Pub Date : 2024-08-05DOI: 10.1016/j.str.2024.07.007
Enzymes facilitating the transfer of phosphate groups constitute the most extensive protein families across all kingdoms of life. They make up approximately 10% of the proteins found in the human genome. Understanding the mechanisms by which enzymes catalyze these reactions is essential in characterizing the processes they regulate. Metal fluorides can be used as multifunctional tools to study these enzymes. These ionic species bear the same charge as phosphate and the transferring phosphoryl group and, in addition, allow the enzyme to be trapped in catalytically important states with spectroscopically sensitive atoms interacting directly with active site residues. The ionic nature of these phosphate surrogates also allows their removal and replacement with other analogs. Here, we describe the best practices to obtain these complexes, their use in NMR, X-ray crystallography, cryo-EM, and SAXS and describe a new metal fluoride, scandium tetrafluoride, which has significant anomalous signal using soft X-rays.
促进磷酸基团转移的酶构成了所有生命王国中最广泛的蛋白质家族。它们约占人类基因组中蛋白质的 10%。了解酶催化这些反应的机制,对于确定它们调控过程的特征至关重要。金属氟化物可用作研究这些酶的多功能工具。这些离子型物质与磷酸盐和转移的磷酸基带有相同的电荷,此外,它们还能使酶处于重要的催化状态,具有光谱敏感性的原子直接与活性位点残基相互作用。这些磷酸盐代用品的离子性质还允许将其移除并用其他类似物替代。在此,我们介绍了获得这些复合物的最佳方法,以及它们在核磁共振、X 射线晶体学、低温电子显微镜和 SAXS 中的应用,并描述了一种新的金属氟化物--四氟化钪,它在使用软 X 射线时具有显著的异常信号。
{"title":"Metal fluorides—multi-functional tools for the study of phosphoryl transfer enzymes, a practical guide","authors":"","doi":"10.1016/j.str.2024.07.007","DOIUrl":"https://doi.org/10.1016/j.str.2024.07.007","url":null,"abstract":"<p>Enzymes facilitating the transfer of phosphate groups constitute the most extensive protein families across all kingdoms of life. They make up approximately 10% of the proteins found in the human genome. Understanding the mechanisms by which enzymes catalyze these reactions is essential in characterizing the processes they regulate. Metal fluorides can be used as multifunctional tools to study these enzymes. These ionic species bear the same charge as phosphate and the transferring phosphoryl group and, in addition, allow the enzyme to be trapped in catalytically important states with spectroscopically sensitive atoms interacting directly with active site residues. The ionic nature of these phosphate surrogates also allows their removal and replacement with other analogs. Here, we describe the best practices to obtain these complexes, their use in NMR, X-ray crystallography, cryo-EM, and SAXS and describe a new metal fluoride, scandium tetrafluoride, which has significant anomalous signal using soft X-rays.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141892169","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 : 2024-07-30DOI: 10.1016/j.str.2024.07.004
Two retinal transcription factors, cone-rod homeobox (CRX) and neural retina leucine zipper (NRL), cooperate functionally and physically to control photoreceptor development and homeostasis. Mutations in CRX and NRL cause severe retinal diseases. Despite the roles of NRL and CRX, insight into their functions at the molecular level is lacking. Here, we have solved the crystal structure of the CRX homeodomain in complex with its cognate response element (Ret4) from the rhodopsin proximal promoter region. The structure reveals an unexpected 2:1 stoichiometry of CRX/Ret4 and unique orientation of CRX molecules on DNA, and it explains the mechanisms of pathogenic mutations in CRX. Mutations R41Q and E42K disrupt the CRX protein-protein contacts based on the structure and reduce the CRX/Ret4 binding stoichiometry, suggesting a novel disease mechanism. Furthermore, we show that NRL alters the stoichiometry and increases affinity of CRX binding at the rhodopsin promoter, which may enhance transcription of rod-specific genes and suppress transcription of cone-specific genes.
{"title":"Molecular basis of CRX/DNA recognition and stoichiometry at the Ret4 response element","authors":"","doi":"10.1016/j.str.2024.07.004","DOIUrl":"https://doi.org/10.1016/j.str.2024.07.004","url":null,"abstract":"<p>Two retinal transcription factors, cone-rod homeobox (CRX) and neural retina leucine zipper (NRL), cooperate functionally and physically to control photoreceptor development and homeostasis. Mutations in CRX and NRL cause severe retinal diseases. Despite the roles of NRL and CRX, insight into their functions at the molecular level is lacking. Here, we have solved the crystal structure of the CRX homeodomain in complex with its cognate response element (Ret4) from the rhodopsin proximal promoter region. The structure reveals an unexpected 2:1 stoichiometry of CRX/Ret4 and unique orientation of CRX molecules on DNA, and it explains the mechanisms of pathogenic mutations in CRX. Mutations R41Q and E42K disrupt the CRX protein-protein contacts based on the structure and reduce the CRX/Ret4 binding stoichiometry, suggesting a novel disease mechanism. Furthermore, we show that NRL alters the stoichiometry and increases affinity of CRX binding at the rhodopsin promoter, which may enhance transcription of rod-specific genes and suppress transcription of cone-specific genes.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141795033","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 : 2024-07-29DOI: 10.1016/j.str.2024.07.003
With the advent of modern technologies for cryo-electron tomography (cryo-ET), high-quality tilt series are more rapidly acquired than processed and analyzed. Thus, a robust and fast-automated alignment for batch processing in cryo-ET is needed. While different software packages have made available several approaches for automated marker-based alignment of tilt series, manual user intervention remains necessary for many datasets, thus preventing high-throughput tomography.
We have developed a MATLAB-based framework integrated into the Dynamo software package for automatic detection of fiducial markers that generates a robust alignment model with minimal input parameters. This approach allows high-throughput, unsupervised volume reconstruction. This new module extends Dynamo with a large repertory of tools for tomographic alignment and reconstruction, as well as specific visualization browsers to rapidly assess the biological relevance of the dataset. Our approach has been successfully tested on a broad range of datasets that include diverse biological samples and cryo-ET modalities.
随着现代低温电子断层扫描(cryo-ET)技术的出现,高质量倾斜序列的获取比处理和分析更加迅速。因此,在低温电子断层扫描中需要一种用于批量处理的强大而快速的自动配准方法。我们开发了一个基于 MATLAB 的框架,集成到 Dynamo 软件包中,用于自动检测靶标,以最小的输入参数生成稳健的配准模型。这种方法可实现高通量、无监督的容积重建。这个新模块扩展了 Dynamo,为断层配准和重建提供了大量工具,还提供了特定的可视化浏览器,以快速评估数据集的生物学相关性。我们的方法已在广泛的数据集上进行了成功测试,这些数据集包括不同的生物样本和低温电子显微镜模式。
{"title":"Automated fiducial-based alignment of cryo-electron tomography tilt series in Dynamo","authors":"","doi":"10.1016/j.str.2024.07.003","DOIUrl":"https://doi.org/10.1016/j.str.2024.07.003","url":null,"abstract":"<p>With the advent of modern technologies for cryo-electron tomography (cryo-ET), high-quality tilt series are more rapidly acquired than processed and analyzed. Thus, a robust and fast-automated alignment for batch processing in cryo-ET is needed. While different software packages have made available several approaches for automated marker-based alignment of tilt series, manual user intervention remains necessary for many datasets, thus preventing high-throughput tomography.</p><p>We have developed a MATLAB-based framework integrated into the Dynamo software package for automatic detection of fiducial markers that generates a robust alignment model with minimal input parameters. This approach allows high-throughput, unsupervised volume reconstruction. This new module extends Dynamo with a large repertory of tools for tomographic alignment and reconstruction, as well as specific visualization browsers to rapidly assess the biological relevance of the dataset. Our approach has been successfully tested on a broad range of datasets that include diverse biological samples and cryo-ET modalities.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141795031","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 : 2024-07-26DOI: 10.1016/j.str.2024.07.002
Phages are the most prevalent and diverse entities in the biosphere and represent the simplest systems that are capable of self-replication. Many fundamental concepts of transcriptional regulation were revealed through phage studies. The replication of phages within bacteria entails the hijacking of the host transcription machinery. Typically, this is accomplished through proteins and RNAs encoded by the phage genome that bind to the host RNA polymerase and modify its characteristics. Understanding these processes offers valuable insights into the mechanisms of bacterial transcription itself. Historically, X-ray crystallography has been the major tool for elucidating the structural basis of phage transcriptional regulation. In recent years, the application of cryoelectron microscopy has not only allowed the exploration of protein-protein and protein-nucleic acid interactions at near-atomic resolution but also captured transient intermediate states, further expanding our mechanistic understanding of phage transcriptional regulation.
{"title":"Structural basis of phage transcriptional regulation","authors":"","doi":"10.1016/j.str.2024.07.002","DOIUrl":"https://doi.org/10.1016/j.str.2024.07.002","url":null,"abstract":"<p>Phages are the most prevalent and diverse entities in the biosphere and represent the simplest systems that are capable of self-replication. Many fundamental concepts of transcriptional regulation were revealed through phage studies. The replication of phages within bacteria entails the hijacking of the host transcription machinery. Typically, this is accomplished through proteins and RNAs encoded by the phage genome that bind to the host RNA polymerase and modify its characteristics. Understanding these processes offers valuable insights into the mechanisms of bacterial transcription itself. Historically, X-ray crystallography has been the major tool for elucidating the structural basis of phage transcriptional regulation. In recent years, the application of cryoelectron microscopy has not only allowed the exploration of protein-protein and protein-nucleic acid interactions at near-atomic resolution but also captured transient intermediate states, further expanding our mechanistic understanding of phage transcriptional regulation.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141764459","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 : 2024-07-25DOI: 10.1016/j.str.2024.07.001
Activated FXII (FXIIa) is the principal initiator of the plasma contact system and can activate both procoagulant and proinflammatory pathways. Its activity is important in the pathophysiology of hereditary angioedema (HAE). Here, we describe a high-resolution cryoelectron microscopy (cryo-EM) structure of the beta-chain from FXIIa (βFXIIa) complexed with the Fab fragment of garadacimab. Garadacimab binds to βFXIIa through an unusually long CDR-H3 that inserts into the S1 pocket in a non-canonical way. This structural mechanism is likely the primary contributor to the inhibition of activated FXIIa proteolytic activity in HAE. Garadacimab Fab-βFXIIa structure also reveals critical determinants of high-affinity binding of garadacimab to activated FXIIa. Structural analysis with other bona fide FXIIa inhibitors, such as benzamidine and C1-INH, reveals a surprisingly similar mechanism of βFXIIa inhibition by garadacimab. In summary, the garadacimab Fab-βFXIIa structure provides crucial insights into its mechanism of action and delineates primary and auxiliary paratopes/epitopes.
{"title":"Structural basis for the inhibition of βFXIIa by garadacimab","authors":"","doi":"10.1016/j.str.2024.07.001","DOIUrl":"https://doi.org/10.1016/j.str.2024.07.001","url":null,"abstract":"<p>Activated FXII (FXIIa) is the principal initiator of the plasma contact system and can activate both procoagulant and proinflammatory pathways. Its activity is important in the pathophysiology of hereditary angioedema (HAE). Here, we describe a high-resolution cryoelectron microscopy (cryo-EM) structure of the beta-chain from FXIIa (βFXIIa) complexed with the Fab fragment of garadacimab. Garadacimab binds to βFXIIa through an unusually long CDR-H3 that inserts into the S1 pocket in a non-canonical way. This structural mechanism is likely the primary contributor to the inhibition of activated FXIIa proteolytic activity in HAE. Garadacimab Fab-βFXIIa structure also reveals critical determinants of high-affinity binding of garadacimab to activated FXIIa. Structural analysis with other bona fide FXIIa inhibitors, such as benzamidine and C1-INH, reveals a surprisingly similar mechanism of βFXIIa inhibition by garadacimab. In summary, the garadacimab Fab-βFXIIa structure provides crucial insights into its mechanism of action and delineates primary and auxiliary paratopes/epitopes.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141754352","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 : 2024-07-22DOI: 10.1016/j.str.2024.06.020
The endothelin receptor type B (ETB) exhibits promiscuous coupling with various heterotrimeric G protein subtypes including Gs, Gi/o, Gq/11, and G12/13. Recent fluorescence and structural studies have raised questions regarding the coupling efficiencies and determinants of these G protein subtypes. Herein, by utilizing an integrative approach, combining hydrogen/deuterium exchange mass spectrometry and NanoLuc Binary Technology-based cellular systems, we investigated conformational changes of Gs, Gi, and Gq triggered by ETB activation. ETB coupled to Gi and Gq but not with Gs. We underscored the critical roles of specific regions, including the C terminus of Gα and intracellular loop 2 (ICL2) of ETB in ETB-Gi1 or ETB-Gq coupling. Although The C terminus of Gα is essential for ETB-Gi1 and ETB-Gq coupling, ETB ICL2 influences Gq-coupling but not Gi1-coupling. Our results suggest a differential coupling efficiency of ETB with Gs, Gi1, and Gq, accompanied by distinct conformational changes in G proteins upon ETB-induced activation.
B 型内皮素受体(ETB)表现出与各种异三聚体 G 蛋白亚型(包括 Gs、Gi/o、Gq/11 和 G12/13)的杂乱耦合。最近的荧光和结构研究提出了有关这些 G 蛋白亚型的耦合效率和决定因素的问题。在此,我们利用一种综合方法,结合氢/氘交换质谱法和基于 NanoLuc 二进制技术的细胞系统,研究了 ETB 激活引发的 Gs、Gi 和 Gq 的构象变化。ETB 与 Gi 和 Gq 相耦合,但不与 Gs 相耦合。我们强调了特定区域在 ETB-Gi1 或 ETB-Gq 偶联中的关键作用,包括 Gα 的 C 末端和 ETB 的胞内环 2(ICL2)。虽然 Gα 的 C 末端对 ETB-Gi1 和 ETB-Gq 耦合至关重要,但 ETB ICL2 会影响 Gq 耦合,而不会影响 Gi1 耦合。我们的研究结果表明,ETB 与 Gs、Gi1 和 Gq 的耦合效率不同,在 ETB 诱导的激活过程中 G 蛋白的构象也会发生不同的变化。
{"title":"Molecular mechanism of the endothelin receptor type B interactions with Gs, Gi, and Gq","authors":"","doi":"10.1016/j.str.2024.06.020","DOIUrl":"https://doi.org/10.1016/j.str.2024.06.020","url":null,"abstract":"<p>The endothelin receptor type B (ET<sub>B</sub>) exhibits promiscuous coupling with various heterotrimeric G protein subtypes including Gs, Gi/o, Gq/11, and G12/13. Recent fluorescence and structural studies have raised questions regarding the coupling efficiencies and determinants of these G protein subtypes. Herein, by utilizing an integrative approach, combining hydrogen/deuterium exchange mass spectrometry and NanoLuc Binary Technology-based cellular systems, we investigated conformational changes of Gs, Gi, and Gq triggered by ET<sub>B</sub> activation. ET<sub>B</sub> coupled to Gi and Gq but not with Gs. We underscored the critical roles of specific regions, including the C terminus of Gα and intracellular loop 2 (ICL2) of ET<sub>B</sub> in ET<sub>B</sub>-Gi1 or ET<sub>B</sub>-Gq coupling. Although The C terminus of Gα is essential for ET<sub>B</sub>-Gi1 and ET<sub>B</sub>-Gq coupling, ET<sub>B</sub> ICL2 influences Gq-coupling but not Gi1-coupling. Our results suggest a differential coupling efficiency of ET<sub>B</sub> with Gs, Gi1, and Gq, accompanied by distinct conformational changes in G proteins upon ET<sub>B</sub>-induced activation.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141746577","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 : 2024-07-19DOI: 10.1016/j.str.2024.06.018
Intracellular tau aggregation requires a local protein concentration increase, referred to as “droplets”. However, the cellular mechanism for droplet formation is poorly understood. Here, we expressed OptoTau, a P301L mutant tau fused with CRY2olig, a light-sensitive protein that can form homo-oligomers. Under blue light exposure, OptoTau increased tau phosphorylation and was sequestered in aggresomes. Suppressing aggresome formation by nocodazole formed tau granular clusters in the cytoplasm. The granular clusters disappeared by discontinuing blue light exposure or 1,6-hexanediol treatment suggesting that intracellular tau droplet formation requires microtubule collapse. Expressing OptoTau-ΔN, a species of N-terminal cleaved tau observed in the Alzheimer’s disease brain, formed 1,6-hexanediol and detergent-resistant tau clusters in the cytoplasm with blue light stimulation. These intracellular stable tau clusters acted as a seed for tau fibrils in vitro. These results suggest that tau droplet formation and N-terminal cleavage are necessary for neurofibrillary tangles formation in neurodegenerative diseases.
细胞内 tau 的聚集需要局部蛋白质浓度的增加,即所谓的 "液滴"。然而,人们对液滴形成的细胞机制知之甚少。在这里,我们表达了OptoTau,一种与CRY2olig融合的P301L突变体tau,CRY2olig是一种对光敏感的蛋白质,可以形成同源异构体。在蓝光照射下,OptoTau增加了tau的磷酸化,并固着在凝集体中。用nocodazole抑制凝集体的形成会在细胞质中形成tau颗粒簇。停止蓝光照射或1,6-己二醇处理后,颗粒簇消失,这表明细胞内tau小滴的形成需要微管塌缩。表达 OptoTau-ΔN(一种在阿尔茨海默病大脑中观察到的 N 端裂解 tau)的细胞在蓝光刺激下会在细胞质中形成抗 1,6- 己二醇和去垢剂的 tau 簇。这些细胞内稳定的 tau 簇在体外可作为 tau 纤维的种子。这些结果表明,在神经退行性疾病中,tau小滴的形成和N端裂解是神经纤维缠结形成的必要条件。
{"title":"Intracellular tau fragment droplets serve as seeds for tau fibrils","authors":"","doi":"10.1016/j.str.2024.06.018","DOIUrl":"https://doi.org/10.1016/j.str.2024.06.018","url":null,"abstract":"<p>Intracellular tau aggregation requires a local protein concentration increase, referred to as “droplets”. However, the cellular mechanism for droplet formation is poorly understood. Here, we expressed OptoTau, a P301L mutant tau fused with CRY2olig, a light-sensitive protein that can form homo-oligomers. Under blue light exposure, OptoTau increased tau phosphorylation and was sequestered in aggresomes. Suppressing aggresome formation by nocodazole formed tau granular clusters in the cytoplasm. The granular clusters disappeared by discontinuing blue light exposure or 1,6-hexanediol treatment suggesting that intracellular tau droplet formation requires microtubule collapse. Expressing OptoTau-ΔN, a species of N-terminal cleaved tau observed in the Alzheimer’s disease brain, formed 1,6-hexanediol and detergent-resistant tau clusters in the cytoplasm with blue light stimulation. These intracellular stable tau clusters acted as a seed for tau fibrils <em>in vitro</em>. These results suggest that tau droplet formation and N-terminal cleavage are necessary for neurofibrillary tangles formation in neurodegenerative diseases.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141726361","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 : 2024-07-19DOI: 10.1016/j.str.2024.06.019
Cag type IV secretion system (CagT4SS) translocates oncoprotein cytotoxin-associated gene A (CagA) into host cells and plays a key role in the pathogenesis of Helicobacter pylori. The structure of the outer membrane core complex (OMCC) in CagT4SS consists of CagX, CagY, CagM, CagT, and Cag3 in a stoichiometric ratio of 1:1:2:2:5 with 14-fold symmetry. However, the assembly pathway of OMCC remains elusive. Here, we report the crystal structures of CagT and Cag3-CagT complex, and the structural dynamics of Cag3 and CagT using hydrogen deuterium exchange-mass spectrometry (HDX-MS). The interwoven interaction of Cag3 and CagT involves conformational changes of CagT and β strand swapping. In conjunction with biochemical and biophysical assays, we further demonstrate the different oligomerization states of Cag3 and Cag3-CagT complex. Additionally, the association with CagM requires the pre-formation of Cag3-CagT complex. These results demonstrate the generation of different intermediate sub-assemblies and their structural flexibility, potentially representing different building blocks for OMCC assembly.
{"title":"Structural insights into the assembly pathway of the Helicobacter pylori CagT4SS outer membrane core complex","authors":"","doi":"10.1016/j.str.2024.06.019","DOIUrl":"https://doi.org/10.1016/j.str.2024.06.019","url":null,"abstract":"<p>Cag type IV secretion system (CagT4SS) translocates oncoprotein cytotoxin-associated gene A (CagA) into host cells and plays a key role in the pathogenesis of <em>Helicobacter pylori</em>. The structure of the outer membrane core complex (OMCC) in CagT4SS consists of CagX, CagY, CagM, CagT, and Cag3 in a stoichiometric ratio of 1:1:2:2:5 with 14-fold symmetry. However, the assembly pathway of OMCC remains elusive. Here, we report the crystal structures of CagT and Cag3-CagT complex, and the structural dynamics of Cag3 and CagT using hydrogen deuterium exchange-mass spectrometry (HDX-MS). The interwoven interaction of Cag3 and CagT involves conformational changes of CagT and β strand swapping. In conjunction with biochemical and biophysical assays, we further demonstrate the different oligomerization states of Cag3 and Cag3-CagT complex. Additionally, the association with CagM requires the pre-formation of Cag3-CagT complex. These results demonstrate the generation of different intermediate sub-assemblies and their structural flexibility, potentially representing different building blocks for OMCC assembly.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141726356","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 : 2024-07-18DOI: 10.1016/j.str.2024.06.017
Complex associating with SET1 (COMPASS) is a histone H3K4 tri-methyltransferase controlled by several regulatory subunits including CXXC zinc finger protein 1 (Cfp1). Prior studies established the structural underpinnings controlling H3K4me3 recognition by the PHD domain of Cfp1’s yeast homolog (Spp1). However, metazoans Cfp1PHD lacks structural elements important for H3K4me3 stabilization in Spp1, suggesting that in metazoans, Cfp1PHD domain binds H3K4me3 differently. The structure of Cfp1PHD in complex with H3K4me3 shows unique features such as non-canonical coordination of the first zinc atom and a disulfide bond forcing the reorientation of Cfp1PHD N-terminus, thereby leading to an atypical H3K4me3 binding pocket. This configuration minimizes Cfp1PHD reliance on canonical residues important for histone binding functions of other PHD domains. Cancer-related mutations in Cfp1PHD impair H3K4me3 binding, implying a potential impact on epigenetic signaling. Our work highlights a potential diversification of PHD histone binding modes and the impact of cancer mutations on Cfp1 functions.
{"title":"Structural insights into an atypical histone binding mechanism by a PHD finger","authors":"","doi":"10.1016/j.str.2024.06.017","DOIUrl":"https://doi.org/10.1016/j.str.2024.06.017","url":null,"abstract":"<p>Complex associating with SET1 (COMPASS) is a histone H3K4 tri-methyltransferase controlled by several regulatory subunits including CXXC zinc finger protein 1 (Cfp1). Prior studies established the structural underpinnings controlling H3K4me3 recognition by the PHD domain of Cfp1’s yeast homolog (Spp1). However, metazoans Cfp1<sup>PHD</sup> lacks structural elements important for H3K4me3 stabilization in Spp1, suggesting that in metazoans, Cfp1<sup>PHD</sup> domain binds H3K4me3 differently. The structure of Cfp1<sup>PHD</sup> in complex with H3K4me3 shows unique features such as non-canonical coordination of the first zinc atom and a disulfide bond forcing the reorientation of Cfp1<sup>PHD</sup> N-terminus, thereby leading to an atypical H3K4me3 binding pocket. This configuration minimizes Cfp1<sup>PHD</sup> reliance on canonical residues important for histone binding functions of other PHD domains. Cancer-related mutations in Cfp1<sup>PHD</sup> impair H3K4me3 binding, implying a potential impact on epigenetic signaling. Our work highlights a potential diversification of PHD histone binding modes and the impact of cancer mutations on Cfp1 functions.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141726369","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 : 2024-07-18DOI: 10.1016/j.str.2024.06.016
Mycobacterial HflX confers resistance against macrolide antibiotics. However, the exact molecular mechanism is poorly understood. To gain further insights, we determined the cryo-EM structures of M. smegmatis (Msm) HflX-50S subunit and 50S subunit-erythromycin (ERY) complexes at a global resolution of approximately 3 Å. A conserved nucleotide A2286 at the gate of nascent peptide exit tunnel (NPET) adopts a swayed conformation in HflX-50S complex and interacts with a loop within the linker helical (LH) domain of MsmHflX that contains an additional 9 residues insertion. Interestingly, the swaying of this nucleotide, which is usually found in the non-swayed conformation, is induced by erythromycin binding. Furthermore, we observed that erythromycin decreases HflX’s ribosome-dependent GTP hydrolysis, resulting in its enhanced binding and anti-association activity on the 50S subunit. Our findings reveal how mycobacterial HflX senses the presence of macrolides at the peptide tunnel entrance and confers antibiotic resistance in mycobacteria.
{"title":"Cryo-EM structures reveal the molecular mechanism of HflX-mediated erythromycin resistance in mycobacteria","authors":"","doi":"10.1016/j.str.2024.06.016","DOIUrl":"https://doi.org/10.1016/j.str.2024.06.016","url":null,"abstract":"<p>Mycobacterial HflX confers resistance against macrolide antibiotics. However, the exact molecular mechanism is poorly understood. To gain further insights, we determined the cryo-EM structures of <em>M. smegmatis</em> (Msm) HflX-50S subunit and 50S subunit-erythromycin (ERY) complexes at a global resolution of approximately 3 Å. A conserved nucleotide A2286 at the gate of nascent peptide exit tunnel (NPET) adopts a swayed conformation in HflX-50S complex and interacts with a loop within the linker helical (LH) domain of MsmHflX that contains an additional 9 residues insertion. Interestingly, the swaying of this nucleotide, which is usually found in the non-swayed conformation, is induced by erythromycin binding. Furthermore, we observed that erythromycin decreases HflX’s ribosome-dependent GTP hydrolysis, resulting in its enhanced binding and anti-association activity on the 50S subunit. Our findings reveal how mycobacterial HflX senses the presence of macrolides at the peptide tunnel entrance and confers antibiotic resistance in mycobacteria.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141726362","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}