Pub Date : 2024-11-04DOI: 10.1016/j.str.2024.10.016
Saskia Lesire, Rodrigo Lata, Yannick Hoogvliets, Kune Herrebosch, Paulien Van De Velde, Anouk Speleers, Frauke Christ, Siska Van Belle, Zeger Debyser
Methyl-CpG-binding protein 2 (MeCP2) is a ubiquitously expressed nuclear protein involved in transcriptional regulation and chromatin remodeling. MeCP2 exists in two isoforms, MeCP2 E1 and E2, which share the same functional domains. Loss-of-function mutations in the MeCP2 gene are the main cause of Rett syndrome (RTT). Previous studies identified a complex formation between MeCP2 and lens epithelium derived growth factor (LEDGF), a transcriptional regulator that exists in two isoforms, LEDGF/p75 and LEDGF/p52. Here, we characterized the molecular and functional interaction between MeCP2 and LEDGF. The NCoR interaction domain (NID) domain in MeCP2 is essential for the direct binding to the PWWP-CR1 region of LEDGF. Introduction of R306C, an RTT mutation in the NID of MeCP2, reduced the interaction with LEDGF. Our data reveal mutual inhibition of MeCP2 and LEDGF multimerization due to overlapping binding sites. Aligning with this observation, LEDGF depletion resulted in larger MeCP2 and DNA foci in NIH3T3 cells, suggesting a role for the MeCP2-LEDGF complex in chromatin organization.
{"title":"LEDGF interacts with the NID domain of MeCP2 and modulates MeCP2 condensates","authors":"Saskia Lesire, Rodrigo Lata, Yannick Hoogvliets, Kune Herrebosch, Paulien Van De Velde, Anouk Speleers, Frauke Christ, Siska Van Belle, Zeger Debyser","doi":"10.1016/j.str.2024.10.016","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.016","url":null,"abstract":"Methyl-CpG-binding protein 2 (MeCP2) is a ubiquitously expressed nuclear protein involved in transcriptional regulation and chromatin remodeling. MeCP2 exists in two isoforms, MeCP2 E1 and E2, which share the same functional domains. Loss-of-function mutations in the MeCP2 gene are the main cause of Rett syndrome (RTT). Previous studies identified a complex formation between MeCP2 and lens epithelium derived growth factor (LEDGF), a transcriptional regulator that exists in two isoforms, LEDGF/p75 and LEDGF/p52. Here, we characterized the molecular and functional interaction between MeCP2 and LEDGF. The NCoR interaction domain (NID) domain in MeCP2 is essential for the direct binding to the PWWP-CR1 region of LEDGF. Introduction of R306C, an RTT mutation in the NID of MeCP2, reduced the interaction with LEDGF. Our data reveal mutual inhibition of MeCP2 and LEDGF multimerization due to overlapping binding sites. Aligning with this observation, LEDGF depletion resulted in larger MeCP2 and DNA foci in NIH3T3 cells, suggesting a role for the MeCP2-LEDGF complex in chromatin organization.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"87 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142574503","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 : 2024-11-04DOI: 10.1016/j.str.2024.10.018
Arthur Neuberger, Alexey Shalygin, Yury A. Trofimov, Irina I. Veretenenko, Kirill D. Nadezhdin, Nikolay A. Krylov, Thomas Gudermann, Roman G. Efremov, Vladimir Chubanov, Alexander I. Sobolevsky
TRPV6 is a Ca2+ selective channel that mediates calcium uptake in the gut and contributes to the development and progression of human cancers. TRPV6 is represented by the ancestral and derived haplotypes that differ by three non-synonymous polymorphisms, located in the N-terminal ankyrin repeat domain (C157R), S1–S2 extracellular loop (M378V), and C-terminus (M681T). The ancestral and derived haplotypes were proposed to serve as genomic factors causing a different outcome for cancer patients of African ancestry. We solved cryoelectron microscopy (cryo-EM) structures of ancestral and derived TRPV6 in the open and calmodulin (CaM)-bound inactivated states. Neither state shows substantial structural differences caused by the non-synonymous polymorphisms. Functional properties assessed by electrophysiological recordings and Ca2+ uptake measurements, and water and ion permeation evaluated by molecular modeling also appear similar between the haplotypes. Therefore, ancestral and derived TRPV6 have similar structure and function, implying that other factors are responsible for the differences in susceptibility to cancer.
TRPV6 是一种 Ca2+ 选择性通道，介导肠道中的钙吸收，并导致人类癌症的发生和发展。TRPV6 由祖先型和衍生单倍型代表，这两种单倍型因三个非同义多态性而不同，分别位于 N 端 ankyrin 重复结构域（C157R）、S1-S2 细胞外环（M378V）和 C 端（M681T）。这些祖先单倍型和衍生单倍型被认为是导致非洲裔癌症患者不同预后的基因组因素。我们利用冷冻电子显微镜（cryo-EM）解决了开放状态和与钙调蛋白（CaM）结合的失活状态下祖先和衍生 TRPV6 的结构。这两种状态都没有显示出非同义多态性导致的实质性结构差异。通过电生理记录和 Ca2+ 摄取测量评估的功能特性，以及通过分子建模评估的水和离子渗透性在单倍型之间似乎也很相似。因此，祖先和衍生的 TRPV6 具有相似的结构和功能，这意味着癌症易感性的差异是由其他因素造成的。
{"title":"Structure-function analyses of human TRPV6 ancestral and derived haplotypes","authors":"Arthur Neuberger, Alexey Shalygin, Yury A. Trofimov, Irina I. Veretenenko, Kirill D. Nadezhdin, Nikolay A. Krylov, Thomas Gudermann, Roman G. Efremov, Vladimir Chubanov, Alexander I. Sobolevsky","doi":"10.1016/j.str.2024.10.018","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.018","url":null,"abstract":"TRPV6 is a Ca<sup>2+</sup> selective channel that mediates calcium uptake in the gut and contributes to the development and progression of human cancers. TRPV6 is represented by the ancestral and derived haplotypes that differ by three non-synonymous polymorphisms, located in the N-terminal ankyrin repeat domain (C157R), S1–S2 extracellular loop (M378V), and C-terminus (M681T). The ancestral and derived haplotypes were proposed to serve as genomic factors causing a different outcome for cancer patients of African ancestry. We solved cryoelectron microscopy (cryo-EM) structures of ancestral and derived TRPV6 in the open and calmodulin (CaM)-bound inactivated states. Neither state shows substantial structural differences caused by the non-synonymous polymorphisms. Functional properties assessed by electrophysiological recordings and Ca<sup>2+</sup> uptake measurements, and water and ion permeation evaluated by molecular modeling also appear similar between the haplotypes. Therefore, ancestral and derived TRPV6 have similar structure and function, implying that other factors are responsible for the differences in susceptibility to cancer.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"67 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142574502","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 : 2024-11-04DOI: 10.1016/j.str.2024.10.003
Bradley M. Readnour, Sheiny Tjia-Fleck, Nathan R. McCann, Yetunde A. Ayinuola, Francis J. Castellino
The importance of human plasminogen (hPg)/plasmin (hPm)/cell receptor complexes in invasiveness of cells has been amply established. The objective of this investigation was to determine a high-resolution structure of a major Group A Streptococcus (GAS) bacterial receptor (PAM) for hPg/hPm when bound on a cell surface to its major ligand, hPg. As a model cell surface with endogenous PAM, we employed engineered PAM-expressing lentivirus (LV) particles. We show that the ectodomain of a PAM-type M-Protein (M-Prt), in complex with hPg, is folded through distinct intra- and inter-domain interactions to a more compact form on the cell surface, thus establishing a new paradigm for membrane-bound M-Prt/ligand structures. These studies provide a framework for addressing the need for treatments of GAS disease by providing a molecular platform to solve structures of virulence-determining membrane proteins.
人类纤溶酶原（hPg）/纤溶酶原（hPm）/细胞受体复合物对细胞侵袭性的重要性已得到充分证实。本研究的目的是确定 A 组链球菌（GAS）细菌主要受体（PAM）在细胞表面与其主要配体 hPg 结合时的高分辨率结构。我们的研究表明，PAM 型 M 蛋白（M-Prt）的外结构域与 hPg 复合物通过不同的域内和域间相互作用折叠成细胞表面更紧凑的形式，从而建立了膜结合 M-Prt/ 配体结构的新范例。这些研究提供了一个解决决定毒力的膜蛋白结构的分子平台，从而为满足治疗 GAS 疾病的需要提供了一个框架。
{"title":"High-resolution cryo-EM analysis of a Streptococcus pyogenes M-protein/human plasminogen complex","authors":"Bradley M. Readnour, Sheiny Tjia-Fleck, Nathan R. McCann, Yetunde A. Ayinuola, Francis J. Castellino","doi":"10.1016/j.str.2024.10.003","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.003","url":null,"abstract":"The importance of human plasminogen (hPg)/plasmin (hPm)/cell receptor complexes in invasiveness of cells has been amply established. The objective of this investigation was to determine a high-resolution structure of a major Group A <em>Streptococcus</em> (GAS) bacterial receptor (PAM) for hPg/hPm when bound on a cell surface to its major ligand, hPg. As a model cell surface with endogenous PAM, we employed engineered PAM-expressing lentivirus (LV) particles. We show that the ectodomain of a PAM-type M-Protein (M-Prt), in complex with hPg, is folded through distinct intra- and inter-domain interactions to a more compact form on the cell surface, thus establishing a new paradigm for membrane-bound M-Prt/ligand structures. These studies provide a framework for addressing the need for treatments of GAS disease by providing a molecular platform to solve structures of virulence-determining membrane proteins.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"9 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142574505","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 : 2024-11-01DOI: 10.1016/j.str.2024.10.013
Jinliang Wang, Christopher K. Williams, Michael A. DeTure, Shino D. Magaki, Dennis W. Dickson, Harry V. Vinters, Paul M. Seidler
Fibril-type aggregates of tau occur in Alzheimer’s disease (AD) and dozens of tauopathies. Fibrils catalyze aggregation by prion-like seeding, which in part underlies disease progression. Seeding by recombinant and brain-derived tau fibrils is measured using biosensor cells that express aggregation-prone tau mutants fused with fluorescent reporter proteins. Seeding results in a punctated phenotype that is well established, but evidence that fluorescent tau fusion proteins from biosensor cells assemble into fibril-type structures is lacking. We investigated the effects of seeding on fibril formation by biosensor cells. Fluorescent punctated cell phenotypes that were catalyzed persisted with varying stabilities. Seeded cells bearing punctated phenotypes yielded sarkosyl-insoluble fibrils, although non-seeded cells did not. ImmunoEM of cell-purified fibrils shows that GFP localizes to the proteolytically sensitive fuzzy coat of tau fibrils. The presented data offer compelling evidence that fluorescent puncta are fibril-type aggregates of tau that result from prion-like seeding.
在阿尔茨海默病（AD）和数十种牛头蛋白病中会出现牛头蛋白的纤维状聚集。纤维通过朊病毒样播种催化聚集，这也是疾病进展的部分原因。重组和脑源性 tau 纤维的播种是通过生物传感器细胞来测量的，这些细胞表达易聚集的 tau 突变体，并与荧光报告蛋白融合。播种会导致点状表型，这一点已经得到证实，但缺乏证据表明生物传感器细胞中的荧光 tau 融合蛋白会组装成纤维状结构。我们研究了播种对生物传感器细胞形成纤维的影响。被催化的荧光点状细胞表型以不同的稳定性持续存在。带有点状表型的播种细胞会产生 sarkosyl 不溶性纤维，而非播种细胞则不会。细胞纯化纤维的免疫组织电镜显示，GFP 定位于对蛋白水解敏感的 tau 纤维的模糊外衣上。这些数据提供了令人信服的证据，表明荧光点是朊病毒样播种产生的 tau 纤维聚集体。
{"title":"Tau seeds catalyze fibril-type structures from GFP tau biosensor cells","authors":"Jinliang Wang, Christopher K. Williams, Michael A. DeTure, Shino D. Magaki, Dennis W. Dickson, Harry V. Vinters, Paul M. Seidler","doi":"10.1016/j.str.2024.10.013","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.013","url":null,"abstract":"Fibril-type aggregates of tau occur in Alzheimer’s disease (AD) and dozens of tauopathies. Fibrils catalyze aggregation by prion-like seeding, which in part underlies disease progression. Seeding by recombinant and brain-derived tau fibrils is measured using biosensor cells that express aggregation-prone tau mutants fused with fluorescent reporter proteins. Seeding results in a punctated phenotype that is well established, but evidence that fluorescent tau fusion proteins from biosensor cells assemble into fibril-type structures is lacking. We investigated the effects of seeding on fibril formation by biosensor cells. Fluorescent punctated cell phenotypes that were catalyzed persisted with varying stabilities. Seeded cells bearing punctated phenotypes yielded sarkosyl-insoluble fibrils, although non-seeded cells did not. ImmunoEM of cell-purified fibrils shows that GFP localizes to the proteolytically sensitive fuzzy coat of tau fibrils. The presented data offer compelling evidence that fluorescent puncta are fibril-type aggregates of tau that result from prion-like seeding.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"67 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142562137","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}
The identification of protein binding residues is essential for understanding their functions in vivo. However, it remains a computational challenge to accurately identify binding sites due to the lack of known residue binding patterns. Local residue spatial distribution and its interactive biophysical environment both determine binding patterns. Previous methods could not capture both information simultaneously, resulting in unsatisfactory performance. Here, we present GeoNet, an interpretable geometric deep learning model for predicting DNA, RNA, and protein binding sites by learning the latent residue binding patterns. GeoNet achieves this by introducing a coordinate-free geometric representation to characterize local residue distributions and generating an eigenspace to depict local interactive biophysical environments. Evaluation shows that GeoNet is superior compared to other leading predictors and it shows a strong interpretability of learned representations. We present three test cases, where interaction interfaces were successfully identified with GeoNet.
{"title":"GeoNet enables the accurate prediction of protein-ligand binding sites through interpretable geometric deep learning","authors":"Jiyun Han, Shizhuo Zhang, Mingming Guan, Qiuyu Li, Xin Gao, Juntao Liu","doi":"10.1016/j.str.2024.10.011","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.011","url":null,"abstract":"The identification of protein binding residues is essential for understanding their functions <em>in vivo</em>. However, it remains a computational challenge to accurately identify binding sites due to the lack of known residue binding patterns. Local residue spatial distribution and its interactive biophysical environment both determine binding patterns. Previous methods could not capture both information simultaneously, resulting in unsatisfactory performance. Here, we present GeoNet, an interpretable geometric deep learning model for predicting DNA, RNA, and protein binding sites by learning the latent residue binding patterns. GeoNet achieves this by introducing a coordinate-free geometric representation to characterize local residue distributions and generating an eigenspace to depict local interactive biophysical environments. Evaluation shows that GeoNet is superior compared to other leading predictors and it shows a strong interpretability of learned representations. We present three test cases, where interaction interfaces were successfully identified with GeoNet.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"136 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142562143","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 : 2024-11-01DOI: 10.1016/j.str.2024.10.012
Zhihan Bo, Thomas Rowntree, Steven Johnson, Hilman Nurmahdi, Richard J. Suckling, Johan Hill, Boguslawa Korona, Philip C. Weisshuhn, Devon Sheppard, Yao Meng, Shaoyan Liang, Edward D. Lowe, Susan M. Lea, Christina Redfield, Penny A. Handford
The Notch receptor is activated by the Delta/Serrate/Lag-2 (DSL) family of ligands. The organization of the extracellular signaling complex is unknown, although structures of Notch/ligand complexes comprising the ligand-binding region (LBR), and negative regulatory region (NRR) region, have been solved. Here, we investigate the human Notch-1 epidermal growth factor-like (EGF) 20-27 region, located between the LBR and NRR, and incorporating the Abruptex (Ax) region, associated with distinctive Drosophila phenotypes. Our analyses, using crystallography, NMR and small angle X-ray scattering (SAXS), support a rigid, elongated organization for EGF20-27 with the EGF20-21 linkage showing Ca2+-dependent flexibility. In functional assays, Notch-1 variants containing Ax substitutions result in reduced ligand-dependent trans-activation. When cis-JAG1 was expressed, Notch activity differences between WT and Ca2+-binding Ax variants were less marked than seen in the trans-activation assays alone, consistent with disruption of cis-inhibition. These data indicate the importance of Ca2+-stabilized structure and suggest the balance of cis- and trans-interactions explains the effects of Drosophila Ax mutations.
{"title":"Structural and functional studies of the EGF20-27 region reveal new features of the human Notch receptor important for optimal activation","authors":"Zhihan Bo, Thomas Rowntree, Steven Johnson, Hilman Nurmahdi, Richard J. Suckling, Johan Hill, Boguslawa Korona, Philip C. Weisshuhn, Devon Sheppard, Yao Meng, Shaoyan Liang, Edward D. Lowe, Susan M. Lea, Christina Redfield, Penny A. Handford","doi":"10.1016/j.str.2024.10.012","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.012","url":null,"abstract":"The Notch receptor is activated by the Delta/Serrate/Lag-2 (DSL) family of ligands. The organization of the extracellular signaling complex is unknown, although structures of Notch/ligand complexes comprising the ligand-binding region (LBR), and negative regulatory region (NRR) region, have been solved. Here, we investigate the human Notch-1 epidermal growth factor-like (EGF) 20-27 region, located between the LBR and NRR, and incorporating the Abruptex (Ax) region, associated with distinctive <em>Drosophila</em> phenotypes. Our analyses, using crystallography, NMR and small angle X-ray scattering (SAXS), support a rigid, elongated organization for EGF20-27 with the EGF20-21 linkage showing Ca<sup>2+</sup>-dependent flexibility. In functional assays, Notch-1 variants containing Ax substitutions result in reduced ligand-dependent <em>trans</em>-activation. When <em>cis</em>-JAG1 was expressed, Notch activity differences between WT and Ca<sup>2+</sup>-binding Ax variants were less marked than seen in the <em>trans</em>-activation assays alone, consistent with disruption of <em>cis</em>-inhibition. These data indicate the importance of Ca<sup>2+</sup>-stabilized structure and suggest the balance of <em>cis</em>- and <em>trans</em>-interactions explains the effects of <em>Drosophila Ax</em> mutations.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"6 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142562136","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 : 2024-10-30DOI: 10.1016/j.str.2024.10.008
Claudia S. Kielkopf, Mikhail M. Shneider, Petr G. Leiman, Nicholas M.I. Taylor
Bacteria use the type VI secretion system (T6SS) to secrete toxins into pro- and eukaryotic cells via machinery consisting of a contractile sheath and a rigid tube. Rearrangement hotspot (Rhs) proteins represent one of the most common T6SS effectors. The Rhs C-terminal toxin domain displays great functional diversity, while the Rhs core is characterized by YD repeats. We elucidate the Rhs core structures of PAAR- and VgrG-linked Rhs proteins from Salmonella bongori and Advenella mimigardefordensis, respectively. The Rhs core forms a large shell of β-sheets with a negatively charged interior and encloses a large volume. The S. bongori Rhs toxin does not lead to ordered density in the Rhs shell, suggesting the toxin is unfolded. Together with bioinformatics analysis showing that Rhs toxins predominantly act intracellularly, this suggests that the Rhs core functions two-fold, as a safety feature for the producer cell and as delivery mechanism for the toxin.
细菌利用 VI 型分泌系统（T6SS），通过由收缩鞘和硬管组成的机械装置向原核和真核细胞分泌毒素。重排热点（Rhs）蛋白是最常见的 T6SS 效应器之一。Rhs C 端毒素结构域显示出极大的功能多样性，而 Rhs 核心则以 YD 重复为特征。我们阐明了分别来自邦戈里沙门氏菌和米氏酵母菌的 PAAR 链接 Rhs 蛋白和 VgrG 链接 Rhs 蛋白的 Rhs 核心结构。Rhs 核心形成了一个由内部带负电荷的 β 片层组成的大外壳，并包围着一个大体积。S. bongori Rhs毒素不会导致Rhs外壳出现有序密度，这表明毒素是未折叠的。生物信息学分析表明，Rhs毒素主要在细胞内发挥作用，这表明Rhs核心具有双重功能，既是生产细胞的安全特征，也是毒素的输送机制。
{"title":"T6SS-associated Rhs toxin-encapsulating shells: Structural and bioinformatical insights into bacterial weaponry and self-protection","authors":"Claudia S. Kielkopf, Mikhail M. Shneider, Petr G. Leiman, Nicholas M.I. Taylor","doi":"10.1016/j.str.2024.10.008","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.008","url":null,"abstract":"Bacteria use the type VI secretion system (T6SS) to secrete toxins into pro- and eukaryotic cells via machinery consisting of a contractile sheath and a rigid tube. Rearrangement hotspot (Rhs) proteins represent one of the most common T6SS effectors. The Rhs C-terminal toxin domain displays great functional diversity, while the Rhs core is characterized by YD repeats. We elucidate the Rhs core structures of PAAR- and VgrG-linked Rhs proteins from <em>Salmonella bongori</em> and <em>Advenella mimigardefordensis</em>, respectively. The Rhs core forms a large shell of β-sheets with a negatively charged interior and encloses a large volume. The <em>S. bongori</em> Rhs toxin does not lead to ordered density in the Rhs shell, suggesting the toxin is unfolded. Together with bioinformatics analysis showing that Rhs toxins predominantly act intracellularly, this suggests that the Rhs core functions two-fold, as a safety feature for the producer cell and as delivery mechanism for the toxin.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"15 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142541574","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 : 2024-10-30DOI: 10.1016/j.str.2024.10.029
Shuvankar Dey, Purba Pahari, Srija Mukherjee, James B. Munro, Dibyendu Kumar Das
(Structure 32, 1–16; November 7, 2024)
(结构 32，1-16；2024 年 11 月 7 日）
{"title":"Conformational dynamics of SARS-CoV-2 Omicron spike trimers during fusion activation at single molecule resolution","authors":"Shuvankar Dey, Purba Pahari, Srija Mukherjee, James B. Munro, Dibyendu Kumar Das","doi":"10.1016/j.str.2024.10.029","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.029","url":null,"abstract":"(Structure <em>32</em>, 1–16; November 7, 2024)","PeriodicalId":22168,"journal":{"name":"Structure","volume":"45 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142542031","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 : 2024-10-28DOI: 10.1016/j.str.2024.10.004
Donggyun Kim, Weijing Liu, Rosa Viner, Vadim Cherezov
G protein-coupled receptors (GPCRs) are essential transmembrane proteins playing key roles in human health and disease. Understanding their atomic-level molecular structure and conformational states is imperative for advancing drug development. Recent breakthroughs in single-particle cryogenic electron microscopy (cryo-EM) have propelled the structural biology of GPCRs into a new era. Nevertheless, the preparation of suitable GPCR samples and their complexes for cryo-EM analysis remains challenging due to their poor stability and highly dynamic nature. Here, we present our online buffer exchange-native MS method combined with Direct Mass Technology (OBE-nMS+DMT) which facilitates high-throughput analysis and guides sample preparation. We applied this method to optimize the GPR119-Gs complex sample prior to cryo-EM analysis, leading to a 3.51 Å resolution structure from only 396 movies collected on a 200 kV Glacios. This study suggests that the OBE-nMS+DMT method emerges as a powerful tool for prescreening sample conditions in cryo-EM studies of GPCRs and other membrane protein complexes.
{"title":"Native mass spectrometry prescreening of G protein-coupled receptor complexes for cryo-EM structure determination","authors":"Donggyun Kim, Weijing Liu, Rosa Viner, Vadim Cherezov","doi":"10.1016/j.str.2024.10.004","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.004","url":null,"abstract":"G protein-coupled receptors (GPCRs) are essential transmembrane proteins playing key roles in human health and disease. Understanding their atomic-level molecular structure and conformational states is imperative for advancing drug development. Recent breakthroughs in single-particle cryogenic electron microscopy (cryo-EM) have propelled the structural biology of GPCRs into a new era. Nevertheless, the preparation of suitable GPCR samples and their complexes for cryo-EM analysis remains challenging due to their poor stability and highly dynamic nature. Here, we present our online buffer exchange-native MS method combined with Direct Mass Technology (OBE-nMS+DMT) which facilitates high-throughput analysis and guides sample preparation. We applied this method to optimize the GPR119-G<sub>s</sub> complex sample prior to cryo-EM analysis, leading to a 3.51 Å resolution structure from only 396 movies collected on a 200 kV Glacios. This study suggests that the OBE-nMS+DMT method emerges as a powerful tool for prescreening sample conditions in cryo-EM studies of GPCRs and other membrane protein complexes.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"19 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142519521","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 : 2024-10-28DOI: 10.1016/j.str.2024.10.002
Ashwini Kedari, Rommel Iheozor-Ejiofor, Petja Salminen, Hasan Uğurlu, Anna R. Mäkelä, Lev Levanov, Olli Vapalahti, Vesa P. Hytönen, Kalle Saksela, Ilona Rissanen
Host-cell entry of the highly pathogenic rabies virus (RABV) is mediated by glycoprotein (G) spikes, which also comprise the primary target for the humoral immune response. RABV glycoprotein (RABV-G) displays several antigenic sites that are targeted by neutralizing monoclonal antibodies (mAbs). In this study, we determined the epitope of a potently neutralizing human mAb, CR57, which we engineered into a diabody format to facilitate crystallization. We report the crystal structure of the CR57 diabody alone at 2.38 Å resolution, and in complex with RABV-G domain III at 2.70 Å resolution. The CR57−RABV-G structure reveals critical interactions at the antigen interface, which target the conserved “KLCGVL” peptide and residues proximal to it on RABV-G. Structural analysis combined with a cell-cell fusion assay demonstrates that CR57 effectively inhibits RABV-G-mediated fusion by obstructing the fusogenic transitions of the spike protein. Altogether, this investigation provides a structural perspective on RABV inhibition by a potently neutralizing human antibody.
高致病性狂犬病病毒（RABV）通过糖蛋白（G）尖峰进入宿主细胞，这也是体液免疫反应的主要靶点。RABV 糖蛋白（RABV-G）有几个抗原位点，是中和单克隆抗体（mAbs）的靶点。在本研究中，我们确定了一种强效中和人类 mAb CR57 的表位，并将其设计成二抗体形式以方便结晶。我们报告了分辨率为 2.38 Å 的 CR57 二抗体单独晶体结构，以及分辨率为 2.70 Å 的 CR57 与 RABV-G 结构域 III 复合物的晶体结构。CR57-RABV-G 结构揭示了抗原界面上的关键相互作用，其目标是 RABV-G 上保守的 "KLCGVL "肽及其近端残基。结构分析结合细胞-细胞融合试验表明，CR57 通过阻碍尖峰蛋白的融合转换，有效抑制了 RABV-G 介导的融合。总之，这项研究从结构角度揭示了一种强效中和人类抗体对 RABV 的抑制作用。
{"title":"Structural insight into rabies virus neutralization revealed by an engineered antibody scaffold","authors":"Ashwini Kedari, Rommel Iheozor-Ejiofor, Petja Salminen, Hasan Uğurlu, Anna R. Mäkelä, Lev Levanov, Olli Vapalahti, Vesa P. Hytönen, Kalle Saksela, Ilona Rissanen","doi":"10.1016/j.str.2024.10.002","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.002","url":null,"abstract":"Host-cell entry of the highly pathogenic rabies virus (RABV) is mediated by glycoprotein (G) spikes, which also comprise the primary target for the humoral immune response. RABV glycoprotein (RABV-G) displays several antigenic sites that are targeted by neutralizing monoclonal antibodies (mAbs). In this study, we determined the epitope of a potently neutralizing human mAb, CR57, which we engineered into a diabody format to facilitate crystallization. We report the crystal structure of the CR57 diabody alone at 2.38 Å resolution, and in complex with RABV-G domain III at 2.70 Å resolution. The CR57−RABV-G structure reveals critical interactions at the antigen interface, which target the conserved “KLCGVL” peptide and residues proximal to it on RABV-G. Structural analysis combined with a cell-cell fusion assay demonstrates that CR57 effectively inhibits RABV-G-mediated fusion by obstructing the fusogenic transitions of the spike protein. Altogether, this investigation provides a structural perspective on RABV inhibition by a potently neutralizing human antibody.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"12 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142519525","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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