Pub Date : 2024-09-08DOI: 10.1101/2024.09.06.611729
Matthew D Mann, Min Wang, Josephine C Ferreon, Michael P Suess, Antrix Jain, Anna Malovannaya, Bruce D Pascal, Raj Kumar, Dean P Edwards, PATRICK R GRIFFIN
The progesterone receptor (PR) is a steroid-responsive nuclear receptor, expressed as two isoforms: PR-A and PR-B. The isoforms display distinct expression patterns and biological actions in reproductive target tissues and disruption of PR-A:PR-B signaling is associated with breast cancer development potentially by altering interactions with oncogenic co-regulatory protein (CoRs). However, the molecular details of isoform-specific PR-CoR interactions that influence progesterone signaling remain poorly understood. We employed structural mass spectrometry in this study to investigate the sequential binding mechanism of purified full-length PR and full-length CoRs, steroid receptor coactivator 3 (SRC3) and p300, as complexes with target DNA. Our findings reveal selective CoR NR-box binding by PR and novel interaction surfaces between PR, SRC3, and p300, which change during complex assembly. This provides a structural model for a sequential priming mechanism that activates PR. Comparisons of PR bound to progesterone agonist versus antagonist challenges the classical model of nuclear receptor activation and repression. Collectively, we offer a peptide-level perspective on the organization of the PR transcriptional complex and elucidate the mechanisms behind the interactions of these proteins, both in active and inactive conformations.
{"title":"Structural proteomics defines a sequential priming mechanism for the progesterone receptor","authors":"Matthew D Mann, Min Wang, Josephine C Ferreon, Michael P Suess, Antrix Jain, Anna Malovannaya, Bruce D Pascal, Raj Kumar, Dean P Edwards, PATRICK R GRIFFIN","doi":"10.1101/2024.09.06.611729","DOIUrl":"https://doi.org/10.1101/2024.09.06.611729","url":null,"abstract":"The progesterone receptor (PR) is a steroid-responsive nuclear receptor, expressed as two isoforms: PR-A and PR-B. The isoforms display distinct expression patterns and biological actions in reproductive target tissues and disruption of PR-A:PR-B signaling is associated with breast cancer development potentially by altering interactions with oncogenic co-regulatory protein (CoRs). However, the molecular details of isoform-specific PR-CoR interactions that influence progesterone signaling remain poorly understood. We employed structural mass spectrometry in this study to investigate the sequential binding mechanism of purified full-length PR and full-length CoRs, steroid receptor coactivator 3 (SRC3) and p300, as complexes with target DNA. Our findings reveal selective CoR NR-box binding by PR and novel interaction surfaces between PR, SRC3, and p300, which change during complex assembly. This provides a structural model for a sequential priming mechanism that activates PR. Comparisons of PR bound to progesterone agonist versus antagonist challenges the classical model of nuclear receptor activation and repression. Collectively, we offer a peptide-level perspective on the organization of the PR transcriptional complex and elucidate the mechanisms behind the interactions of these proteins, both in active and inactive conformations.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"106 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176052","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-07DOI: 10.1101/2024.09.06.611769
Asma Waris, Ali Raza Awan, Muhammad Wasim, Abu Saeed Hashmi, Naeem Rashid, Sehrish Firyal, Aisha Khalid, Muhammad Tayyab
The current study was planned keeping in view the significance, industrial impact and import of xylanase to Pakistan. In this study, a thermostable recombinant xylanase from Thermotoga naphthophila was produced and characterized. The PCR product (1.1 kb) was purified, ligated in the pTZ57R/T and was used for transformation of DH5α cells. The presence of the gene in the recombinant pTZ57R/T was confirmed by restriction analysis. The gene was sub-cloned in pET21a and expression was examined using BL21 CodonPlus (DE3) cells. The recombinant xylanase was expressed as an intracellular soluble protein. SDS-PAGE demonstrated the purified recombinant xylanase as 37 kDa protein. Xylanase showed its optimal activity at 90°C and pH 7. The enzyme was found thermostable and retained 67% activity after an incubation of 1.5h at 90°C in the presence of Mn2+. The xylanase activity was enhanced in the presence of Triton X-I00 while the presence of SDS, Tween 20 and Tween 80 showed a declined impact on the activity. Kinetics studies showed the Vmax and Km values of 2313 μmol/mg/min and 3.3 mg/ml respectively. The 3D structure analysis demonstrated the presence of a conserved active site comprised of two glutamate and substrate accommodate sites comprised of + 1, +2 and -1, -2 xylose binding sites in the structure of xylanase. The ability of this thermostable xylanase to work at a wide range of temperatures and pH makes it a suitable candidate for industrial applications.
{"title":"3D modeling of thermostable xylanase from Thermotoga naphthophila a member of GH10 family: characterization studies of recombinant xylanase","authors":"Asma Waris, Ali Raza Awan, Muhammad Wasim, Abu Saeed Hashmi, Naeem Rashid, Sehrish Firyal, Aisha Khalid, Muhammad Tayyab","doi":"10.1101/2024.09.06.611769","DOIUrl":"https://doi.org/10.1101/2024.09.06.611769","url":null,"abstract":"The current study was planned keeping in view the significance, industrial impact and import of xylanase to Pakistan. In this study, a thermostable recombinant xylanase from Thermotoga naphthophila was produced and characterized. The PCR product (1.1 kb) was purified, ligated in the pTZ57R/T and was used for transformation of DH5α cells. The presence of the gene in the recombinant pTZ57R/T was confirmed by restriction analysis. The gene was sub-cloned in pET21a and expression was examined using BL21 CodonPlus (DE3) cells. The recombinant xylanase was expressed as an intracellular soluble protein. SDS-PAGE demonstrated the purified recombinant xylanase as 37 kDa protein. Xylanase showed its optimal activity at 90°C and pH 7. The enzyme was found thermostable and retained 67% activity after an incubation of 1.5h at 90°C in the presence of Mn2+. The xylanase activity was enhanced in the presence of Triton X-I00 while the presence of SDS, Tween 20 and Tween 80 showed a declined impact on the activity. Kinetics studies showed the Vmax and Km values of 2313 μmol/mg/min and 3.3 mg/ml respectively. The 3D structure analysis demonstrated the presence of a conserved active site comprised of two glutamate and substrate accommodate sites comprised of + 1, +2 and -1, -2 xylose binding sites in the structure of xylanase. The ability of this thermostable xylanase to work at a wide range of temperatures and pH makes it a suitable candidate for industrial applications.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"8 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176061","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-07DOI: 10.1101/2024.09.07.611787
Francesca Chandler, Poli Adi Narayana Reddy, Smita Bhutda, Rebecca L. Ross, Miriam Walden, Kieran Walker, Stefano Di Donato, Joel A. Cassel, Michael A. Prakesch, Ahmed Aman, Alessandro Datti, Lisa J. Campbell, Martina Foglizzo, Lillie Bell, Daniel N. Stein, James R. Ault, Rima S. Al-awar, Antonio N. Calabrese, Frank Sicheri, Francesco Del Galdo, Joseph M. Salvino, Roger A. Greenberg, Elton Zeqiraj
Deubiquitylases (DUBs) play a pivotal role in cell signalling and are often regulated by homo- or hetero-interactions within protein complexes. The BRCC36 isopeptidase complex (BRISC) regulates inflammatory signalling by selectively cleaving K63-linked polyubiquitin chains on Type I interferon receptors (IFNAR1). BRCC36 is a Zn2+-dependent JAMM/MPN DUB, a challenging ubiquitin protease class for the design of selective inhibitors. We identified first-in-class DUB inhibitors that act as BRISC molecular glues (BLUEs). BLUEs inhibit DUB activity by stabilising a BRISC dimer consisting of 16 subunits. The BLUE-stabilised BRISC dimer is an autoinhibited conformation, whereby the active sites and interactions with the recruiting subunit SHMT2 are blocked. This unique mode of action leads to highly selective inhibitors for BRISC over related complexes with the same catalytic subunit, splice variants and other JAMM/MPN DUBs. Structure-guided inhibitor resistant mutants confirm BLUEs on-target activity in cells, and BLUE treatment results in reduced interferon-stimulated gene (ISG) expression in human peripheral blood mononuclear cells from Scleroderma patients, a disease linked with aberrant IFNAR1 activation. BLUEs represent a new class of molecules with potential utility in Type I interferon-mediated diseases and a template for designing selective inhibitors of large protein complexes by promoting protein-protein interactions instead of blocking them.
{"title":"Molecular glues that inhibit specific Zn2+-dependent DUB activity and inflammation","authors":"Francesca Chandler, Poli Adi Narayana Reddy, Smita Bhutda, Rebecca L. Ross, Miriam Walden, Kieran Walker, Stefano Di Donato, Joel A. Cassel, Michael A. Prakesch, Ahmed Aman, Alessandro Datti, Lisa J. Campbell, Martina Foglizzo, Lillie Bell, Daniel N. Stein, James R. Ault, Rima S. Al-awar, Antonio N. Calabrese, Frank Sicheri, Francesco Del Galdo, Joseph M. Salvino, Roger A. Greenberg, Elton Zeqiraj","doi":"10.1101/2024.09.07.611787","DOIUrl":"https://doi.org/10.1101/2024.09.07.611787","url":null,"abstract":"Deubiquitylases (DUBs) play a pivotal role in cell signalling and are often regulated by homo- or hetero-interactions within protein complexes. The BRCC36 isopeptidase complex (BRISC) regulates inflammatory signalling by selectively cleaving K63-linked polyubiquitin chains on Type I interferon receptors (IFNAR1). BRCC36 is a Zn2+-dependent JAMM/MPN DUB, a challenging ubiquitin protease class for the design of selective inhibitors. We identified first-in-class DUB inhibitors that act as BRISC molecular glues (BLUEs). BLUEs inhibit DUB activity by stabilising a BRISC dimer consisting of 16 subunits. The BLUE-stabilised BRISC dimer is an autoinhibited conformation, whereby the active sites and interactions with the recruiting subunit SHMT2 are blocked. This unique mode of action leads to highly selective inhibitors for BRISC over related complexes with the same catalytic subunit, splice variants and other JAMM/MPN DUBs. Structure-guided inhibitor resistant mutants confirm BLUEs on-target activity in cells, and BLUE treatment results in reduced interferon-stimulated gene (ISG) expression in human peripheral blood mononuclear cells from Scleroderma patients, a disease linked with aberrant IFNAR1 activation. BLUEs represent a new class of molecules with potential utility in Type I interferon-mediated diseases and a template for designing selective inhibitors of large protein complexes by promoting protein-protein interactions instead of blocking them.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"67 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176059","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-07DOI: 10.1101/2024.09.05.611252
Daniella Pretorius, James W Murray
Motivation: Tandem repeat proteins consist of repetitive sequence and structure motifs and have diverse roles in Nature for molecular recognition and signalling. The architecture of repeat proteins can be described using simple helical parameters. Understanding these structural features can inform both the function of these proteins and be used to parametrically design new proteins. Despite their importance, no existing program is capable of completely parameterising repeat proteins. Results: Here we describe a novel repeat protein parameterisation algorithm, RepeatParam, and a comprehensive repeat protein dataset. RepeatParam determines a helix that defines the global protein architecture and a superhelix that describes the relationship between consecutive repeats. We analyse the relationships between helical parameters and families of different repeat proteins.
{"title":"RepeatParam: Algorithm for Parameterising Repeat Proteins and Analysis of Repeat Protein Architectures","authors":"Daniella Pretorius, James W Murray","doi":"10.1101/2024.09.05.611252","DOIUrl":"https://doi.org/10.1101/2024.09.05.611252","url":null,"abstract":"<strong>Motivation</strong>: Tandem repeat proteins consist of repetitive sequence and structure motifs and have diverse roles in Nature for molecular recognition and signalling. The architecture of repeat proteins can be described using simple helical parameters. Understanding these structural features can inform both the function of these proteins and be used to parametrically design new proteins. Despite their importance, no existing program is capable of completely parameterising repeat proteins. <strong>Results</strong>: Here we describe a novel repeat protein parameterisation algorithm, RepeatParam, and a comprehensive repeat protein dataset. RepeatParam determines a helix that defines the global protein architecture and a superhelix that describes the relationship between consecutive repeats. We analyse the relationships between helical parameters and families of different repeat proteins.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"36 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176055","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-07DOI: 10.1101/2024.09.05.611419
Paulina Fischer, Matthias Thoms, Benjamin Lau, Timo Denk, Maria Kuvshinova, Otto Berninghausen, Dirk Flemming, Ed Hurt, Roland Beckmann
During synthesis, modification and maturation of the ribosomal RNA, correct subdomain folding without additional guidance poses a major challenge. An essential H/ACA snoRNP, snR30, was observed to be outsourced from the core of the 90S, the earliest known pre-ribosome, to form a "satellite particle". This particle interacts with the so-called 18S rRNA platform subdomain, guiding its independent folding through localized structural interactions. Once this subdomain achieves its proper conformation, it is integrated into the core pre-ribosome, contributing to the overall maturation of the 18S rRNA. By temporarily segregating the folding process within a specialized satellite particle, the correct assembly of individual components is facilitated, thereby reducing the risk of errors during the complex process of ribosome assembly. In a broader sense, molecular outsourcing in cellular processes, where transient subcomplexes handle specific tasks before rejoining the main assembly line, can enhance efficiency and accuracy of complex molecular processes like ribosome biogenesis.
{"title":"H/ACA snoRNP guides ribosomal RNA subdomain folding in a satellite particle before joining the core 90S pre-ribosome","authors":"Paulina Fischer, Matthias Thoms, Benjamin Lau, Timo Denk, Maria Kuvshinova, Otto Berninghausen, Dirk Flemming, Ed Hurt, Roland Beckmann","doi":"10.1101/2024.09.05.611419","DOIUrl":"https://doi.org/10.1101/2024.09.05.611419","url":null,"abstract":"During synthesis, modification and maturation of the ribosomal RNA, correct subdomain folding without additional guidance poses a major challenge. An essential H/ACA snoRNP, snR30, was observed to be outsourced from the core of the 90S, the earliest known pre-ribosome, to form a \"satellite particle\". This particle interacts with the so-called 18S rRNA platform subdomain, guiding its independent folding through localized structural interactions. Once this subdomain achieves its proper conformation, it is integrated into the core pre-ribosome, contributing to the overall maturation of the 18S rRNA. By temporarily segregating the folding process within a specialized satellite particle, the correct assembly of individual components is facilitated, thereby reducing the risk of errors during the complex process of ribosome assembly. In a broader sense, molecular outsourcing in cellular processes, where transient subcomplexes handle specific tasks before rejoining the main assembly line, can enhance efficiency and accuracy of complex molecular processes like ribosome biogenesis.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176057","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Conventional knowledge of mitochondrial iron metabolism talks about the export of iron in its doubly charged state once it is reduced inside the cell until it reaches the mitochondria. At physiological oxygen tension and pH 7.4, the comparatively soluble Fe(II) is easily available. Fe(III) hydrolyses to generate insoluble ferric hydroxides. Iron must be regularly chaperoned because of its near insolubility and potential toxicity due to redox activity. All tissues pick up iron through the binding of transferrin (Tf) to the transferrin receptor 1 (TfR1), followed by the complex's internalisation through receptor-mediated endocytosis. The low pH created by the operation of a proton pump within the endosome reduces Tf's affinity for iron. Importantly, the TfR1 promotes iron escape from Tf in the pH range (pH 5-5.5) reached by the endosome. A "trap," such as pyrophosphate, is needed in vitro for iron release from Tf. However, a physiological chelator that can play this role has not yet been discovered. Fe(III) is hypothesised to be reduced to Fe(II) in erythroid cells by a ferrireductase known as the six-transmembrane epithelial antigen of the prostate 3 in the endosomal membrane after being released from Tf in the endosome. Following this, the divalent metal transporter-1 (DMT1) transports Fe(II) through the endosomal membrane and, it is generally accepted that this generates the cytosolic labile or chelatable iron pool. This reservoir of iron is believed to provide the metal for metabolic requirements, including as iron intake by the mitochondrion for haem and ISC synthesis, as well as storage in the cytosolic protein ferritin. The possibility of Fe(III) entering the mitochondria has not been explored before. A series of dockings shows that binding of the alpha subunit to a complex of Fe(III) with enterobactin is as stable as the binding with of the same chelator with Fe(II). Enterobactin is a bacterial iron chelator thought to play an integral role in host iron metabolism. Our results suggest an interesting possibility of iron being trafficked to the mitochondria as Fe(III). We propose a potential mechanism of Fe(III) trafficking and subsequent reduction to Fe(II) inside the mitochondria.
{"title":"A novel mechanism for the uptake of enterobactin-chelated ferric ions by the mitochondria and subsequent reduction to ferrous ions inside","authors":"Abeg Dutta, Anutthaman Parthasarathy, Namasivayam Ganesh Pandian","doi":"10.1101/2024.09.05.611381","DOIUrl":"https://doi.org/10.1101/2024.09.05.611381","url":null,"abstract":"Conventional knowledge of mitochondrial iron metabolism talks about the export of iron in its doubly charged state once it is reduced inside the cell until it reaches the mitochondria. At physiological oxygen tension and pH 7.4, the comparatively soluble Fe(II) is easily available. Fe(III) hydrolyses to generate insoluble ferric hydroxides. Iron must be regularly chaperoned because of its near insolubility and potential toxicity due to redox activity. All tissues pick up iron through the binding of transferrin (Tf) to the transferrin receptor 1 (TfR1), followed by the complex's internalisation through receptor-mediated endocytosis. The low pH created by the operation of a proton pump within the endosome reduces Tf's affinity for iron. Importantly, the TfR1 promotes iron escape from Tf in the pH range (pH 5-5.5) reached by the endosome. A \"trap,\" such as pyrophosphate, is needed in vitro for iron release from Tf. However, a physiological chelator that can play this role has not yet been discovered. Fe(III) is hypothesised to be reduced to Fe(II) in erythroid cells by a ferrireductase known as the six-transmembrane epithelial antigen of the prostate 3 in the endosomal membrane after being released from Tf in the endosome. Following this, the divalent metal transporter-1 (DMT1) transports Fe(II) through the endosomal membrane and, it is generally accepted that this generates the cytosolic labile or chelatable iron pool. This reservoir of iron is believed to provide the metal for metabolic requirements, including as iron intake by the mitochondrion for haem and ISC synthesis, as well as storage in the cytosolic protein ferritin. The possibility of Fe(III) entering the mitochondria has not been explored before. A series of dockings shows that binding of the alpha subunit to a complex of Fe(III) with enterobactin is as stable as the binding with of the same chelator with Fe(II). Enterobactin is a bacterial iron chelator thought to play an integral role in host iron metabolism. Our results suggest an interesting possibility of iron being trafficked to the mitochondria as Fe(III). We propose a potential mechanism of Fe(III) trafficking and subsequent reduction to Fe(II) inside the mitochondria.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176053","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-07DOI: 10.1101/2024.09.06.611712
Oleksii Zdorevskyi, Johannes Laukkanen, Vivek Sharma
Redox chemistry of quinones is an essential component of life on earth. In the mitochondrial electron transport chain, ubiquinone molecule is reduced to ubiquinol by respiratory complex I to drive the synthesis of ATP. By performing both classical and hybrid QM/MM simulations on high-resolution cryo-EM structures, including quantitative free energy calculations, we show that semiquinone species in complex I is anionic in nature and is trapped in the active site chamber for its subsequent reduction. Two-electron reduction of ubiquinone yields a metastable ubiquinol anion, which is electrostatically pushed by 15-20 Å towards the exit of the ubiquinone binding chamber to drive the proton pump of complex I. As part of the two-electron reduction of ubiquinone, protonic rearrangements take place in the active site in which a highly conserved histidine converts from its one tautomeric state to another. The combined findings provide a detailed and testable mechanistic picture of proton-coupled electron transfer reaction at the active site of complex I in wild-type as well as mutant conditions.
醌的氧化还原化学是地球生命的重要组成部分。在线粒体电子传递链中，泛醌分子被呼吸复合体 I 还原成泛醌醇，从而推动 ATP 的合成。通过对高分辨率低温电子显微镜结构进行经典模拟和混合 QM/MM 模拟，包括定量自由能计算，我们发现复合物 I 中的半醌类物质具有阴离子性质，并被困在活性位点室中进行后续还原。泛醌的双电子还原产生了可转移的泛醌醇阴离子，该阴离子被 15-20 安培的静电力推向泛醌结合室的出口，以驱动复合物 I 的质子泵。在泛醌的双电子还原过程中，活性位点会发生质子重排，其中高度保守的组氨酸会从一种同素异形体状态转化为另一种同素异形体状态。这些发现为野生型和突变型条件下复合物 I 活性位点的质子耦合电子传递反应提供了详细的、可检验的机理图景。
{"title":"Catalytic relevance of quinol anion in biological energy conversion by respiratory complex I","authors":"Oleksii Zdorevskyi, Johannes Laukkanen, Vivek Sharma","doi":"10.1101/2024.09.06.611712","DOIUrl":"https://doi.org/10.1101/2024.09.06.611712","url":null,"abstract":"Redox chemistry of quinones is an essential component of life on earth. In the mitochondrial electron transport chain, ubiquinone molecule is reduced to ubiquinol by respiratory complex I to drive the synthesis of ATP. By performing both classical and hybrid QM/MM simulations on high-resolution cryo-EM structures, including quantitative free energy calculations, we show that semiquinone species in complex I is anionic in nature and is trapped in the active site chamber for its subsequent reduction. Two-electron reduction of ubiquinone yields a metastable ubiquinol anion, which is electrostatically pushed by 15-20 &Aring towards the exit of the ubiquinone binding chamber to drive the proton pump of complex I. As part of the two-electron reduction of ubiquinone, protonic rearrangements take place in the active site in which a highly conserved histidine converts from its one tautomeric state to another. The combined findings provide a detailed and testable mechanistic picture of proton-coupled electron transfer reaction at the active site of complex I in wild-type as well as mutant conditions.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176054","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-07DOI: 10.1101/2024.09.06.611651
Torsten Krude, Jiaming Bi, Rachel Doran, Rebecca A Jones, James C Smith
DNA replication initiates at tens of thousands of sites on the human genome during each S phase. However, no consensus DNA sequence has been found that specifies the locations of these replication origins. Here, we investigate modifications of human genomic DNA by density equilibrium centrifugation and DNA sequencing. We identified short discrete sites with increased density during quiescence and G1 phase that overlap with DNA replication origins before their activation in S phase. The increased density is due to the oxidation of 5-methyl-deoxycytidines by TET enzymes at GC-rich domains. Reversible inhibition of de novo methylation and of subsequent oxidation of deoxycytidines results in a reversible inhibition of DNA replication and of cell proliferation. Our findings suggest a mechanism for the epigenetic specification and semiconservative inheritance of DNA replication origin sites in human cells that also provides a stable integral DNA replication licence to support once-per-cell cycle control of origin activation.
在每个 S 期，人类基因组上有数以万计的 DNA 复制起始点。然而，目前还没有找到一个共识的 DNA 序列来指定这些复制起源的位置。在这里，我们通过密度平衡离心和 DNA 测序研究了人类基因组 DNA 的修饰。我们确定了在静止期和 G1 期密度增加的短离散位点，这些位点在 S 期激活之前与 DNA 复制起源重叠。密度增加的原因是 TET 酶在富含 GC 的结构域氧化了 5-甲基脱氧胞嘧啶。对从头甲基化和随后的脱氧胞嘧啶氧化的可逆抑制会导致 DNA 复制和细胞增殖的可逆抑制。我们的研究结果表明，在人类细胞中，DNA 复制起源位点的表观遗传学规范和半保守遗传机制还提供了稳定的整体 DNA 复制许可，以支持每细胞周期一次的起源激活控制。
{"title":"Human DNA replication initiation sites are specified epigenetically by oxidation of 5-methyl-deoxycytidine","authors":"Torsten Krude, Jiaming Bi, Rachel Doran, Rebecca A Jones, James C Smith","doi":"10.1101/2024.09.06.611651","DOIUrl":"https://doi.org/10.1101/2024.09.06.611651","url":null,"abstract":"DNA replication initiates at tens of thousands of sites on the human genome during each S phase. However, no consensus DNA sequence has been found that specifies the locations of these replication origins. Here, we investigate modifications of human genomic DNA by density equilibrium centrifugation and DNA sequencing. We identified short discrete sites with increased density during quiescence and G1 phase that overlap with DNA replication origins before their activation in S phase. The increased density is due to the oxidation of 5-methyl-deoxycytidines by TET enzymes at GC-rich domains. Reversible inhibition of de novo methylation and of subsequent oxidation of deoxycytidines results in a reversible inhibition of DNA replication and of cell proliferation. Our findings suggest a mechanism for the epigenetic specification and semiconservative inheritance of DNA replication origin sites in human cells that also provides a stable integral DNA replication licence to support once-per-cell cycle control of origin activation.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"27 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176058","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-07DOI: 10.1101/2024.09.07.611824
Henry Wienkers, Han Han, Frank Whitby, Christopher Hill
The ESCRT pathway's AAA+ ATPase, Vps4p, remodels ESCRT-III complexes to drive membrane fission. Here, we use peptide binding assays to further the understanding of substrate specificity and the mechanism of autoinhibition. Our results reveal unexpected sequence preference to the substrate binding groove and an elegant mechanism of regulation that couples localization to substrate with release from autoinhibition.
{"title":"Vps4 substrate binding and coupled mechanisms of Vps4p substrate recruitment and release from autoinhibition","authors":"Henry Wienkers, Han Han, Frank Whitby, Christopher Hill","doi":"10.1101/2024.09.07.611824","DOIUrl":"https://doi.org/10.1101/2024.09.07.611824","url":null,"abstract":"The ESCRT pathway's AAA+ ATPase, Vps4p, remodels ESCRT-III complexes to drive membrane fission. Here, we use peptide binding assays to further the understanding of substrate specificity and the mechanism of autoinhibition. Our results reveal unexpected sequence preference to the substrate binding groove and an elegant mechanism of regulation that couples localization to substrate with release from autoinhibition.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"150 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176056","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-06DOI: 10.1101/2024.09.05.611425
Ankita Chadda, Binh Nguyen, Timothy M. Lohman, Eric A Galburt
UvrD-family helicases are superfamily 1A motor proteins that function during DNA replication, recombination, repair, and transcription. UvrD family monomers translocate along single stranded (ss) DNA but need to be activated by dimerization to unwind DNA in the absence of force or accessory factors. However, prior structural studies have only revealed monomeric complexes. Here, we report the first structures of a dimeric UvrD-family helicase, Mycobacterium tuberculosis UvrD1, both free and bound to a DNA junction. In each structure, the dimer interface occurs between the 2B subdomains of each subunit. The apo UvrD1 dimer is observed in symmetric compact and extended forms indicating substantial flexibility. This symmetry is broken in the DNA-bound dimer complex with leading and trailing subunits adopting distinct conformations. Biochemical experiments reveal that the E. coli UvrD dimer shares the same 2B-2B interface. In contrast to the dimeric structures, an inactive, auto-inhibited UvrD1 DNA-bound monomer structure reveals 2B subdomain-DNA contacts that are likely inhibitory. The major re-orientation of the 2B subdomains that occurs upon UvrD1 dimerization prevents these duplex DNA interactions, thus relieving the auto-inhibition. These structures reveal that the 2B subdomain serves a major regulatory role rather than participating directly in DNA unwinding.
UvrD 家族螺旋酶是超家族 1A 运动蛋白，在 DNA 复制、重组、修复和转录过程中发挥作用。UvrD 家族单体沿着单链（ss）DNA 转移，但需要通过二聚化激活，才能在没有外力或辅助因子的情况下解开 DNA。然而，之前的结构研究只揭示了单体复合物。在这里，我们首次报道了一种二聚体 UvrD 家族螺旋酶（结核分枝杆菌 UvrD1）的自由结构和与 DNA 连接结合的结构。在每种结构中，二聚体界面都位于每个亚基的 2B 亚域之间。观察发现，apo UvrD1 二聚体有对称的紧凑型和延伸型，这表明其具有很大的灵活性。这种对称性在与 DNA 结合的二聚体复合物中被打破，前导亚基和后导亚基采用不同的构象。生化实验显示，大肠杆菌 UvrD 二聚体具有相同的 2B-2B 接口。与二聚体结构相反，非活性、自动抑制的 UvrD1 DNA 结合单体结构显示，2B 亚域与 DNA 的接触可能具有抑制作用。UvrD1 二聚化时 2B 亚域发生的重大重新定向阻止了这些双工 DNA 相互作用，从而缓解了自动抑制作用。这些结构揭示了 2B 亚域的主要调节作用，而不是直接参与 DNA 的解旋。
{"title":"Structural Basis for Dimerization and Activation of UvrD-family Helicases","authors":"Ankita Chadda, Binh Nguyen, Timothy M. Lohman, Eric A Galburt","doi":"10.1101/2024.09.05.611425","DOIUrl":"https://doi.org/10.1101/2024.09.05.611425","url":null,"abstract":"UvrD-family helicases are superfamily 1A motor proteins that function during DNA replication, recombination, repair, and transcription. UvrD family monomers translocate along single stranded (ss) DNA but need to be activated by dimerization to unwind DNA in the absence of force or accessory factors. However, prior structural studies have only revealed monomeric complexes. Here, we report the first structures of a dimeric UvrD-family helicase, Mycobacterium tuberculosis UvrD1, both free and bound to a DNA junction. In each structure, the dimer interface occurs between the 2B subdomains of each subunit. The apo UvrD1 dimer is observed in symmetric compact and extended forms indicating substantial flexibility. This symmetry is broken in the DNA-bound dimer complex with leading and trailing subunits adopting distinct conformations. Biochemical experiments reveal that the E. coli UvrD dimer shares the same 2B-2B interface. In contrast to the dimeric structures, an inactive, auto-inhibited UvrD1 DNA-bound monomer structure reveals 2B subdomain-DNA contacts that are likely inhibitory. The major re-orientation of the 2B subdomains that occurs upon UvrD1 dimerization prevents these duplex DNA interactions, thus relieving the auto-inhibition. These structures reveal that the 2B subdomain serves a major regulatory role rather than participating directly in DNA unwinding.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176090","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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