Pub Date : 2024-12-05DOI: 10.1016/j.str.2024.11.007
Wei-Feng Xue
Atomic force microscopy (AFM) is a powerful and increasingly accessible technology that has a wide range of bio-imaging applications. AFM is capable of producing detailed three-dimensional topographical images with high signal-to-noise ratio, which enables the structural features of individual molecules to be studied without the need for ensemble averaging. Here, a software tool Trace_y, designed to reconstruct the three-dimensional surface envelopes of individual helical filament structures from topographical AFM images, is presented. Workflow using Trace_y is demonstrated on the structural analysis of individual helical amyloid protein fibrils where the assembly mechanism of heterogeneous, complex and diverse fibril populations due to structural polymorphism is not understood. The algorithms presented here allow structural information encoded in topographical AFM height images to be extracted and understood as three-dimensional (3D) contact point clouds. This approach will facilitate the use of AFM in structural biology to understand molecular structures and behaviors at individual molecule level.
{"title":"Trace_y: Software algorithms for structural analysis of individual helical filaments by three-dimensional contact point reconstruction atomic force microscopy","authors":"Wei-Feng Xue","doi":"10.1016/j.str.2024.11.007","DOIUrl":"https://doi.org/10.1016/j.str.2024.11.007","url":null,"abstract":"Atomic force microscopy (AFM) is a powerful and increasingly accessible technology that has a wide range of bio-imaging applications. AFM is capable of producing detailed three-dimensional topographical images with high signal-to-noise ratio, which enables the structural features of individual molecules to be studied without the need for ensemble averaging. Here, a software tool Trace_y, designed to reconstruct the three-dimensional surface envelopes of individual helical filament structures from topographical AFM images, is presented. Workflow using Trace_y is demonstrated on the structural analysis of individual helical amyloid protein fibrils where the assembly mechanism of heterogeneous, complex and diverse fibril populations due to structural polymorphism is not understood. The algorithms presented here allow structural information encoded in topographical AFM height images to be extracted and understood as three-dimensional (3D) contact point clouds. This approach will facilitate the use of AFM in structural biology to understand molecular structures and behaviors at individual molecule level.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"12 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142776742","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.2024.11.003
In this issue of Structure, Kielkopf et al.1 report the crystal structures of Rhs proteins that are genetically fused to the type VI secretion system …
在这一期的《结构》中，Kielkopf et al.1报道了与VI型分泌系统基因融合的Rhs蛋白的晶体结构。
{"title":"The universal Rhs shell structure accommodates various toxins inside and different functional decorations on the outside","authors":"","doi":"10.1016/j.str.2024.11.003","DOIUrl":"https://doi.org/10.1016/j.str.2024.11.003","url":null,"abstract":"In this issue of Structure, Kielkopf et al.1 report the crystal structures of Rhs proteins that are genetically fused to the type VI secretion system …","PeriodicalId":22168,"journal":{"name":"Structure","volume":"3 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142776459","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}
Phthalate diesters are important pollutants and act as endocrine disruptors. While certain bacterial esterases have been identified for phthalate diesters degradation to monoesters, their structural and mechanistic characteristics remain largely unexplored. Here, we highlight the potential of the thermostable and pH-tolerant EstS1 esterase from Sulfobacillus acidophilus DSM10332 to degrade high molecular weight bis(2-ethylhexyl) phthalate (DEHP) by combining biophysical and biochemical approaches along with high-resolution EstS1 crystal structures of the apo form and with bound substrates, products, and their analogs to elucidate its mechanism. The catalytic tunnel mediates entry and exit of the substrate and product, respectively. The centralized Ser-His-Asp triad performs catalysis by a bi-bi ping-pong mechanism, forming a tetrahedral intermediate. Mutagenesis analysis showed that the Met207Ala mutation abolished DEHP binding at the active site, confirming its essential role in supporting catalysis. These findings underscore EstS1 as a promising tool for advancing technologies aimed at phthalate diesters biodegradation.
{"title":"Mechanistic and structural insights into EstS1 esterase: A potent broad-spectrum phthalate diester degrading enzyme","authors":"Shalja Verma, Shweta Choudhary, Kamble Amith Kumar, Jai Krishna Mahto, Anil Kumar Vamsi K, Ishani Mishra, Vellanki Bhanu Prakash, Debabrata Sircar, Shailly Tomar, Ashwani Kumar Sharma, Jitin Singla, Pravindra Kumar","doi":"10.1016/j.str.2024.11.006","DOIUrl":"https://doi.org/10.1016/j.str.2024.11.006","url":null,"abstract":"Phthalate diesters are important pollutants and act as endocrine disruptors. While certain bacterial esterases have been identified for phthalate diesters degradation to monoesters, their structural and mechanistic characteristics remain largely unexplored. Here, we highlight the potential of the thermostable and pH-tolerant EstS1 esterase from <em>Sulfobacillus acidophilus</em> DSM10332 to degrade high molecular weight bis(2-ethylhexyl) phthalate (DEHP) by combining biophysical and biochemical approaches along with high-resolution EstS1 crystal structures of the apo form and with bound substrates, products, and their analogs to elucidate its mechanism. The catalytic tunnel mediates entry and exit of the substrate and product, respectively. The centralized Ser-His-Asp triad performs catalysis by a bi-bi ping-pong mechanism, forming a tetrahedral intermediate. Mutagenesis analysis showed that the Met207Ala mutation abolished DEHP binding at the active site, confirming its essential role in supporting catalysis. These findings underscore EstS1 as a promising tool for advancing technologies aimed at phthalate diesters biodegradation.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"31 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142776457","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.2024.11.002
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}
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