue Dong, Enrico Guarnera, Igor N Berezovsky
The thermodynamic stability of proteins and regulation of their functional activity can be described within the energy landscape framework, where the former is provided by a unique native conformational ensemble separated by an energy gap from misfolded structures, and the latter is based on conformational transitions between structural states in the native ensemble. This work investigates the relationship between fundamentals of structural stability and dynamics-driven allosteric regulation. We describe here general proteomic trends and fold/function-specific determinants of protein stability. The intricate relationship between stability and allostery has been observed, showing how requirements on stability and thermal adaptation drive and shape the protein's "structural platform", while complementary sequence-structure determinants control the allosteric signaling and regulation. We illustrate our findings using four groups of proteins - inorganic pyrophosphatase and β-glucosidase representing hydrolases, the CheY signaling protein, and adenylate kinase - obtained from host organisms spanning from psychrophiles to hyperthermophiles. We also show that allosteric effects of mutations in adenylate kinase account for experimentally observed changes in organismal fitness expressed in bacterial growth rates. Epistasis arising from the effects of these mutations is another important phenomenon, resulting in unexpected non-additive changes in fitness that could not be explained by the stability changes alone. The findings in this work and options for further investigations of the stability-signaling relationship are provided by the sequence-dependent model of allostery employed here and implemented in AlloSigMA 3 - the latest update of our AlloSigMA web-server (https://allosigma.bii.a-star.edu.sg).
{"title":"On the Relationship Between Protein Stability, Thermostability, and Allosteric Signaling.","authors":"Raechell, Wei-Ven Tee, Bingxue Dong, Enrico Guarnera, Igor N Berezovsky","doi":"10.1016/j.jmb.2025.169537","DOIUrl":"10.1016/j.jmb.2025.169537","url":null,"abstract":"<p><p>The thermodynamic stability of proteins and regulation of their functional activity can be described within the energy landscape framework, where the former is provided by a unique native conformational ensemble separated by an energy gap from misfolded structures, and the latter is based on conformational transitions between structural states in the native ensemble. This work investigates the relationship between fundamentals of structural stability and dynamics-driven allosteric regulation. We describe here general proteomic trends and fold/function-specific determinants of protein stability. The intricate relationship between stability and allostery has been observed, showing how requirements on stability and thermal adaptation drive and shape the protein's \"structural platform\", while complementary sequence-structure determinants control the allosteric signaling and regulation. We illustrate our findings using four groups of proteins - inorganic pyrophosphatase and β-glucosidase representing hydrolases, the CheY signaling protein, and adenylate kinase - obtained from host organisms spanning from psychrophiles to hyperthermophiles. We also show that allosteric effects of mutations in adenylate kinase account for experimentally observed changes in organismal fitness expressed in bacterial growth rates. Epistasis arising from the effects of these mutations is another important phenomenon, resulting in unexpected non-additive changes in fitness that could not be explained by the stability changes alone. The findings in this work and options for further investigations of the stability-signaling relationship are provided by the sequence-dependent model of allostery employed here and implemented in AlloSigMA 3 - the latest update of our AlloSigMA web-server (https://allosigma.bii.a-star.edu.sg).</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169537"},"PeriodicalIF":4.5,"publicationDate":"2025-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145501318","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 : 2025-11-10DOI: 10.1016/j.jmb.2025.169534
Evan Pacheco , Aron A Shoara , Logan W Donaldson
Slf1 and Sro9 are paralogous RNA-binding proteins in Saccharomyces cerevisiae that belong to the LARP1 (La-related protein 1) subgroup of the greater La family. These proteins function as translational regulators during cellular stress, acting through either direct mRNA binding or interactions with ribosomal factors. In this study, we characterized the structural and RNA-binding properties of the La-motif (LaM) domains of Slf1 and Sro9 using a combination of nuclear magnetic resonance (NMR) spectroscopy, calorimetry, and molecular dynamics (MD) simulations. Both LaM domains exhibited micromolar affinity for RNA ligands, including poly(A). Notably, the Sro9 LaM domain displayed a thermal denaturation midpoint of 36 °C suggesting a potential regulatory mechanism for this protein during hyperthermic stress. An NMR analysis of the Slf1 LaM domain revealed that its RNA binding platform undergoes widespread conformational sampling on the micro- to millisecond timescale, even in the presence of RNA. Molecular dynamics simulations corroborated these experimental NMR observations and highlighted the role of transient aromatic stacking during RNA binding. Furthermore, a glutamine substitution mutant (Q278A in Slf1) known to impair RNA binding also destabilized the protein-RNA interaction in molecular simulations. Collectively, our findings confirm that RNA binding by LaM domains is an evolutionarily conserved feature among eukaryotes and provide critical insights into the structural and dynamic mechanisms underlying Slf1 and Sro9 function in yeast.
{"title":"RNA Binding by the Yeast Slf1 and Sro9 La-motif Domains","authors":"Evan Pacheco , Aron A Shoara , Logan W Donaldson","doi":"10.1016/j.jmb.2025.169534","DOIUrl":"10.1016/j.jmb.2025.169534","url":null,"abstract":"<div><div>Slf1 and Sro9 are paralogous RNA-binding proteins in <em>Saccharomyces cerevisiae</em> that belong to the LARP1 (La-related protein 1) subgroup of the greater La family. These proteins function as translational regulators during cellular stress, acting through either direct mRNA binding or interactions with ribosomal factors. In this study, we characterized the structural and RNA-binding properties of the La-motif (LaM) domains of Slf1 and Sro9 using a combination of nuclear magnetic resonance (NMR) spectroscopy, calorimetry, and molecular dynamics (MD) simulations. Both LaM domains exhibited micromolar affinity for RNA ligands, including poly(A). Notably, the Sro9 LaM domain displayed a thermal denaturation midpoint of 36 °C suggesting a potential regulatory mechanism for this protein during hyperthermic stress. An NMR analysis of the Slf1 LaM domain revealed that its RNA binding platform undergoes widespread conformational sampling on the micro- to millisecond timescale, even in the presence of RNA. Molecular dynamics simulations corroborated these experimental NMR observations and highlighted the role of transient aromatic stacking during RNA binding. Furthermore, a glutamine substitution mutant (Q278A in Slf1) known to impair RNA binding also destabilized the protein-RNA interaction in molecular simulations. Collectively, our findings confirm that RNA binding by LaM domains is an evolutionarily conserved feature among eukaryotes and provide critical insights into the structural and dynamic mechanisms underlying Slf1 and Sro9 function in yeast.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 24","pages":"Article 169534"},"PeriodicalIF":4.5,"publicationDate":"2025-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145501332","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}
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