Pub Date : 2024-05-31DOI: 10.1016/j.str.2024.05.007
Wen-Wen Kong, Yun Zhu, Heng-Rui Zhao, Kang Du, Rui-Qian Zhou, Bo Li, Feng Yang, Pu Hou, Xia-He Huang, Yuxing Chen, Ying-Chun Wang, Fei Sun, Yong-Liang Jiang, Cong-Zhao Zhou
Carboxysomes are large self-assembled microcompartments that serve as the central machinery of a CO2-concentrating mechanism (CCM). Biogenesis of carboxysome requires the fine organization of thousands of individual proteins; however, the packaging pattern of internal RuBisCOs remains largely unknown. Here we purified the intact β-carboxysomes from Synechococcus elongatus PCC 7942 and identified the protein components by mass spectrometry. Cryo-electron tomography combined with subtomogram averaging revealed the general organization pattern of internal RuBisCOs, in which the adjacent RuBisCOs are mainly arranged in three distinct manners: head-to-head, head-to-side, and side-by-side. The RuBisCOs in the outermost layer are regularly aligned along the shell, the majority of which directly interact with the shell. Moreover, statistical analysis enabled us to propose an ideal packaging model of RuBisCOs in the β-carboxysome. These results provide new insights into the biogenesis of β-carboxysomes and also advance our understanding of the efficient carbon fixation functionality of carboxysomes.
{"title":"Cryo-electron tomography reveals the packaging pattern of RuBisCOs in Synechococcus β-carboxysome","authors":"Wen-Wen Kong, Yun Zhu, Heng-Rui Zhao, Kang Du, Rui-Qian Zhou, Bo Li, Feng Yang, Pu Hou, Xia-He Huang, Yuxing Chen, Ying-Chun Wang, Fei Sun, Yong-Liang Jiang, Cong-Zhao Zhou","doi":"10.1016/j.str.2024.05.007","DOIUrl":"https://doi.org/10.1016/j.str.2024.05.007","url":null,"abstract":"<p>Carboxysomes are large self-assembled microcompartments that serve as the central machinery of a CO<sub>2</sub>-concentrating mechanism (CCM). Biogenesis of carboxysome requires the fine organization of thousands of individual proteins; however, the packaging pattern of internal RuBisCOs remains largely unknown. Here we purified the intact β-carboxysomes from <em>Synechococcus elongatus</em> PCC 7942 and identified the protein components by mass spectrometry. Cryo-electron tomography combined with subtomogram averaging revealed the general organization pattern of internal RuBisCOs, in which the adjacent RuBisCOs are mainly arranged in three distinct manners: head-to-head, head-to-side, and side-by-side. The RuBisCOs in the outermost layer are regularly aligned along the shell, the majority of which directly interact with the shell. Moreover, statistical analysis enabled us to propose an ideal packaging model of RuBisCOs in the β-carboxysome. These results provide new insights into the biogenesis of β-carboxysomes and also advance our understanding of the efficient carbon fixation functionality of carboxysomes.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141182651","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 : 2024-05-31DOI: 10.1016/j.str.2024.05.006
Ranhao Zhang, Yuan Shen, Xueming Li
Contrast transfer function (CTF) estimation is a necessary step in the cryo-electron tomography (cryoET) workflow and essential for high-resolution in situ structural determination. However, the low signal-to-noise ratio and continuous defocus variation in micrographs of cryoET tilt series make accurate CTF estimation challenging. Here, we report a tilt-series-based joint CTF estimation method implemented in the new software CTFMeasure. The joint estimation method combines all Thon-ring signals in a tilt series to improve the estimation accuracy. By using an objective function involving the CTF parameters and geometric parameters of a cryoET tilt series, CTFMeasure can estimate the CTF parameters of each micrograph and the absolute tilt angle offset of the lamellar sample relative to the sample stage plane, which is usually the glancing angle used during focused ion beam (FIB) milling. Tests on both synthetic and experimental data, as well as subtomogram averaging, demonstrated the accurate CTF estimation of cryoET tilt series by CTFMeasure.
{"title":"Tilt-series-based joint CTF estimation for cryo-electron tomography","authors":"Ranhao Zhang, Yuan Shen, Xueming Li","doi":"10.1016/j.str.2024.05.006","DOIUrl":"https://doi.org/10.1016/j.str.2024.05.006","url":null,"abstract":"<p>Contrast transfer function (CTF) estimation is a necessary step in the cryo-electron tomography (cryoET) workflow and essential for high-resolution <em>in situ</em> structural determination. However, the low signal-to-noise ratio and continuous defocus variation in micrographs of cryoET tilt series make accurate CTF estimation challenging. Here, we report a tilt-series-based joint CTF estimation method implemented in the new software CTFMeasure. The joint estimation method combines all Thon-ring signals in a tilt series to improve the estimation accuracy. By using an objective function involving the CTF parameters and geometric parameters of a cryoET tilt series, CTFMeasure can estimate the CTF parameters of each micrograph and the absolute tilt angle offset of the lamellar sample relative to the sample stage plane, which is usually the glancing angle used during focused ion beam (FIB) milling. Tests on both synthetic and experimental data, as well as subtomogram averaging, demonstrated the accurate CTF estimation of cryoET tilt series by CTFMeasure.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141182536","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}
Multidrug and toxin extrusion (MATE) family transporters excrete toxic compounds coupled to Na+/H+ influx. Although structures of MATE transporters are available, the mechanism by which substrate export is coupled to ion influx remains unknown. To address this issue, we conducted a structural analysis of Pyrococcus furiosus MATE (PfMATE) using solution nuclear magnetic resonance (NMR). The NMR analysis, along with thorough substitutions of all non-exposed acidic residues, confirmed that PfMATE is under an equilibrium between inward-facing (IF) and outward-facing (OF) conformations, dictated by the Glu163 protonation. Importantly, we found that only the IF conformation exhibits a mid-μM affinity for substrate recognition. In contrast, the OF conformation exhibited only weak mM substrate affinity, suitable for releasing substrate to the extracellular side. These results indicate that PfMATE is an affinity-directed H+ antiporter where substrates selectively bind to the protonated IF conformation in the equilibrium, and subsequent proton release mechanistically ensures H+-coupled substrate excretion by the transporter.
多药和毒素挤出(MATE)家族转运体排出有毒化合物与 Na+/H+ 流入有关。虽然已有 MATE 转运体的结构,但底物输出与离子流入耦合的机制仍然未知。为了解决这个问题,我们利用溶液核磁共振(NMR)对暴怒双球菌 MATE(PfMATE)进行了结构分析。核磁共振分析以及对所有未暴露的酸性残基进行的彻底置换证实,PfMATE 在由 Glu163 质子化决定的内向型(IF)和外向型(OF)构象之间处于平衡状态。重要的是,我们发现只有 IF 构象对底物的识别具有中等微米的亲和力。相比之下,OF 构象只表现出微弱的 mM 底物亲和力,适合将底物释放到胞外侧。这些结果表明,PfMATE 是一种亲和力导向的 H+ 反转运体,底物在平衡状态下选择性地与质子化的 IF 构象结合,随后的质子释放从机理上确保转运体排出 H+ 耦合的底物。
{"title":"Affinity-directed substrate/H+-antiport by a MATE transporter","authors":"Koh Takeuchi, Takumi Ueda, Misaki Imai, Miwa Fujisaki, Mie Shimura, Yuji Tokunaga, Yutaka Kofuku, Ichio Shimada","doi":"10.1016/j.str.2024.05.004","DOIUrl":"https://doi.org/10.1016/j.str.2024.05.004","url":null,"abstract":"<p>Multidrug and toxin extrusion (MATE) family transporters excrete toxic compounds coupled to Na<sup>+</sup>/H<sup>+</sup> influx. Although structures of MATE transporters are available, the mechanism by which substrate export is coupled to ion influx remains unknown. To address this issue, we conducted a structural analysis of <em>Pyrococcus furiosus</em> MATE (PfMATE) using solution nuclear magnetic resonance (NMR). The NMR analysis, along with thorough substitutions of all non-exposed acidic residues, confirmed that PfMATE is under an equilibrium between inward-facing (IF) and outward-facing (OF) conformations, dictated by the Glu163 protonation. Importantly, we found that only the IF conformation exhibits a mid-μM affinity for substrate recognition. In contrast, the OF conformation exhibited only weak mM substrate affinity, suitable for releasing substrate to the extracellular side. These results indicate that PfMATE is an affinity-directed H<sup>+</sup> antiporter where substrates selectively bind to the protonated IF conformation in the equilibrium, and subsequent proton release mechanistically ensures H<sup>+</sup>-coupled substrate excretion by the transporter.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141177888","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 : 2024-05-29DOI: 10.1016/j.str.2024.05.003
Chenghai Wang, Min Li, Yufan Wang, Qiang Ding, Shilong Fan, Jun Lan
Langya virus (LayV) was recently detected in patients with acute pneumonic diseases in China. Genome alignment indicated that LayV is a type of zoonotic henipavirus (HNV) that might also infect domestic animals. Previous studies revealed that HNVs mainly use ephrin-B1, ephrin-B2, or ephrin-B3 as cell receptors and the attachment glycoprotein (G) is the host cell receptor-binding protein. However, the LayV receptor remains unknown. Here, we present the 2.77 Å crystal structure of the LayV G C-terminal domain (CTD). We show that the LayV G protein CTD possesses a similar architecture as the Mojiang virus (MojV) G protein but is markedly different from the Nipah virus (NiV), Hendra virus (HeV), and Cedar virus (CedV) G proteins. Surface plasmon resonance (SPR) experiments indicate that LayV G does not bind ephrin-B proteins. Steric hindrance may prevent interactions between LayV G and ephrin-B. Our data might facilitate drug development targeting LayV.
中国最近在急性肺炎患者中发现了琅琊病毒(LayV)。基因组比对表明,琅琊病毒是一种人畜共患的鸡病毒(HNV),也可能感染家畜。以往的研究表明,HNV主要以ephrin-B1、ephrin-B2或ephrin-B3作为细胞受体,附着糖蛋白(G)是宿主细胞受体结合蛋白。然而,LayV受体仍然未知。在这里,我们展示了 LayV G C 端结构域(CTD)的 2.77 Å 晶体结构。我们发现,LayV G 蛋白 CTD 具有与墨江病毒 (MojV) G 蛋白相似的结构,但与尼帕病毒 (NiV)、亨德拉病毒 (HeV) 和雪松病毒 (CedV) G 蛋白有明显不同。表面等离子体共振(SPR)实验表明,LayV G 不会与 ephrin-B 蛋白结合。立体阻碍可能阻止了 LayV G 与 ephrin-B 之间的相互作用。我们的数据可能有助于开发针对 LayV 的药物。
{"title":"Structural insights into the Langya virus attachment glycoprotein","authors":"Chenghai Wang, Min Li, Yufan Wang, Qiang Ding, Shilong Fan, Jun Lan","doi":"10.1016/j.str.2024.05.003","DOIUrl":"https://doi.org/10.1016/j.str.2024.05.003","url":null,"abstract":"<p>Langya virus (LayV) was recently detected in patients with acute pneumonic diseases in China. Genome alignment indicated that LayV is a type of zoonotic henipavirus (HNV) that might also infect domestic animals. Previous studies revealed that HNVs mainly use ephrin-B1, ephrin-B2, or ephrin-B3 as cell receptors and the attachment glycoprotein (G) is the host cell receptor-binding protein. However, the LayV receptor remains unknown. Here, we present the 2.77 Å crystal structure of the LayV G C-terminal domain (CTD). We show that the LayV G protein CTD possesses a similar architecture as the Mojiang virus (MojV) G protein but is markedly different from the Nipah virus (NiV), Hendra virus (HeV), and Cedar virus (CedV) G proteins. Surface plasmon resonance (SPR) experiments indicate that LayV G does not bind ephrin-B proteins. Steric hindrance may prevent interactions between LayV G and ephrin-B. Our data might facilitate drug development targeting LayV.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141177852","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 : 2024-05-29DOI: 10.1016/j.str.2024.05.005
Gamma Chi, Dawid Jaślan, Veronika Kudrina, Julia Böck, Huanyu Li, Ashley C.W. Pike, Susanne Rautenberg, Einar Krogsaeter, Tina Bohstedt, Dong Wang, Gavin McKinley, Alejandra Fernandez-Cid, Shubhashish M.M. Mukhopadhyay, Nicola A. Burgess-Brown, Marco Keller, Franz Bracher, Christian Grimm, Katharina L. Dürr
Two pore channels are lysosomal cation channels with crucial roles in tumor angiogenesis and viral release from endosomes. Inhibition of the two-pore channel 2 (TPC2) has emerged as potential therapeutic strategy for the treatment of cancers and viral infections, including Ebola and COVID-19. Here, we demonstrate that antagonist SG-094, a synthetic analog of the Chinese alkaloid medicine tetrandrine with increased potency and reduced toxicity, induces asymmetrical structural changes leading to a single binding pocket at only one intersubunit interface within the asymmetrical dimer. Supported by functional characterization of mutants by Ca2+ imaging and patch clamp experiments, we identify key residues in S1 and S4 involved in compound binding to the voltage sensing domain II. SG-094 arrests IIS4 in a downward shifted state which prevents pore opening via the IIS4/S5 linker, hence resembling gating modifiers of canonical VGICs. These findings may guide the rational development of new therapeutics antagonizing TPC2 activity.
{"title":"Structural basis for inhibition of the lysosomal two-pore channel TPC2 by a small molecule antagonist","authors":"Gamma Chi, Dawid Jaślan, Veronika Kudrina, Julia Böck, Huanyu Li, Ashley C.W. Pike, Susanne Rautenberg, Einar Krogsaeter, Tina Bohstedt, Dong Wang, Gavin McKinley, Alejandra Fernandez-Cid, Shubhashish M.M. Mukhopadhyay, Nicola A. Burgess-Brown, Marco Keller, Franz Bracher, Christian Grimm, Katharina L. Dürr","doi":"10.1016/j.str.2024.05.005","DOIUrl":"https://doi.org/10.1016/j.str.2024.05.005","url":null,"abstract":"<p>Two pore channels are lysosomal cation channels with crucial roles in tumor angiogenesis and viral release from endosomes. Inhibition of the two-pore channel 2 (TPC2) has emerged as potential therapeutic strategy for the treatment of cancers and viral infections, including Ebola and COVID-19. Here, we demonstrate that antagonist SG-094, a synthetic analog of the Chinese alkaloid medicine tetrandrine with increased potency and reduced toxicity, induces asymmetrical structural changes leading to a single binding pocket at only one intersubunit interface within the asymmetrical dimer. Supported by functional characterization of mutants by Ca<sup>2+</sup> imaging and patch clamp experiments, we identify key residues in S1 and S4 involved in compound binding to the voltage sensing domain II. SG-094 arrests IIS4 in a downward shifted state which prevents pore opening via the IIS4/S5 linker, hence resembling gating modifiers of canonical VGICs. These findings may guide the rational development of new therapeutics antagonizing TPC2 activity.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141177864","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 : 2024-05-22DOI: 10.1016/j.str.2024.04.020
Kateřina Krejčová, Petra Krafcikova, Martin Klima, Dominika Chalupska, Karel Chalupsky, Eva Zilecka, Evzen Boura
Flaviviruses are single-stranded positive-sense RNA (+RNA) viruses that are responsible for several (re)emerging diseases such as yellow, dengue, or West Nile fevers. The Zika epidemic highlighted their dangerousness when a relatively benign virus known since the 1950s turned into a deadly pathogen. The central protein for their replication is NS5 (non-structural protein 5), which is composed of the N-terminal methyltransferase (MTase) domain and the C-terminal RNA-dependent RNA-polymerase (RdRp) domain. It is responsible for both RNA replication and installation of the 5′ RNA cap. We structurally and biochemically analyzed the Ntaya virus MTase and RdRp domains and we compared their properties to other flaviviral NS5s. The enzymatic centers are well conserved across Flaviviridae, suggesting that the development of drugs targeting all flaviviruses is feasible. However, the enzymatic activities of the isolated proteins were significantly different for the MTase domains.
{"title":"Structural and functional insights in flavivirus NS5 proteins gained by the structure of Ntaya virus polymerase and methyltransferase","authors":"Kateřina Krejčová, Petra Krafcikova, Martin Klima, Dominika Chalupska, Karel Chalupsky, Eva Zilecka, Evzen Boura","doi":"10.1016/j.str.2024.04.020","DOIUrl":"https://doi.org/10.1016/j.str.2024.04.020","url":null,"abstract":"<p>Flaviviruses are single-stranded positive-sense RNA (+RNA) viruses that are responsible for several (re)emerging diseases such as yellow, dengue, or West Nile fevers. The Zika epidemic highlighted their dangerousness when a relatively benign virus known since the 1950s turned into a deadly pathogen. The central protein for their replication is NS5 (non-structural protein 5), which is composed of the N-terminal methyltransferase (MTase) domain and the C-terminal RNA-dependent RNA-polymerase (RdRp) domain. It is responsible for both RNA replication and installation of the 5′ RNA cap. We structurally and biochemically analyzed the Ntaya virus MTase and RdRp domains and we compared their properties to other flaviviral NS5s. The enzymatic centers are well conserved across <em>Flaviviridae</em>, suggesting that the development of drugs targeting all flaviviruses is feasible. However, the enzymatic activities of the isolated proteins were significantly different for the MTase domains.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141085832","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 : 2024-05-15DOI: 10.1016/j.str.2024.04.017
Jerry Gao, Maxwell Tong, Chinkyu Lee, Jacek Gaertig, Thibault Legal, Khanh Huy Bui
Cryoelectron microscopy (cryo-EM) has revolutionized the structural determination of macromolecular complexes. With the paradigm shift to structure determination of highly complex endogenous macromolecular complexes ex vivo and in situ structural biology, there are an increasing number of structures of native complexes. These complexes often contain unidentified proteins, related to different cellular states or processes. Identifying proteins at resolutions lower than 4 Å remains challenging because side chains cannot be visualized reliably. Here, we present DomainFit, a program for semi-automated domain-level protein identification from cryo-EM maps, particularly at resolutions lower than 4 Å. By fitting domains from AlphaFold2-predicted models into cryo-EM maps, the program performs statistical analyses and attempts to identify the domains and protein candidates forming the density. Using DomainFit, we identified two microtubule inner proteins, one of which contains a CCDC81 domain and is exclusively localized in the proximal region of the doublet microtubule in Tetrahymena thermophila.
冷冻电镜(cryo-EM)为大分子复合物的结构测定带来了革命性的变化。随着体内外高难度内源大分子复合物结构测定和原位结构生物学模式的转变,原生复合物的结构越来越多。这些复合物通常含有与不同细胞状态或过程相关的未识别蛋白质。由于侧链不能被可靠地可视化,因此以低于 4 Å 的分辨率识别蛋白质仍然具有挑战性。通过将 AlphaFold2 预测模型中的结构域拟合到低温电子显微镜图中,该程序会执行统计分析,并尝试识别形成密度的结构域和候选蛋白质。利用 DomainFit,我们确定了两种微管内部蛋白,其中一种含有 CCDC81 结构域,专门定位于嗜热四膜虫双微管的近端区域。
{"title":"DomainFit: Identification of protein domains in cryo-EM maps at intermediate resolution using AlphaFold2-predicted models","authors":"Jerry Gao, Maxwell Tong, Chinkyu Lee, Jacek Gaertig, Thibault Legal, Khanh Huy Bui","doi":"10.1016/j.str.2024.04.017","DOIUrl":"https://doi.org/10.1016/j.str.2024.04.017","url":null,"abstract":"<p>Cryoelectron microscopy (cryo-EM) has revolutionized the structural determination of macromolecular complexes. With the paradigm shift to structure determination of highly complex endogenous macromolecular complexes <em>ex vivo</em> and <em>in situ</em> structural biology, there are an increasing number of structures of native complexes. These complexes often contain unidentified proteins, related to different cellular states or processes. Identifying proteins at resolutions lower than 4 Å remains challenging because side chains cannot be visualized reliably. Here, we present DomainFit, a program for semi-automated domain-level protein identification from cryo-EM maps, particularly at resolutions lower than 4 Å. By fitting domains from AlphaFold2-predicted models into cryo-EM maps, the program performs statistical analyses and attempts to identify the domains and protein candidates forming the density. Using DomainFit, we identified two microtubule inner proteins, one of which contains a CCDC81 domain and is exclusively localized in the proximal region of the doublet microtubule in <em>Tetrahymena thermophila</em>.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140942960","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 : 2024-05-14DOI: 10.1016/j.str.2024.04.016
Alexej Dick, Vasilii Mikirtumov, Jonas Fuchs, Ferdinand Krupp, Daniel Olal, Elias Bendl, Thiemo Sprink, Christoph Diebolder, Mikhail Kudryashev, Georg Kochs, Yvette Roske, Oliver Daumke
Orthomyxoviruses, such as influenza and thogotoviruses, are important human and animal pathogens. Their segmented viral RNA genomes are wrapped by viral nucleoproteins (NPs) into helical ribonucleoprotein complexes (RNPs). NP structures of several influenza viruses have been reported. However, there are still contradictory models of how orthomyxovirus RNPs are assembled. Here, we characterize the crystal structure of Thogoto virus (THOV) NP and found striking similarities to structures of influenza viral NPs, including a two-lobed domain architecture, a positively charged RNA-binding cleft, and a tail loop important for trimerization and viral transcription. A low-resolution cryo-electron tomography reconstruction of THOV RNPs elucidates a left-handed double helical assembly. By providing a model for RNP assembly of THOV, our study suggests conserved NP assembly and RNA encapsidation modes for thogoto- and influenza viruses.
{"title":"Structural characterization of Thogoto Virus nucleoprotein provides insights into viral RNA encapsidation and RNP assembly","authors":"Alexej Dick, Vasilii Mikirtumov, Jonas Fuchs, Ferdinand Krupp, Daniel Olal, Elias Bendl, Thiemo Sprink, Christoph Diebolder, Mikhail Kudryashev, Georg Kochs, Yvette Roske, Oliver Daumke","doi":"10.1016/j.str.2024.04.016","DOIUrl":"https://doi.org/10.1016/j.str.2024.04.016","url":null,"abstract":"<p>Orthomyxoviruses, such as influenza and thogotoviruses, are important human and animal pathogens. Their segmented viral RNA genomes are wrapped by viral nucleoproteins (NPs) into helical ribonucleoprotein complexes (RNPs). NP structures of several influenza viruses have been reported. However, there are still contradictory models of how orthomyxovirus RNPs are assembled. Here, we characterize the crystal structure of Thogoto virus (THOV) NP and found striking similarities to structures of influenza viral NPs, including a two-lobed domain architecture, a positively charged RNA-binding cleft, and a tail loop important for trimerization and viral transcription. A low-resolution cryo-electron tomography reconstruction of THOV RNPs elucidates a left-handed double helical assembly. By providing a model for RNP assembly of THOV, our study suggests conserved NP assembly and RNA encapsidation modes for thogoto- and influenza viruses.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140920016","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 : 2024-05-10DOI: 10.1016/j.str.2024.04.014
Jose M. Martínez Felices, Yan Borges Barreto, Chancievan Thangaratnarajah, Jacob J. Whittaker, Adriano M. Alencar, Albert Guskov, Dirk J. Slotboom
BtuM is a bacterial cobalamin transporter that binds the transported substrate in the base-off state, with a cysteine residue providing the α-axial coordination of the central cobalt ion via a sulfur-cobalt bond. Binding leads to decyanation of cobalamin variants with a cyano group as the β-axial ligand. Here, we report the crystal structures of untagged BtuM bound to two variants of cobalamin, hydroxycobalamin and cyanocobalamin, and unveil the native residue responsible for the β-axial coordination, His28. This coordination had previously been obscured by non-native histidines of His-tagged BtuM. A model in which BtuM initially binds cobinamide reversibly with low affinity (KD = 4.0 μM), followed by the formation of a covalent bond (rate constant of 0.163 s−1), fits the kinetics data of substrate binding and decyanation of the cobalamin precursor cobinamide by BtuM. The covalent binding mode suggests a mechanism not used by any other transport protein.
{"title":"Cobalamin decyanation by the membrane transporter BtuM","authors":"Jose M. Martínez Felices, Yan Borges Barreto, Chancievan Thangaratnarajah, Jacob J. Whittaker, Adriano M. Alencar, Albert Guskov, Dirk J. Slotboom","doi":"10.1016/j.str.2024.04.014","DOIUrl":"https://doi.org/10.1016/j.str.2024.04.014","url":null,"abstract":"<p>BtuM is a bacterial cobalamin transporter that binds the transported substrate in the base-off state, with a cysteine residue providing the α-axial coordination of the central cobalt ion via a sulfur-cobalt bond. Binding leads to decyanation of cobalamin variants with a cyano group as the β-axial ligand. Here, we report the crystal structures of untagged BtuM bound to two variants of cobalamin, hydroxycobalamin and cyanocobalamin, and unveil the native residue responsible for the β-axial coordination, His28. This coordination had previously been obscured by non-native histidines of His-tagged BtuM. A model in which BtuM initially binds cobinamide reversibly with low affinity (K<sub>D</sub> = 4.0 μM), followed by the formation of a covalent bond (rate constant of 0.163 s<sup>−1</sup>), fits the kinetics data of substrate binding and decyanation of the cobalamin precursor cobinamide by BtuM. The covalent binding mode suggests a mechanism not used by any other transport protein.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140903344","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 : 2024-05-10DOI: 10.1016/j.str.2024.04.015
Rhoda Tano-Menka, Nishant K. Singh, Itai Muzhingi, Xiaolong Li, Michael V. Mandanas, Clarety Kaseke, Charles R. Crain, Angela Zhang, Funsho J. Ogunshola, Liza Vecchiarello, Alicja Piechocka-Trocha, Arman Bashirova, Michael E. Birnbaum, Mary Carrington, Bruce D. Walker, Gaurav D. Gaiha
Immunogenetic studies have shown that specific HLA-B residues (67, 70, 97, and 156) mediate the impact of HLA class I on HIV infection, but the molecular basis is not well understood. Here we evaluate the function of these residues within the protective HLA-B∗5701 allele. While mutation of Met67, Ser70, and Leu156 disrupt CD8+ T cell recognition, substitution of Val97 had no significant impact. Thermal denaturation of HLA-B∗5701-peptide complexes revealed that Met67 and Leu156 maintain HLA-peptide stability, while Ser70 and Leu156 facilitate T cell receptor (TCR) interactions. Analyses of existing structures and structural models suggested that Val97 mediates HLA-peptide binding to inhibitory KIR3DL1 molecules, which was confirmed by experimental assays. These data thereby demonstrate that the genetic basis by which host immunity impacts HIV outcomes occurs by modulating HLA-B-peptide stability and conformation for interaction with TCR and killer immunoglobulin receptor (KIR) molecules. Moreover, they indicate a key role for epitope specificity and HLA-KIR interactions to HIV control.
免疫遗传学研究表明,特定的 HLA-B 残基(67、70、97 和 156)介导 HLA I 类对 HIV 感染的影响,但其分子基础尚不十分清楚。在这里,我们评估了这些残基在保护性 HLA-B∗5701 等位基因中的功能。Met67、Ser70和Leu156的突变会破坏CD8+ T细胞的识别,而Val97的替代则没有明显影响。HLA-B∗5701-肽复合物的热变性显示,Met67和Leu156维持了HLA-肽的稳定性,而Ser70和Leu156促进了T细胞受体(TCR)的相互作用。对现有结构和结构模型的分析表明,Val97 介导了 HLA 肽与具有抑制作用的 KIR3DL1 分子的结合,这一点在实验测定中得到了证实。这些数据由此证明,宿主免疫通过调节 HLA-B 肽的稳定性和构象与 TCR 和杀伤性免疫球蛋白受体 (KIR) 分子相互作用,从而影响 HIV 的结局。此外,它们还表明了表位特异性和 HLA-KIR 相互作用对艾滋病毒控制的关键作用。
{"title":"Polymorphic residues in HLA-B that mediate HIV control distinctly modulate peptide interactions with both TCR and KIR molecules","authors":"Rhoda Tano-Menka, Nishant K. Singh, Itai Muzhingi, Xiaolong Li, Michael V. Mandanas, Clarety Kaseke, Charles R. Crain, Angela Zhang, Funsho J. Ogunshola, Liza Vecchiarello, Alicja Piechocka-Trocha, Arman Bashirova, Michael E. Birnbaum, Mary Carrington, Bruce D. Walker, Gaurav D. Gaiha","doi":"10.1016/j.str.2024.04.015","DOIUrl":"https://doi.org/10.1016/j.str.2024.04.015","url":null,"abstract":"<p>Immunogenetic studies have shown that specific HLA-B residues (67, 70, 97, and 156) mediate the impact of HLA class I on HIV infection, but the molecular basis is not well understood. Here we evaluate the function of these residues within the protective <em>HLA-B<sup>∗</sup>5701</em> allele. While mutation of Met67, Ser70, and Leu156 disrupt CD8<sup>+</sup> T cell recognition, substitution of Val97 had no significant impact. Thermal denaturation of HLA-B<sup>∗</sup>5701-peptide complexes revealed that Met67 and Leu156 maintain HLA-peptide stability, while Ser70 and Leu156 facilitate T cell receptor (TCR) interactions. Analyses of existing structures and structural models suggested that Val97 mediates HLA-peptide binding to inhibitory KIR3DL1 molecules, which was confirmed by experimental assays. These data thereby demonstrate that the genetic basis by which host immunity impacts HIV outcomes occurs by modulating HLA-B-peptide stability and conformation for interaction with TCR and killer immunoglobulin receptor (KIR) molecules. Moreover, they indicate a key role for epitope specificity and HLA-KIR interactions to HIV control.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":5.7,"publicationDate":"2024-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140903406","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}