Pub Date : 2024-07-01DOI: 10.1038/s41594-024-01336-0
Wenxin Hu, Amit Kumar, Syed Faraz Ahmed, Shijiao Qi, David K. G. Ma, Honglin Chen, Gurjeet J. Singh, Joshua M. L. Casan, Michelle Haber, Ilia Voskoboinik, Matthew R. McKay, Joseph A. Trapani, Paul G. Ekert, Mohamed Fareh
The development of precise RNA-editing tools is essential for the advancement of RNA therapeutics. CRISPR (clustered regularly interspaced short palindromic repeats) PspCas13b is a programmable RNA nuclease predicted to offer superior specificity because of its 30-nucleotide spacer sequence. However, its design principles and its on-target, off-target and collateral activities remain poorly characterized. Here, we present single-base tiled screening and computational analyses that identify key design principles for potent and highly selective RNA recognition and cleavage in human cells. We show that the de novo design of spacers containing guanosine bases at precise positions can greatly enhance the catalytic activity of inefficient CRISPR RNAs (crRNAs). These validated design principles (integrated into an online tool, https://cas13target.azurewebsites.net/) can predict highly effective crRNAs with ~90% accuracy. Furthermore, the comprehensive spacer–target mutagenesis revealed that PspCas13b can tolerate only up to four mismatches and requires ~26-nucleotide base pairing with the target to activate its nuclease domains, highlighting its superior specificity compared to other RNA or DNA interference tools. On the basis of this targeting resolution, we predict an extremely low probability of PspCas13b having off-target effects on other cellular transcripts. Proteomic analysis validated this prediction and showed that, unlike other Cas13 orthologs, PspCas13b exhibits potent on-target activity and lacks collateral effects.
{"title":"Single-base tiled screen unveils design principles of PspCas13b for potent and off-target-free RNA silencing","authors":"Wenxin Hu, Amit Kumar, Syed Faraz Ahmed, Shijiao Qi, David K. G. Ma, Honglin Chen, Gurjeet J. Singh, Joshua M. L. Casan, Michelle Haber, Ilia Voskoboinik, Matthew R. McKay, Joseph A. Trapani, Paul G. Ekert, Mohamed Fareh","doi":"10.1038/s41594-024-01336-0","DOIUrl":"https://doi.org/10.1038/s41594-024-01336-0","url":null,"abstract":"<p>The development of precise RNA-editing tools is essential for the advancement of RNA therapeutics. CRISPR (clustered regularly interspaced short palindromic repeats) PspCas13b is a programmable RNA nuclease predicted to offer superior specificity because of its 30-nucleotide spacer sequence. However, its design principles and its on-target, off-target and collateral activities remain poorly characterized. Here, we present single-base tiled screening and computational analyses that identify key design principles for potent and highly selective RNA recognition and cleavage in human cells. We show that the de novo design of spacers containing guanosine bases at precise positions can greatly enhance the catalytic activity of inefficient CRISPR RNAs (crRNAs). These validated design principles (integrated into an online tool, https://cas13target.azurewebsites.net/) can predict highly effective crRNAs with ~90% accuracy. Furthermore, the comprehensive spacer–target mutagenesis revealed that PspCas13b can tolerate only up to four mismatches and requires ~26-nucleotide base pairing with the target to activate its nuclease domains, highlighting its superior specificity compared to other RNA or DNA interference tools. On the basis of this targeting resolution, we predict an extremely low probability of PspCas13b having off-target effects on other cellular transcripts. Proteomic analysis validated this prediction and showed that, unlike other Cas13 orthologs, PspCas13b exhibits potent on-target activity and lacks collateral effects.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141475334","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 : 2024-07-01DOI: 10.1038/s41594-024-01349-9
Ruth M. Saecker, Andreas U. Mueller, Brandon Malone, James Chen, William C. Budell, Venkata P. Dandey, Kashyap Maruthi, Joshua H. Mendez, Nina Molina, Edward T. Eng, Laura Y. Yen, Clinton S. Potter, Bridget Carragher, Seth A. Darst
During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ70–RNAP and the λPR promoter. Cryo-EM snapshots revealed that the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As the nt-strand ‘read-out’ extends, the RNAP clamp closes, expelling an inhibitory σ70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation.
在细菌 RNA 聚合酶(RNAPs)形成转录功能开放复合物(RPo)的过程中,瞬时中间产物在克服限速步骤之前会堆积起来。目前还没有关于这些相互转化的实时结构描述。为了填补这一空白,我们在此使用时间分辨低温电子显微镜(cryo-EM)捕捉大肠杆菌σ70-RNAP与λPR启动子混合后120毫秒或500毫秒内产生的四个中间产物。低温电子显微镜快照显示,转录泡的上游边缘迅速解除配对,随后两个保守的非模板链(nt-strand)碱基逐步插入 RNAP 口袋。随着 nt 链 "读出 "的延伸,RNAP 夹闭,将抑制性 σ70 结构域从活性位点裂隙中排出。模板链在 120 毫秒前完全未配对,但仍保持动态,这表明在随后的步骤中,未知的构象变化完成了 RPo 的形成。鉴于这些事件很可能描述了许多细菌启动子的 DNA 开启过程,本研究提供了有关 DNA 序列如何调控 RPo 形成步骤的见解。
{"title":"Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy","authors":"Ruth M. Saecker, Andreas U. Mueller, Brandon Malone, James Chen, William C. Budell, Venkata P. Dandey, Kashyap Maruthi, Joshua H. Mendez, Nina Molina, Edward T. Eng, Laura Y. Yen, Clinton S. Potter, Bridget Carragher, Seth A. Darst","doi":"10.1038/s41594-024-01349-9","DOIUrl":"https://doi.org/10.1038/s41594-024-01349-9","url":null,"abstract":"<p>During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing <i>Escherichia coli</i> σ<sup>70</sup>–RNAP and the λP<sub>R</sub> promoter. Cryo-EM snapshots revealed that the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As the nt-strand ‘read-out’ extends, the RNAP clamp closes, expelling an inhibitory σ<sup>70</sup> domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141475119","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 : 2024-07-01DOI: 10.1038/s41594-024-01352-0
Julian R. Braxton, Hao Shao, Eric Tse, Jason E. Gestwicki, Daniel R. Southworth
The mitochondrial chaperonin, mitochondrial heat shock protein 60 (mtHsp60), promotes the folding of newly imported and transiently misfolded proteins in the mitochondrial matrix, assisted by its co-chaperone mtHsp10. Despite its essential role in mitochondrial proteostasis, structural insights into how this chaperonin progresses through its ATP-dependent client folding cycle are not clear. Here, we determined cryo-EM structures of a hyperstable disease-associated human mtHsp60 mutant, V72I. Client density is identified in three distinct states, revealing interactions with the mtHsp60 apical domains and C termini that coordinate client positioning in the folding chamber. We further identify an asymmetric arrangement of the apical domains in the ATP state, in which an alternating up/down configuration positions interaction surfaces for simultaneous recruitment of mtHsp10 and client retention. Client is then fully encapsulated in mtHsp60–10, revealing prominent contacts at two discrete sites that potentially support maturation. These results identify distinct roles for the apical domains in coordinating client capture and progression through the chaperone cycle, supporting a conserved mechanism of group I chaperonin function.
线粒体合子蛋白--线粒体热休克蛋白 60(mtHsp60)在其辅助合子 mtHsp10 的协助下,促进线粒体基质中新导入蛋白质和瞬时折叠错误蛋白质的折叠。尽管它在线粒体蛋白稳态中发挥着重要作用,但人们对这种伴侣蛋白如何通过其依赖 ATP 的客户折叠周期进行折叠的结构研究并不清楚。在这里,我们测定了与疾病相关的人类 mtHsp60 超稳定突变体 V72I 的冷冻电镜结构。在三种不同的状态下确定了客户密度,揭示了与 mtHsp60 顶端结构域和 C 末端的相互作用,这些相互作用协调了客户在折叠室中的定位。我们进一步确定了顶端结构域在 ATP 状态下的不对称排列,在这种状态下,上下交替的配置定位了相互作用表面,以便同时招募 mtHsp10 和保留客户。然后,客户被完全包裹在 mtHsp60-10 中,显示出两个离散位点的突出接触,这两个位点可能支持成熟。这些结果确定了顶端结构域在协调客户捕获和伴侣循环过程中的不同作用,支持了 I 组伴侣素功能的保守机制。
{"title":"Asymmetric apical domain states of mitochondrial Hsp60 coordinate substrate engagement and chaperonin assembly","authors":"Julian R. Braxton, Hao Shao, Eric Tse, Jason E. Gestwicki, Daniel R. Southworth","doi":"10.1038/s41594-024-01352-0","DOIUrl":"https://doi.org/10.1038/s41594-024-01352-0","url":null,"abstract":"<p>The mitochondrial chaperonin, mitochondrial heat shock protein 60 (mtHsp60), promotes the folding of newly imported and transiently misfolded proteins in the mitochondrial matrix, assisted by its co-chaperone mtHsp10. Despite its essential role in mitochondrial proteostasis, structural insights into how this chaperonin progresses through its ATP-dependent client folding cycle are not clear. Here, we determined cryo-EM structures of a hyperstable disease-associated human mtHsp60 mutant, V72I. Client density is identified in three distinct states, revealing interactions with the mtHsp60 apical domains and C termini that coordinate client positioning in the folding chamber. We further identify an asymmetric arrangement of the apical domains in the ATP state, in which an alternating up/down configuration positions interaction surfaces for simultaneous recruitment of mtHsp10 and client retention. Client is then fully encapsulated in mtHsp60–10, revealing prominent contacts at two discrete sites that potentially support maturation. These results identify distinct roles for the apical domains in coordinating client capture and progression through the chaperone cycle, supporting a conserved mechanism of group I chaperonin function.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141475327","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 : 2024-06-25DOI: 10.1038/s41594-024-01341-3
Philipp Throll, Luciano G. Dolce, Palma Rico-Lastres, Katharina Arnold, Laura Tengo, Shibom Basu, Stefanie Kaiser, Robert Schneider, Eva Kowalinski
Methylation of cytosine 32 in the anticodon loop of tRNAs to 3-methylcytosine (m3C) is crucial for cellular translation fidelity. Misregulation of the RNA methyltransferases setting this modification can cause aggressive cancers and metabolic disturbances. Here, we report the cryo-electron microscopy structure of the human m3C tRNA methyltransferase METTL6 in complex with seryl-tRNA synthetase (SerRS) and their common substrate tRNASer. Through the complex structure, we identify the tRNA-binding domain of METTL6. We show that SerRS acts as the tRNASer substrate selection factor for METTL6. We demonstrate that SerRS augments the methylation activity of METTL6 and that direct contacts between METTL6 and SerRS are necessary for efficient tRNASer methylation. Finally, on the basis of the structure of METTL6 in complex with SerRS and tRNASer, we postulate a universal tRNA-binding mode for m3C RNA methyltransferases, including METTL2 and METTL8, suggesting that these mammalian paralogs use similar ways to engage their respective tRNA substrates and cofactors.
{"title":"Structural basis of tRNA recognition by the m3C RNA methyltransferase METTL6 in complex with SerRS seryl-tRNA synthetase","authors":"Philipp Throll, Luciano G. Dolce, Palma Rico-Lastres, Katharina Arnold, Laura Tengo, Shibom Basu, Stefanie Kaiser, Robert Schneider, Eva Kowalinski","doi":"10.1038/s41594-024-01341-3","DOIUrl":"https://doi.org/10.1038/s41594-024-01341-3","url":null,"abstract":"<p>Methylation of cytosine 32 in the anticodon loop of tRNAs to 3-methylcytosine (m<sup>3</sup>C) is crucial for cellular translation fidelity. Misregulation of the RNA methyltransferases setting this modification can cause aggressive cancers and metabolic disturbances. Here, we report the cryo-electron microscopy structure of the human m<sup>3</sup>C tRNA methyltransferase METTL6 in complex with seryl-tRNA synthetase (SerRS) and their common substrate tRNA<sup>Ser</sup>. Through the complex structure, we identify the tRNA-binding domain of METTL6. We show that SerRS acts as the tRNA<sup>Ser</sup> substrate selection factor for METTL6. We demonstrate that SerRS augments the methylation activity of METTL6 and that direct contacts between METTL6 and SerRS are necessary for efficient tRNA<sup>Ser</sup> methylation. Finally, on the basis of the structure of METTL6 in complex with SerRS and tRNA<sup>Ser</sup>, we postulate a universal tRNA-binding mode for m<sup>3</sup>C RNA methyltransferases, including METTL2 and METTL8, suggesting that these mammalian paralogs use similar ways to engage their respective tRNA substrates and cofactors.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141448130","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 : 2024-06-25DOI: 10.1038/s41594-024-01343-1
Overexpression of the RNA methyltransferase METTL6 leads to increased proliferation and promotes cancer. Our cryo-electron microscopy (cryo-EM) and biochemical analyses reveal that METTL6 requires seryl-tRNA synthetase as a cofactor to efficiently generate 3-methyl-cytosine in serine tRNAs.
{"title":"The tRNA methyltransferase METTL6 requires seryl-tRNA synthetase for tRNASer targeting","authors":"","doi":"10.1038/s41594-024-01343-1","DOIUrl":"https://doi.org/10.1038/s41594-024-01343-1","url":null,"abstract":"Overexpression of the RNA methyltransferase METTL6 leads to increased proliferation and promotes cancer. Our cryo-electron microscopy (cryo-EM) and biochemical analyses reveal that METTL6 requires seryl-tRNA synthetase as a cofactor to efficiently generate 3-methyl-cytosine in serine tRNAs.","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141448111","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 : 2024-06-25DOI: 10.1038/s41594-024-01338-y
Elias Adriaenssens, Thanh Ngoc Nguyen, Justyna Sawa-Makarska, Grace Khuu, Martina Schuschnig, Stephen Shoebridge, Marvin Skulsuppaisarn, Emily Maria Watts, Kitti Dora Csalyi, Benjamin Scott Padman, Michael Lazarou, Sascha Martens
Mitophagy preserves overall mitochondrial fitness by selectively targeting damaged mitochondria for degradation. The regulatory mechanisms that prevent PTEN-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase Parkin (PINK1/Parkin)-dependent mitophagy and other selective autophagy pathways from overreacting while ensuring swift progression once initiated are largely elusive. Here, we demonstrate how the TBK1 (TANK-binding kinase 1) adaptors NAP1 (NAK-associated protein 1) and SINTBAD (similar to NAP1 TBK1 adaptor) restrict the initiation of OPTN (optineurin)-driven mitophagy by competing with OPTN for TBK1. Conversely, they promote the progression of nuclear dot protein 52 (NDP52)-driven mitophagy by recruiting TBK1 to NDP52 and stabilizing its interaction with FIP200. Notably, OPTN emerges as the primary recruiter of TBK1 during mitophagy initiation, which in return boosts NDP52-mediated mitophagy. Our results thus define NAP1 and SINTBAD as cargo receptor rheostats, elevating the threshold for mitophagy initiation by OPTN while promoting the progression of the pathway once set in motion by supporting NDP52. These findings shed light on the cellular strategy to prevent pathway hyperactivity while still ensuring efficient progression.
{"title":"Control of mitophagy initiation and progression by the TBK1 adaptors NAP1 and SINTBAD","authors":"Elias Adriaenssens, Thanh Ngoc Nguyen, Justyna Sawa-Makarska, Grace Khuu, Martina Schuschnig, Stephen Shoebridge, Marvin Skulsuppaisarn, Emily Maria Watts, Kitti Dora Csalyi, Benjamin Scott Padman, Michael Lazarou, Sascha Martens","doi":"10.1038/s41594-024-01338-y","DOIUrl":"https://doi.org/10.1038/s41594-024-01338-y","url":null,"abstract":"<p>Mitophagy preserves overall mitochondrial fitness by selectively targeting damaged mitochondria for degradation. The regulatory mechanisms that prevent PTEN-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase Parkin (PINK1/Parkin)-dependent mitophagy and other selective autophagy pathways from overreacting while ensuring swift progression once initiated are largely elusive. Here, we demonstrate how the TBK1 (TANK-binding kinase 1) adaptors NAP1 (NAK-associated protein 1) and SINTBAD (similar to NAP1 TBK1 adaptor) restrict the initiation of OPTN (optineurin)-driven mitophagy by competing with OPTN for TBK1. Conversely, they promote the progression of nuclear dot protein 52 (NDP52)-driven mitophagy by recruiting TBK1 to NDP52 and stabilizing its interaction with FIP200. Notably, OPTN emerges as the primary recruiter of TBK1 during mitophagy initiation, which in return boosts NDP52-mediated mitophagy. Our results thus define NAP1 and SINTBAD as cargo receptor rheostats, elevating the threshold for mitophagy initiation by OPTN while promoting the progression of the pathway once set in motion by supporting NDP52. These findings shed light on the cellular strategy to prevent pathway hyperactivity while still ensuring efficient progression.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141448223","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 : 2024-06-25DOI: 10.1038/s41594-024-01342-2
Huasong Ai, Zaozhen He, Zhiheng Deng, Guo-Chao Chu, Qiang Shi, Zebin Tong, Jia-Bin Li, Man Pan, Lei Liu
Epigenetic regulators have a crucial effect on gene expression based on their manipulation of histone modifications. Histone H2AK119 monoubiquitination (H2AK119Ub), a well-established hallmark in transcription repression, is dynamically regulated by the opposing activities of Polycomb repressive complex 1 (PRC1) and nucleosome deubiquitinases including the primary human USP16 and Polycomb repressive deubiquitinase (PR-DUB) complex. Recently, the catalytic mechanism for the multi-subunit PR-DUB complex has been described, but how the single-subunit USP16 recognizes the H2AK119Ub nucleosome and cleaves the ubiquitin (Ub) remains unknown. Here we report the cryo-EM structure of USP16–H2AK119Ub nucleosome complex, which unveils a fundamentally distinct mode of H2AK119Ub deubiquitination compared to PR-DUB, encompassing the nucleosome recognition pattern independent of the H2A–H2B acidic patch and the conformational heterogeneity in the Ub motif and the histone H2A C-terminal tail. Our work highlights the mechanism diversity of H2AK119Ub deubiquitination and provides a structural framework for understanding the disease-causing mutations of USP16.
{"title":"Structural and mechanistic basis for nucleosomal H2AK119 deubiquitination by single-subunit deubiquitinase USP16","authors":"Huasong Ai, Zaozhen He, Zhiheng Deng, Guo-Chao Chu, Qiang Shi, Zebin Tong, Jia-Bin Li, Man Pan, Lei Liu","doi":"10.1038/s41594-024-01342-2","DOIUrl":"https://doi.org/10.1038/s41594-024-01342-2","url":null,"abstract":"<p>Epigenetic regulators have a crucial effect on gene expression based on their manipulation of histone modifications. Histone H2AK119 monoubiquitination (H2AK119Ub), a well-established hallmark in transcription repression, is dynamically regulated by the opposing activities of Polycomb repressive complex 1 (PRC1) and nucleosome deubiquitinases including the primary human USP16 and Polycomb repressive deubiquitinase (PR-DUB) complex. Recently, the catalytic mechanism for the multi-subunit PR-DUB complex has been described, but how the single-subunit USP16 recognizes the H2AK119Ub nucleosome and cleaves the ubiquitin (Ub) remains unknown. Here we report the cryo-EM structure of USP16–H2AK119Ub nucleosome complex, which unveils a fundamentally distinct mode of H2AK119Ub deubiquitination compared to PR-DUB, encompassing the nucleosome recognition pattern independent of the H2A–H2B acidic patch and the conformational heterogeneity in the Ub motif and the histone H2A C-terminal tail. Our work highlights the mechanism diversity of H2AK119Ub deubiquitination and provides a structural framework for understanding the disease-causing mutations of USP16.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141448217","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 : 2024-06-19DOI: 10.1038/s41594-024-01339-x
Duc-Duy Vu, Alessio Bonucci, Manon Brenière, Metztli Cisneros-Aguirre, Philippe Pelupessy, Ziqing Wang, Ludovic Carlier, Guillaume Bouvignies, Patricia Cortes, Aneel K. Aggarwal, Martin Blackledge, Zoher Gueroui, Valérie Belle, Jeremy M. Stark, Mauro Modesti, Fabien Ferrage
In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70–Ku80 heterodimer (Ku), X-ray repair cross complementing 4 (XRCC4) in complex with DNA ligase 4 (X4L4) and XRCC4-like factor (XLF) form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were recently obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at residue resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs lead to the formation of XLF and X4L4 condensates in vitro, which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome-editing strategies.
{"title":"Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro","authors":"Duc-Duy Vu, Alessio Bonucci, Manon Brenière, Metztli Cisneros-Aguirre, Philippe Pelupessy, Ziqing Wang, Ludovic Carlier, Guillaume Bouvignies, Patricia Cortes, Aneel K. Aggarwal, Martin Blackledge, Zoher Gueroui, Valérie Belle, Jeremy M. Stark, Mauro Modesti, Fabien Ferrage","doi":"10.1038/s41594-024-01339-x","DOIUrl":"https://doi.org/10.1038/s41594-024-01339-x","url":null,"abstract":"<p>In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70–Ku80 heterodimer (Ku), X-ray repair cross complementing 4 (XRCC4) in complex with DNA ligase 4 (X4L4) and XRCC4-like factor (XLF) form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were recently obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at residue resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs lead to the formation of XLF and X4L4 condensates in vitro, which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome-editing strategies.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141425193","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 : 2024-06-19DOI: 10.1038/s41594-024-01335-1
Anshumali Mittal, Matthew F. Martin, Elena J. Levin, Christopher Adams, Meng Yang, Laurent Provins, Adrian Hall, Martin Procter, Marie Ledecq, Alexander Hillisch, Christian Wolff, Michel Gillard, Peter S. Horanyi, Jonathan A. Coleman
Epilepsy is a common neurological disorder characterized by abnormal activity of neuronal networks, leading to seizures. The racetam class of anti-seizure medications bind specifically to a membrane protein found in the synaptic vesicles of neurons called synaptic vesicle protein 2 (SV2) A (SV2A). SV2A belongs to an orphan subfamily of the solute carrier 22 organic ion transporter family that also includes SV2B and SV2C. The molecular basis for how anti-seizure medications act on SV2s remains unknown. Here we report cryo-electron microscopy structures of SV2A and SV2B captured in a luminal-occluded conformation complexed with anticonvulsant ligands. The conformation bound by anticonvulsants resembles an inhibited transporter with closed luminal and intracellular gates. Anticonvulsants bind to a highly conserved central site in SV2s. These structures provide blueprints for future drug design and will facilitate future investigations into the biological function of SV2s.
{"title":"Structures of synaptic vesicle protein 2A and 2B bound to anticonvulsants","authors":"Anshumali Mittal, Matthew F. Martin, Elena J. Levin, Christopher Adams, Meng Yang, Laurent Provins, Adrian Hall, Martin Procter, Marie Ledecq, Alexander Hillisch, Christian Wolff, Michel Gillard, Peter S. Horanyi, Jonathan A. Coleman","doi":"10.1038/s41594-024-01335-1","DOIUrl":"https://doi.org/10.1038/s41594-024-01335-1","url":null,"abstract":"<p>Epilepsy is a common neurological disorder characterized by abnormal activity of neuronal networks, leading to seizures. The racetam class of anti-seizure medications bind specifically to a membrane protein found in the synaptic vesicles of neurons called synaptic vesicle protein 2 (SV2) A (SV2A). SV2A belongs to an orphan subfamily of the solute carrier 22 organic ion transporter family that also includes SV2B and SV2C. The molecular basis for how anti-seizure medications act on SV2s remains unknown. Here we report cryo-electron microscopy structures of SV2A and SV2B captured in a luminal-occluded conformation complexed with anticonvulsant ligands. The conformation bound by anticonvulsants resembles an inhibited transporter with closed luminal and intracellular gates. Anticonvulsants bind to a highly conserved central site in SV2s. These structures provide blueprints for future drug design and will facilitate future investigations into the biological function of SV2s.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141425279","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 : 2024-06-18DOI: 10.1038/s41594-024-01319-1
Qian Li, Jun Zhang, Cory Haluska, Xiang Zhang, Lei Wang, Guangfeng Liu, Zhaoning Wang, Duo Jin, Tong Cheng, Hongxia Wang, Yuan Tian, Xiangxi Wang, Lei Sun, Xiaolan Zhao, Zhenguo Chen, Lanfeng Wang
Smc5/6 is a member of the eukaryotic structural maintenance of chromosomes (SMC) family of complexes with important roles in genome maintenance and viral restriction. However, limited structural understanding of Smc5/6 hinders the elucidation of its diverse functions. Here, we report cryo-EM structures of the budding yeast Smc5/6 complex in eight-subunit, six-subunit and five-subunit states. Structural maps throughout the entire length of these complexes reveal modularity and key elements in complex assembly. We show that the non-SMC element (Nse)2 subunit supports the overall shape of the complex and uses a wedge motif to aid the stability and function of the complex. The Nse6 subunit features a flexible hook region for attachment to the Smc5 and Smc6 arm regions, contributing to the DNA repair roles of the complex. Our results also suggest a structural basis for the opposite effects of the Nse1–3–4 and Nse5–6 subcomplexes in regulating Smc5/6 ATPase activity. Collectively, our integrated structural and functional data provide a framework for understanding Smc5/6 assembly and function.
{"title":"Cryo-EM structures of Smc5/6 in multiple states reveal its assembly and functional mechanisms","authors":"Qian Li, Jun Zhang, Cory Haluska, Xiang Zhang, Lei Wang, Guangfeng Liu, Zhaoning Wang, Duo Jin, Tong Cheng, Hongxia Wang, Yuan Tian, Xiangxi Wang, Lei Sun, Xiaolan Zhao, Zhenguo Chen, Lanfeng Wang","doi":"10.1038/s41594-024-01319-1","DOIUrl":"https://doi.org/10.1038/s41594-024-01319-1","url":null,"abstract":"<p>Smc5/6 is a member of the eukaryotic structural maintenance of chromosomes (SMC) family of complexes with important roles in genome maintenance and viral restriction. However, limited structural understanding of Smc5/6 hinders the elucidation of its diverse functions. Here, we report cryo-EM structures of the budding yeast Smc5/6 complex in eight-subunit, six-subunit and five-subunit states. Structural maps throughout the entire length of these complexes reveal modularity and key elements in complex assembly. We show that the non-SMC element (Nse)2 subunit supports the overall shape of the complex and uses a wedge motif to aid the stability and function of the complex. The Nse6 subunit features a flexible hook region for attachment to the Smc5 and Smc6 arm regions, contributing to the DNA repair roles of the complex. Our results also suggest a structural basis for the opposite effects of the Nse1–3–4 and Nse5–6 subcomplexes in regulating Smc5/6 ATPase activity. Collectively, our integrated structural and functional data provide a framework for understanding Smc5/6 assembly and function.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141334155","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}