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":null,"pages":null},"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":null,"pages":null},"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":null,"pages":null},"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":null,"pages":null},"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":null,"pages":null},"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}
Lytic podophages (VP1–VP5) play crucial roles in subtyping Vibrio cholerae O1 biotype El Tor. However, until now no structures of these phages have been available, which hindered our understanding of the molecular mechanisms of infection and DNA release. Here, we determined the cryoelectron microscopy (cryo-EM) structures of mature and DNA-ejected VP1 structures at near-atomic and subnanometer resolutions, respectively. The VP1 head is composed of 415 copies of the major capsid protein gp7 and 11 turret-shaped spikes. The VP1 tail consists of an adapter, a nozzle, a slender ring, and a tail needle, and is flanked by three extended fibers I and six trimeric fibers II. Conformational changes of fiber II in DNA-ejected VP1 may cause the release of the tail needle and core proteins, forming an elongated tail channel. Our structures provide insights into the molecular mechanisms of infection and DNA release for podophages with a tail needle.
溶解性荚膜噬菌体（VP1-VP5）在霍乱弧菌 O1 生物型 El Tor 的亚型鉴定中起着至关重要的作用。然而，到目前为止，这些噬菌体还没有任何结构，这阻碍了我们对感染和 DNA 释放的分子机制的了解。在这里，我们用冷冻电子显微镜（cryo-EM）测定了成熟的 VP1 结构和 DNA 释放的 VP1 结构，其分辨率分别接近原子和亚纳米。VP1 头部由 415 个拷贝的主要帽状蛋白 gp7 和 11 个塔形尖峰组成。VP1 尾部由一个适配器、一个喷嘴、一个细长的环和一个尾针组成，两侧是三个延伸纤维 I 和六个三聚体纤维 II。在 DNA 射出的 VP1 中，纤维 II 的构象变化可能会导致尾针和核心蛋白的释放，从而形成一个拉长的尾部通道。我们的结构让我们了解了带有尾针的荚膜病毒感染和 DNA 释放的分子机制。
{"title":"Three-dimensional structures of Vibrio cholerae typing podophage VP1 in two states","authors":"Hao Pang, Fenxia Fan, Jing Zheng, Hao Xiao, Zhixue Tan, Jingdong Song, Biao Kan, Hongrong Liu","doi":"10.1016/j.str.2024.10.005","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.005","url":null,"abstract":"Lytic podophages (VP1–VP5) play crucial roles in subtyping <em>Vibrio cholerae</em> O1 biotype El Tor. However, until now no structures of these phages have been available, which hindered our understanding of the molecular mechanisms of infection and DNA release. Here, we determined the cryoelectron microscopy (cryo-EM) structures of mature and DNA-ejected VP1 structures at near-atomic and subnanometer resolutions, respectively. The VP1 head is composed of 415 copies of the major capsid protein gp7 and 11 turret-shaped spikes. The VP1 tail consists of an adapter, a nozzle, a slender ring, and a tail needle, and is flanked by three extended fibers I and six trimeric fibers II. Conformational changes of fiber II in DNA-ejected VP1 may cause the release of the tail needle and core proteins, forming an elongated tail channel. Our structures provide insights into the molecular mechanisms of infection and DNA release for podophages with a tail needle.","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142519519","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 : 2024-10-26DOI: 10.1016/j.str.2024.10.027
Martyna W. Pastok, Charles W.E. Tomlinson, Shannon Turberville, Abbey M. Butler, Arnaud Baslé, Martin E.M. Noble, Jane A. Endicott, Ehmke Pohl, Natalie J. Tatum
(Structure 32, 1–15; December 5, 2024)
(结构 32，1-15；2024 年 12 月 5 日）
{"title":"Structural requirements for the specific binding of CRABP2 to cyclin D3","authors":"Martyna W. Pastok, Charles W.E. Tomlinson, Shannon Turberville, Abbey M. Butler, Arnaud Baslé, Martin E.M. Noble, Jane A. Endicott, Ehmke Pohl, Natalie J. Tatum","doi":"10.1016/j.str.2024.10.027","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.027","url":null,"abstract":"(Structure <em>32</em>, 1–15; December 5, 2024)","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142490568","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 : 2024-10-25DOI: 10.1016/j.str.2024.10.001
Kevin Michalewicz, Mauricio Barahona, Barbara Bravi
The high binding affinity of antibodies toward their cognate targets is key to eliciting effective immune responses, as well as to the use of antibodies as research and therapeutic tools. Here, we propose ANTIPASTI, a convolutional neural network model that achieves state-of-the-art performance in the prediction of antibody binding affinity using as input a representation of antibody-antigen structures in terms of normal mode correlation maps derived from elastic network models. This representation captures not only structural features but energetic patterns of local and global residue fluctuations. The learnt representations are interpretable: they reveal similarities of binding patterns among antibodies targeting the same antigen type, and can be used to quantify the importance of antibody regions contributing to binding affinity. Our results show the importance of the antigen imprint in the normal mode landscape, and the dominance of cooperative effects and long-range correlations between antibody regions to determine binding affinity.
{"title":"ANTIPASTI: Interpretable prediction of antibody binding affinity exploiting normal modes and deep learning","authors":"Kevin Michalewicz, Mauricio Barahona, Barbara Bravi","doi":"10.1016/j.str.2024.10.001","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.001","url":null,"abstract":"The high binding affinity of antibodies toward their cognate targets is key to eliciting effective immune responses, as well as to the use of antibodies as research and therapeutic tools. Here, we propose ANTIPASTI, a convolutional neural network model that achieves state-of-the-art performance in the prediction of antibody binding affinity using as input a representation of antibody-antigen structures in terms of normal mode correlation maps derived from elastic network models. This representation captures not only structural features but energetic patterns of local and global residue fluctuations. The learnt representations are interpretable: they reveal similarities of binding patterns among antibodies targeting the same antigen type, and can be used to quantify the importance of antibody regions contributing to binding affinity. Our results show the importance of the antigen imprint in the normal mode landscape, and the dominance of cooperative effects and long-range correlations between antibody regions to determine binding affinity.","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142489632","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 : 2024-10-24DOI: 10.1016/j.str.2024.09.025
Molly S.C. Gravett, David P. Klebl, Oliver G. Harlen, Daniel J. Read, Stephen P. Muench, Sarah A. Harris, Michelle Peckham
Myosin 5a (Myo5a) is a dimeric processive motor protein that transports cellular cargos along filamentous actin (F-actin). Its long lever is responsible for its large power-stroke, step size, and load-bearing ability. Little is known about the levers’ structure and physical properties, and how they contribute to walking mechanics. Using cryoelectron microscopy (cryo-EM) and molecular dynamics (MD) simulations, we resolved the structure of monomeric Myo5a, comprising the motor domain and full-length lever, bound to F-actin. The range of its lever conformations revealed its physical properties, how stiffness varies along its length and predicts a large, 35 nm, working stroke. Thus, the newly released trail head in a dimeric Myo5a would only need to perform a small diffusive search for its new binding site on F-actin, and stress would only be generated across the dimer once phosphate is released from the lead head, revealing new insight into the walking behavior of Myo5a.
{"title":"Exploiting cryo-EM structures of actomyosin-5a to reveal the physical properties of its lever","authors":"Molly S.C. Gravett, David P. Klebl, Oliver G. Harlen, Daniel J. Read, Stephen P. Muench, Sarah A. Harris, Michelle Peckham","doi":"10.1016/j.str.2024.09.025","DOIUrl":"https://doi.org/10.1016/j.str.2024.09.025","url":null,"abstract":"Myosin 5a (Myo5a) is a dimeric processive motor protein that transports cellular cargos along filamentous actin (F-actin). Its long lever is responsible for its large power-stroke, step size, and load-bearing ability. Little is known about the levers’ structure and physical properties, and how they contribute to walking mechanics. Using cryoelectron microscopy (cryo-EM) and molecular dynamics (MD) simulations, we resolved the structure of monomeric Myo5a, comprising the motor domain and full-length lever, bound to F-actin. The range of its lever conformations revealed its physical properties, how stiffness varies along its length and predicts a large, 35 nm, working stroke. Thus, the newly released trail head in a dimeric Myo5a would only need to perform a small diffusive search for its new binding site on F-actin, and stress would only be generated across the dimer once phosphate is released from the lead head, revealing new insight into the walking behavior of Myo5a.","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142489057","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 : 2024-10-22DOI: 10.1016/j.str.2024.09.024
William J. Bradshaw, Gemma Harris, Opher Gileadi, Vittorio L. Katis
Spleen tyrosine kinase (SYK) is central to adaptive and innate immune signaling. It features a regulatory region containing tandem SH2 (tSH2) domains separated by a helical “hinge” segment keeping SYK inactive by associating with the kinase domain. SYK activation is triggered when the tSH2 domains bind to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) found on receptor tails. Past mutational studies have indicated that ITAM binding disrupts the hinge-kinase interaction, leading to SYK phosphorylation and activation. However, the mechanism of this process is unclear, as the ITAM interaction occurs far from the hinge region. We have determined crystal structures of three phospho-ITAMs in complex with the tSH2 domains, revealing a highly conserved binding mechanism. These structures, together with mutational studies and biophysical analyses, reveal that phospho-ITAM binding restricts SH2 domain movement and causes allosteric changes in the hinge region. These changes are not compatible with the association of the kinase domain, leading to kinase activation.
{"title":"The mechanism of allosteric activation of SYK kinase derived from multiple phospho-ITAM-bound structures","authors":"William J. Bradshaw, Gemma Harris, Opher Gileadi, Vittorio L. Katis","doi":"10.1016/j.str.2024.09.024","DOIUrl":"https://doi.org/10.1016/j.str.2024.09.024","url":null,"abstract":"Spleen tyrosine kinase (SYK) is central to adaptive and innate immune signaling. It features a regulatory region containing tandem SH2 (tSH2) domains separated by a helical “hinge” segment keeping SYK inactive by associating with the kinase domain. SYK activation is triggered when the tSH2 domains bind to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) found on receptor tails. Past mutational studies have indicated that ITAM binding disrupts the hinge-kinase interaction, leading to SYK phosphorylation and activation. However, the mechanism of this process is unclear, as the ITAM interaction occurs far from the hinge region. We have determined crystal structures of three phospho-ITAMs in complex with the tSH2 domains, revealing a highly conserved binding mechanism. These structures, together with mutational studies and biophysical analyses, reveal that phospho-ITAM binding restricts SH2 domain movement and causes allosteric changes in the hinge region. These changes are not compatible with the association of the kinase domain, leading to kinase activation.","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142486612","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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