Pub Date : 2024-12-05DOI: 10.1016/j.str.2024.11.002
Mineyuki Mizuguchi
In this issue of Structure, Nguyen et al.1 reveal that amyloid fibrils of the transthyretin (TTR) V30M variant from the heart and nerves of the same patient exhibit structural homogeneity. This finding is crucial for advancing our understanding of V30M-TTR amyloid deposition, which leads to fatal ATTRv amyloidosis.
{"title":"Does the structure of transthyretin amyloid fibrils vary depending on the organ of accumulation?","authors":"Mineyuki Mizuguchi","doi":"10.1016/j.str.2024.11.002","DOIUrl":"https://doi.org/10.1016/j.str.2024.11.002","url":null,"abstract":"In this issue of <em>Structure</em>, Nguyen et al.<span><span><sup>1</sup></span></span> reveal that amyloid fibrils of the transthyretin (TTR) V30M variant from the heart and nerves of the same patient exhibit structural homogeneity. This finding is crucial for advancing our understanding of V30M-TTR amyloid deposition, which leads to fatal ATTRv amyloidosis.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"78 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142776810","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 : 2024-12-05DOI: 10.1016/j.str.2024.11.001
David Neuhaus, Katherine Stott
In this issue of Structure, Viennet et al.1 report a study characterizing the DNA binding by a three-zinc-finger fragment from the transcription factor BCL11A, with the unusual feature that an interfinger interaction in the free protein is disrupted during binding, which provides a positive entropic contribution that enhances the affinity.
{"title":"A finger that gets in the way: When binding isn’t just about the bound state","authors":"David Neuhaus, Katherine Stott","doi":"10.1016/j.str.2024.11.001","DOIUrl":"https://doi.org/10.1016/j.str.2024.11.001","url":null,"abstract":"In this issue of <em>Structure</em>, Viennet et al.<span><span><sup>1</sup></span></span> report a study characterizing the DNA binding by a three-zinc-finger fragment from the transcription factor BCL11A, with the unusual feature that an interfinger interaction in the free protein is disrupted during binding, which provides a positive entropic contribution that enhances the affinity.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"5 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142776458","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 : 2024-12-04DOI: 10.1016/j.str.2024.11.005
Peter Rory Hall, Thibault Jouen-Tachoire, Marcus Schewe, Peter Proks, Thomas Baukrowitz, Elisabeth P. Carpenter, Simon Newstead, Karin E.J. Rödström, Stephen J. Tucker
TASK-1 and TASK-3 are pH-sensitive two-pore domain (K2P/KCNK) K+ channels. Their functional roles make them promising targets for treatment of multiple disorders including sleep apnea, pain, and atrial fibrillation. Mutations in these channels are also associated with neurodevelopmental and hypertensive disorders. A previous crystal structure of TASK-1 revealed a lower “X-gate” as a hotspot for missense gain-of-function (GoF) mutations associated with DDSA (developmental delay with sleep apnea). However, the mechanisms of gating in TASK channels are still not fully understood. Here, we resolve structures for both human TASK-1 and TASK-3 by cryoelectron microscopy (cryo-EM), as well as a recurrent TASK-3 variant (G236R) associated with KCNK9 imprinting syndrome (KIS) (formerly known as Birk-Barel syndrome). Combined with functional studies of the X-gating mechanism, we provide evidence for how a highly conserved gating mechanism becomes defective in disease, and also provide further insight into the pathway of conformational changes that underlie the pH-dependent inhibition of TASK channel activity.
{"title":"Structures of TASK-1 and TASK-3 K2P channels provide insight into their gating and dysfunction in disease","authors":"Peter Rory Hall, Thibault Jouen-Tachoire, Marcus Schewe, Peter Proks, Thomas Baukrowitz, Elisabeth P. Carpenter, Simon Newstead, Karin E.J. Rödström, Stephen J. Tucker","doi":"10.1016/j.str.2024.11.005","DOIUrl":"https://doi.org/10.1016/j.str.2024.11.005","url":null,"abstract":"TASK-1 and TASK-3 are pH-sensitive two-pore domain (K2P/<em>KCNK</em>) K<sup>+</sup> channels. Their functional roles make them promising targets for treatment of multiple disorders including sleep apnea, pain, and atrial fibrillation. Mutations in these channels are also associated with neurodevelopmental and hypertensive disorders. A previous crystal structure of TASK-1 revealed a lower “X-gate” as a hotspot for missense gain-of-function (GoF) mutations associated with DDSA (developmental delay with sleep apnea). However, the mechanisms of gating in TASK channels are still not fully understood. Here, we resolve structures for both human TASK-1 and TASK-3 by cryoelectron microscopy (cryo-EM), as well as a recurrent TASK-3 variant (G236R) associated with <em>KCNK9</em> imprinting syndrome (KIS) (formerly known as Birk-Barel syndrome). Combined with functional studies of the X-gating mechanism, we provide evidence for how a highly conserved gating mechanism becomes defective in disease, and also provide further insight into the pathway of conformational changes that underlie the pH-dependent inhibition of TASK channel activity.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"37 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142763464","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 : 2024-11-29DOI: 10.1016/j.str.2024.11.004
Elnaz Khalili Samani, S.M. Naimul Hasan, Matthew Waas, Alexander F.A. Keszei, Xiaoxiao Xu, Mahtab Heydari, Mary Elizabeth Hill, JoAnne McLaurin, Thomas Kislinger, Mohammad T. Mazhab-Jafari
Studying native protein structures at near-atomic resolution in a crowded environment presents challenges. Consequently, understanding the structural intricacies of proteins within pathologically affected tissues often relies on mass spectrometry and proteomic analysis. Here, we utilized cryoelectron microscopy (cryo-EM) and the Build and Retrieve (BaR) method to investigate protein complexes’ structural characteristics such as post-translational modification, active site occupancy, and arrested conformational state in Alzheimer’s disease (AD) using brain lysate from a rat model (TgF344-AD). Our findings reveal novel insights into the architecture of these complexes, corroborated through mass spectrometry analysis. Interestingly, it has been shown that the dysfunction of these protein complexes extends beyond AD, implicating them in cancer, as well as other neurodegenerative disorders such as Parkinson’s disease, Huntington’s disease, and schizophrenia. By elucidating these structural details, our work not only enhances our understanding of disease pathology but also suggests new avenues for future approaches in therapeutic intervention.
{"title":"Unveiling the structural proteome of an Alzheimer’s disease rat brain model","authors":"Elnaz Khalili Samani, S.M. Naimul Hasan, Matthew Waas, Alexander F.A. Keszei, Xiaoxiao Xu, Mahtab Heydari, Mary Elizabeth Hill, JoAnne McLaurin, Thomas Kislinger, Mohammad T. Mazhab-Jafari","doi":"10.1016/j.str.2024.11.004","DOIUrl":"https://doi.org/10.1016/j.str.2024.11.004","url":null,"abstract":"Studying native protein structures at near-atomic resolution in a crowded environment presents challenges. Consequently, understanding the structural intricacies of proteins within pathologically affected tissues often relies on mass spectrometry and proteomic analysis. Here, we utilized cryoelectron microscopy (cryo-EM) and the Build and Retrieve (BaR) method to investigate protein complexes’ structural characteristics such as post-translational modification, active site occupancy, and arrested conformational state in Alzheimer’s disease (AD) using brain lysate from a rat model (TgF344-AD). Our findings reveal novel insights into the architecture of these complexes, corroborated through mass spectrometry analysis. Interestingly, it has been shown that the dysfunction of these protein complexes extends beyond AD, implicating them in cancer, as well as other neurodegenerative disorders such as Parkinson’s disease, Huntington’s disease, and schizophrenia. By elucidating these structural details, our work not only enhances our understanding of disease pathology but also suggests new avenues for future approaches in therapeutic intervention.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"9 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142742585","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 : 2024-11-25DOI: 10.1016/j.str.2024.10.032
Jingxia Chen, Xueyin Zhou, Yuqi Yang, Long Li
Protein translocation systems are essential for distributing proteins across various lipid membranes in cells. Cellular membranes, such as the endoplasmic reticulum (ER) membrane and mitochondrial inner membrane, require highly regulated protein translocation machineries that specifically allow the passage of protein polypeptides while blocking smaller molecules like ions and water. Key translocation systems include the Sec translocation channel, the protein insertases of the Oxa1 superfamily, and the translocases of the mitochondrial inner membrane (TIM). These machineries utilize different mechanisms to create pathways for proteins to move across membranes while preventing ion leakage during the dynamic translocation processes. In this review, we highlight recent advances in our understanding of these α-helical translocation machineries and examine their structures, mechanisms, and regulation. We also discuss the therapeutic potential of these translocation pathways and summarize the progress in drug development targeting these systems for treating diseases.
{"title":"Protein translocation through α-helical channels and insertases","authors":"Jingxia Chen, Xueyin Zhou, Yuqi Yang, Long Li","doi":"10.1016/j.str.2024.10.032","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.032","url":null,"abstract":"Protein translocation systems are essential for distributing proteins across various lipid membranes in cells. Cellular membranes, such as the endoplasmic reticulum (ER) membrane and mitochondrial inner membrane, require highly regulated protein translocation machineries that specifically allow the passage of protein polypeptides while blocking smaller molecules like ions and water. Key translocation systems include the Sec translocation channel, the protein insertases of the Oxa1 superfamily, and the translocases of the mitochondrial inner membrane (TIM). These machineries utilize different mechanisms to create pathways for proteins to move across membranes while preventing ion leakage during the dynamic translocation processes. In this review, we highlight recent advances in our understanding of these α-helical translocation machineries and examine their structures, mechanisms, and regulation. We also discuss the therapeutic potential of these translocation pathways and summarize the progress in drug development targeting these systems for treating diseases.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"80 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142696627","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 : 2024-11-22DOI: 10.1016/j.str.2024.10.031
Patryk Ludzia, Midori Ishii, Gauri Deák, Christos Spanos, Marcus D. Wilson, Christina Redfield, Bungo Akiyoshi
The kinetochore is the macromolecular protein machine that drives chromosome segregation in eukaryotes. In an evolutionarily divergent group of organisms called kinetoplastids, kinetochores are built using a unique set of proteins (KKT1–25 and KKIP1–12). KKT23 is a constitutively localized kinetochore protein containing a C-terminal acetyltransferase domain of unknown function. Here, using X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, we have determined the structure and dynamics of the KKT23 acetyltransferase domain from Trypanosoma brucei and found that it is structurally similar to the GCN5 histone acetyltransferase domain. We find that KKT23 can acetylate the C-terminal tail of histone H2A and that knockdown of KKT23 results in decreased H2A acetylation levels in T. brucei. Finally, we have determined the crystal structure of the N-terminal region of KKT23 and shown that it interacts with KKT22. Our study provides important insights into the structure and function of the unique kinetochore acetyltransferase in trypanosomes.
动核是真核生物中驱动染色体分离的大分子蛋白质机器。在进化过程中出现分化的一类生物（称为动点细胞）中，动点由一组独特的蛋白质（KKT1-25 和 KKIP1-12）构建。KKT23 是一种组成型定位的动点核蛋白，含有一个功能未知的 C 端乙酰转移酶结构域。在这里，我们利用 X 射线晶体学和核磁共振（NMR）光谱测定了布氏锥虫 KKT23 乙酰转移酶结构域的结构和动力学，发现它在结构上与 GCN5 组蛋白乙酰转移酶结构域相似。我们发现 KKT23 能使组蛋白 H2A 的 C 端尾乙酰化，而且敲除 KKT23 会导致布氏锥虫 H2A 乙酰化水平下降。最后，我们测定了 KKT23 N 端区域的晶体结构，并证明它与 KKT22 相互作用。我们的研究对锥虫中独特的动点核乙酰转移酶的结构和功能提供了重要的见解。
{"title":"The kinetoplastid kinetochore protein KKT23 acetyltransferase is a structural homolog of GCN5 that acetylates the histone H2A C-terminal tail","authors":"Patryk Ludzia, Midori Ishii, Gauri Deák, Christos Spanos, Marcus D. Wilson, Christina Redfield, Bungo Akiyoshi","doi":"10.1016/j.str.2024.10.031","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.031","url":null,"abstract":"The kinetochore is the macromolecular protein machine that drives chromosome segregation in eukaryotes. In an evolutionarily divergent group of organisms called kinetoplastids, kinetochores are built using a unique set of proteins (KKT1–25 and KKIP1–12). KKT23 is a constitutively localized kinetochore protein containing a C-terminal acetyltransferase domain of unknown function. Here, using X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, we have determined the structure and dynamics of the KKT23 acetyltransferase domain from <em>Trypanosoma brucei</em> and found that it is structurally similar to the GCN5 histone acetyltransferase domain. We find that KKT23 can acetylate the C-terminal tail of histone H2A and that knockdown of KKT23 results in decreased H2A acetylation levels in <em>T. brucei</em>. Finally, we have determined the crystal structure of the N-terminal region of KKT23 and shown that it interacts with KKT22. Our study provides important insights into the structure and function of the unique kinetochore acetyltransferase in trypanosomes.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"11 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142690706","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 : 2024-11-21DOI: 10.1016/j.str.2024.10.030
Joao Ramos, Valerie Laux, Sax A. Mason, Marie-Hélène Lemée, Matthew W. Bowler, Kay Diederichs, Michael Haertlein, V. Trevor Forsyth, Estelle Mossou, Sine Larsen, Annette E. Langkilde
Hen egg-white lysozyme (HEWL) is a widely used model protein in crystallographic studies and its enzymatic mechanism has been extensively investigated for decades. Despite this, the interaction between the reaction intermediate and the catalytic Asp52, as well as the orientation of Asn44 and Asn46 side chains, remain ambiguous. Here, we report the crystal structures of perdeuterated HEWL and D2O buffer-exchanged HEWL from 0.91 and 1.1 Å resolution neutron diffraction data, respectively. These structures were obtained at room temperature and acidic pH, representing the active state of the enzyme. The unambiguous assignment of hydrogen positions based on the neutron scattering length density maps elucidates the roles of Asn44, Asn46, Asn59, and nearby water molecules in the stabilization of Asp52. Additionally, the identification of hydrogen positions reveals unique details of lysozyme’s folding, hydrogen (H)/deuterium (D) exchange, and side chain disorder.
{"title":"Structure and dynamics of the active site of hen egg-white lysozyme from atomic resolution neutron crystallography","authors":"Joao Ramos, Valerie Laux, Sax A. Mason, Marie-Hélène Lemée, Matthew W. Bowler, Kay Diederichs, Michael Haertlein, V. Trevor Forsyth, Estelle Mossou, Sine Larsen, Annette E. Langkilde","doi":"10.1016/j.str.2024.10.030","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.030","url":null,"abstract":"Hen egg-white lysozyme (HEWL) is a widely used model protein in crystallographic studies and its enzymatic mechanism has been extensively investigated for decades. Despite this, the interaction between the reaction intermediate and the catalytic Asp52, as well as the orientation of Asn44 and Asn46 side chains, remain ambiguous. Here, we report the crystal structures of perdeuterated HEWL and D<sub>2</sub>O buffer-exchanged HEWL from 0.91 and 1.1 Å resolution neutron diffraction data, respectively. These structures were obtained at room temperature and acidic pH, representing the active state of the enzyme. The unambiguous assignment of hydrogen positions based on the neutron scattering length density maps elucidates the roles of Asn44, Asn46, Asn59, and nearby water molecules in the stabilization of Asp52. Additionally, the identification of hydrogen positions reveals unique details of lysozyme’s folding, hydrogen (H)/deuterium (D) exchange, and side chain disorder.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"99 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142678316","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 : 2024-11-19DOI: 10.1016/j.str.2024.10.028
Anna Yudenko, Sergey Bukhdruker, Pavel Shishkin, Sergey Rodin, Anastasia Burtseva, Aleksandr Petrov, Natalia Pigareva, Alexey Sokolov, Egor Zinovev, Igor Eliseev, Alina Remeeva, Egor Marin, Alexey Mishin, Valentin Gordeliy, Ivan Gushchin, Aleksandr Ischenko, Valentin Borshchevskiy
Interleukin-6 (IL-6) is a multifaceted cytokine essential in many immune system processes and their regulation. It also plays a key role in hematopoiesis, and in triggering the acute phase reaction. IL-6 overproduction is critical in chronic inflammation associated with autoimmune diseases like rheumatoid arthritis and contributes to cytokine storms in COVID-19 patients. Over 20 years ago, researchers proposed that IL-6, which is typically monomeric, can also form dimers via a domain-swap mechanism, with indirect evidence supporting their existence. The physiological significance of IL-6 dimers was shown in B-cell chronic lymphocytic leukemia. However, no structures have been reported so far. Here, we present the crystal structure of an IL-6 domain-swapped dimer that computational approaches could not predict. The structure explains why the IL-6 dimer is antagonistic to the IL-6 monomer in signaling complex formation and provides insights for IL-6 targeted therapies.
{"title":"Structural basis of signaling complex inhibition by IL-6 domain-swapped dimers","authors":"Anna Yudenko, Sergey Bukhdruker, Pavel Shishkin, Sergey Rodin, Anastasia Burtseva, Aleksandr Petrov, Natalia Pigareva, Alexey Sokolov, Egor Zinovev, Igor Eliseev, Alina Remeeva, Egor Marin, Alexey Mishin, Valentin Gordeliy, Ivan Gushchin, Aleksandr Ischenko, Valentin Borshchevskiy","doi":"10.1016/j.str.2024.10.028","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.028","url":null,"abstract":"Interleukin-6 (IL-6) is a multifaceted cytokine essential in many immune system processes and their regulation. It also plays a key role in hematopoiesis, and in triggering the acute phase reaction. IL-6 overproduction is critical in chronic inflammation associated with autoimmune diseases like rheumatoid arthritis and contributes to cytokine storms in COVID-19 patients. Over 20 years ago, researchers proposed that IL-6, which is typically monomeric, can also form dimers via a domain-swap mechanism, with indirect evidence supporting their existence. The physiological significance of IL-6 dimers was shown in B-cell chronic lymphocytic leukemia. However, no structures have been reported so far. Here, we present the crystal structure of an IL-6 domain-swapped dimer that computational approaches could not predict. The structure explains why the IL-6 dimer is antagonistic to the IL-6 monomer in signaling complex formation and provides insights for IL-6 targeted therapies.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"13 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142670678","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 : 2024-11-13DOI: 10.1016/j.str.2024.10.025
María Ángeles Márquez-Moñino, Clara M. Santiveri, Patricia de León, Sergio Camero, Ramón Campos-Olivas, M. Ángeles Jiménez, Margarita Sáiz, Beatriz González, José Manuel Pérez-Cañadillas
Nucleoproteins (N) play an essential role in virus assembly and are less prone to mutation than other viral structural proteins, making them attractive targets for drug discovery. Using an NMR fragment-based drug discovery approach, we identified the 1,3-benzothiazol-2-amine (BZT) group as a scaffold to develop potential antivirals for SARS-CoV-2 nucleocapsid (N) protein. A thorough characterization of BZT derivatives using NMR, X-ray crystallography, antiviral activity assays, and intrinsic fluorescence measurements revealed their binding in the C-terminal domain (CTD) domain of the N protein, to residues Arg 259, Trp 330, and Lys 338, coinciding with the nucleotide binding site. Our most effective compound exhibits a slightly better affinity than GTP and the ALS drug riluzole, also identified during the screening, and displays notable viral inhibition activity. A virtual screening of 218 BZT-based compounds revealed a potential extended binding site that could be exploited for the future development of new SARS-CoV-2 antivirals.
核蛋白（N）在病毒组装过程中起着至关重要的作用，而且与其他病毒结构蛋白相比不易发生变异，因此成为具有吸引力的药物发现目标。利用基于核磁共振片段的药物发现方法，我们发现 1,3-苯并噻唑-2-胺（BZT）基团是开发 SARS-CoV-2 核苷酸（N）蛋白潜在抗病毒药物的支架。利用核磁共振、X 射线晶体学、抗病毒活性测定和本征荧光测量法对 BZT 衍生物进行的全面表征显示，它们与 N 蛋白的 C 端结构域 (CTD) 的 Arg 259、Trp 330 和 Lys 338 残基结合，与核苷酸结合位点相吻合。我们最有效的化合物比 GTP 和 ALS 药物利鲁唑（也是在筛选过程中发现的）的亲和力稍强，并具有显著的病毒抑制活性。对 218 种基于 BZT 的化合物进行的虚拟筛选发现了一个潜在的扩展结合位点，可用于未来开发新的 SARS-CoV-2 抗病毒药物。
{"title":"The ALS drug riluzole binds to the C-terminal domain of SARS-CoV-2 nucleocapsid protein and has antiviral activity","authors":"María Ángeles Márquez-Moñino, Clara M. Santiveri, Patricia de León, Sergio Camero, Ramón Campos-Olivas, M. Ángeles Jiménez, Margarita Sáiz, Beatriz González, José Manuel Pérez-Cañadillas","doi":"10.1016/j.str.2024.10.025","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.025","url":null,"abstract":"Nucleoproteins (N) play an essential role in virus assembly and are less prone to mutation than other viral structural proteins, making them attractive targets for drug discovery. Using an NMR fragment-based drug discovery approach, we identified the 1,3-benzothiazol-2-amine (BZT) group as a scaffold to develop potential antivirals for SARS-CoV-2 nucleocapsid (N) protein. A thorough characterization of BZT derivatives using NMR, X-ray crystallography, antiviral activity assays, and intrinsic fluorescence measurements revealed their binding in the C-terminal domain (CTD) domain of the N protein, to residues Arg 259, Trp 330, and Lys 338, coinciding with the nucleotide binding site. Our most effective compound exhibits a slightly better affinity than GTP and the ALS drug riluzole, also identified during the screening, and displays notable viral inhibition activity. A virtual screening of 218 BZT-based compounds revealed a potential extended binding site that could be exploited for the future development of new SARS-CoV-2 antivirals.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"41 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142601000","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}
Anti-CRISPR (Acr) proteins are natural inhibitors of CRISPR-Cas systems, found in bacteriophages and other genetic elements. AcrIE3, identified in a Pseudomonas phage, inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa by engaging with the Cascade complex. However, its precise inhibition mechanism has remained elusive. In this study, we present a comprehensive structural and biochemical analysis of AcrIE3, providing mechanistic insight into its anti-CRISPR function. Our results reveal that AcrIE3 selectively binds to the Cas8e subunit of the Cascade complex. The crystal structure of AcrIE3 exhibits an all-helical fold with a negatively charged surface. Through extensive mutational analyses, we show that AcrIE3 interacts with the protospacer adjacent motif (PAM) recognition site in Cas8e through its negatively charged surface residues. These findings enhance our understanding of the structure and function of type I-E Acr proteins, suggesting PAM interaction sites as primary targets for divergent Acr inhibitors.
{"title":"Structural and biochemical insights into the mechanism of the anti-CRISPR protein AcrIE3","authors":"Jasung Koo, Gyujin Lee, Changkon Park, Hyejin Oh, Sung-Hyun Hong, Jeong-Yong Suh, Euiyoung Bae","doi":"10.1016/j.str.2024.10.024","DOIUrl":"https://doi.org/10.1016/j.str.2024.10.024","url":null,"abstract":"Anti-CRISPR (Acr) proteins are natural inhibitors of CRISPR-Cas systems, found in bacteriophages and other genetic elements. AcrIE3, identified in a <em>Pseudomonas</em> phage, inactivates the type I-E CRISPR-Cas system in <em>Pseudomonas aeruginosa</em> by engaging with the Cascade complex. However, its precise inhibition mechanism has remained elusive. In this study, we present a comprehensive structural and biochemical analysis of AcrIE3, providing mechanistic insight into its anti-CRISPR function. Our results reveal that AcrIE3 selectively binds to the Cas8e subunit of the Cascade complex. The crystal structure of AcrIE3 exhibits an all-helical fold with a negatively charged surface. Through extensive mutational analyses, we show that AcrIE3 interacts with the protospacer adjacent motif (PAM) recognition site in Cas8e through its negatively charged surface residues. These findings enhance our understanding of the structure and function of type I-E Acr proteins, suggesting PAM interaction sites as primary targets for divergent Acr inhibitors.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"95 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142601436","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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