Pub Date : 2024-06-01Epub Date: 2024-06-05DOI: 10.1107/S2059798324004480
Shumeng Ma, Shymaa Damfo, Matthew W Bowler, Vitaliy Mykhaylyk, Frank Kozielski
Fragment-based drug design using X-ray crystallography is a powerful technique to enable the development of new lead compounds, or probe molecules, against biological targets. This study addresses the need to determine fragment binding orientations for low-occupancy fragments with incomplete electron density, an essential step before further development of the molecule. Halogen atoms play multiple roles in drug discovery due to their unique combination of electronegativity, steric effects and hydrophobic properties. Fragments incorporating halogen atoms serve as promising starting points in hit-to-lead development as they often establish halogen bonds with target proteins, potentially enhancing binding affinity and selectivity, as well as counteracting drug resistance. Here, the aim was to unambiguously identify the binding orientations of fragment hits for SARS-CoV-2 nonstructural protein 1 (nsp1) which contain a combination of sulfur and/or chlorine, bromine and iodine substituents. The binding orientations of carefully selected nsp1 analogue hits were focused on by employing their anomalous scattering combined with Pan-Dataset Density Analysis (PanDDA). Anomalous difference Fourier maps derived from the diffraction data collected at both standard and long-wavelength X-rays were compared. The discrepancies observed in the maps of iodine-containing fragments collected at different energies were attributed to site-specific radiation-damage stemming from the strong X-ray absorption of I atoms, which is likely to cause cleavage of the C-I bond. A reliable and effective data-collection strategy to unambiguously determine the binding orientations of low-occupancy fragments containing sulfur and/or halogen atoms while mitigating radiation damage is presented.
利用 X 射线晶体学技术进行基于片段的药物设计是一项强大的技术,可以针对生物靶标开发新的先导化合物或探针分子。这项研究解决了确定电子密度不完全的低占位片段结合方向的需求,这是进一步开发分子前的重要步骤。卤素原子具有独特的电负性、立体效应和疏水特性,因此在药物研发中发挥着多重作用。含有卤素原子的片段在 "先导-命中 "研发过程中是很有希望的起点,因为它们通常会与靶蛋白建立卤素键,从而有可能增强结合亲和力和选择性,并抵消耐药性。本研究的目的是明确确定含有硫和/或氯、溴和碘取代基组合的 SARS-CoV-2 非结构蛋白 1(nsp1)片段的结合方向。通过采用异常散射和泛数据集密度分析(PanDDA)技术,重点研究了精心挑选的 nsp1 类似物的结合方向。比较了从标准 X 射线和长波长 X 射线收集的衍射数据中得出的异常差分傅立叶图。在不同能量下采集的含碘片段图谱中观察到的差异被归因于特定位点的辐射损伤,这种辐射损伤源于 I 原子对 X 射线的强烈吸收,很可能导致 C-I 键的裂解。本文介绍了一种可靠有效的数据收集策略,既能明确确定含硫和/或卤原子的低占位片段的结合方向,又能减轻辐射损伤。
{"title":"High-confidence placement of low-occupancy fragments into electron density using the anomalous signal of sulfur and halogen atoms.","authors":"Shumeng Ma, Shymaa Damfo, Matthew W Bowler, Vitaliy Mykhaylyk, Frank Kozielski","doi":"10.1107/S2059798324004480","DOIUrl":"10.1107/S2059798324004480","url":null,"abstract":"<p><p>Fragment-based drug design using X-ray crystallography is a powerful technique to enable the development of new lead compounds, or probe molecules, against biological targets. This study addresses the need to determine fragment binding orientations for low-occupancy fragments with incomplete electron density, an essential step before further development of the molecule. Halogen atoms play multiple roles in drug discovery due to their unique combination of electronegativity, steric effects and hydrophobic properties. Fragments incorporating halogen atoms serve as promising starting points in hit-to-lead development as they often establish halogen bonds with target proteins, potentially enhancing binding affinity and selectivity, as well as counteracting drug resistance. Here, the aim was to unambiguously identify the binding orientations of fragment hits for SARS-CoV-2 nonstructural protein 1 (nsp1) which contain a combination of sulfur and/or chlorine, bromine and iodine substituents. The binding orientations of carefully selected nsp1 analogue hits were focused on by employing their anomalous scattering combined with Pan-Dataset Density Analysis (PanDDA). Anomalous difference Fourier maps derived from the diffraction data collected at both standard and long-wavelength X-rays were compared. The discrepancies observed in the maps of iodine-containing fragments collected at different energies were attributed to site-specific radiation-damage stemming from the strong X-ray absorption of I atoms, which is likely to cause cleavage of the C-I bond. A reliable and effective data-collection strategy to unambiguously determine the binding orientations of low-occupancy fragments containing sulfur and/or halogen atoms while mitigating radiation damage is presented.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":"80 Pt 6","pages":"451-463"},"PeriodicalIF":2.2,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11154595/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141260530","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-01Epub Date: 2024-05-28DOI: 10.1107/S2059798324004066
Marta Alberti, Giulio Poli, Luca Broggini, Stefano Sainas, Menico Rizzi, Donatella Boschi, Davide M Ferraris, Elena Martino, Stefano Ricagno, Tiziano Tuccinardi, Marco L Lolli, Riccardo Miggiano
Over the years, human dihydroorotate dehydrogenase (hDHODH), which is a key player in the de novo pyrimidine-biosynthesis pathway, has been targeted in the treatment of several conditions, including autoimmune disorders and acute myelogenous leukaemia, as well as in host-targeted antiviral therapy. A molecular exploration of its inhibitor-binding behaviours yielded promising candidates for innovative drug design. A detailed description of the enzymatic pharmacophore drove the decoration of well-established inhibitory scaffolds, thus gaining further in vitro and in vivo efficacy. In the present work, using X-ray crystallography, an atypical rearrangement was identified in the binding pose of a potent inhibitor characterized by a polar pyridine-based moiety (compound 18). The crystal structure shows that upon binding compound 18 the dynamics of a protein loop involved in a gating mechanism at the cofactor-binding site is modulated by the presence of three water molecules, thus fine-tuning the polarity/hydrophobicity of the binding pocket. These solvent molecules are engaged in the formation of a hydrogen-bond mesh in which one of them establishes a direct contact with the pyridine moiety of compound 18, thus paving the way for a reappraisal of the inhibition of hDHODH. Using an integrated approach, the thermodynamics of such a modulation is described by means of isothermal titration calorimetry coupled with molecular modelling. These structural insights will guide future drug design to obtain a finer Kd/logD7.4 balance and identify membrane-permeable molecules with a drug-like profile in terms of water solubility.
{"title":"An alternative conformation of the N-terminal loop of human dihydroorotate dehydrogenase drives binding to a potent antiproliferative agent.","authors":"Marta Alberti, Giulio Poli, Luca Broggini, Stefano Sainas, Menico Rizzi, Donatella Boschi, Davide M Ferraris, Elena Martino, Stefano Ricagno, Tiziano Tuccinardi, Marco L Lolli, Riccardo Miggiano","doi":"10.1107/S2059798324004066","DOIUrl":"10.1107/S2059798324004066","url":null,"abstract":"<p><p>Over the years, human dihydroorotate dehydrogenase (hDHODH), which is a key player in the de novo pyrimidine-biosynthesis pathway, has been targeted in the treatment of several conditions, including autoimmune disorders and acute myelogenous leukaemia, as well as in host-targeted antiviral therapy. A molecular exploration of its inhibitor-binding behaviours yielded promising candidates for innovative drug design. A detailed description of the enzymatic pharmacophore drove the decoration of well-established inhibitory scaffolds, thus gaining further in vitro and in vivo efficacy. In the present work, using X-ray crystallography, an atypical rearrangement was identified in the binding pose of a potent inhibitor characterized by a polar pyridine-based moiety (compound 18). The crystal structure shows that upon binding compound 18 the dynamics of a protein loop involved in a gating mechanism at the cofactor-binding site is modulated by the presence of three water molecules, thus fine-tuning the polarity/hydrophobicity of the binding pocket. These solvent molecules are engaged in the formation of a hydrogen-bond mesh in which one of them establishes a direct contact with the pyridine moiety of compound 18, thus paving the way for a reappraisal of the inhibition of hDHODH. Using an integrated approach, the thermodynamics of such a modulation is described by means of isothermal titration calorimetry coupled with molecular modelling. These structural insights will guide future drug design to obtain a finer K<sub>d</sub>/logD<sub>7.4</sub> balance and identify membrane-permeable molecules with a drug-like profile in terms of water solubility.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":" ","pages":"386-396"},"PeriodicalIF":2.2,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141157416","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-01Epub Date: 2024-05-28DOI: 10.1107/S2059798324003723
William C Gasper, Sarah Gardner, Adam Ross, Sarah A Oppelt, Karen N Allen, Dean R Tolan
Over the past forty years there has been a drastic increase in fructose-related diseases, including obesity, heart disease and diabetes. Ketohexokinase (KHK), the first enzyme in the liver fructolysis pathway, catalyzes the ATP-dependent phosphorylation of fructose to fructose 1-phosphate. Understanding the role of KHK in disease-related processes is crucial for the management and prevention of this growing epidemic. Molecular insight into the structure-function relationship in ligand binding and catalysis by KHK is needed for the design of therapeutic inhibitory ligands. Ketohexokinase has two isoforms: ketohexokinase A (KHK-A) is produced ubiquitously at low levels, whereas ketohexokinase C (KHK-C) is found at much higher levels, specifically in the liver, kidneys and intestines. Structures of the unliganded and liganded human isoforms KHK-A and KHK-C are known, as well as structures of unliganded and inhibitor-bound mouse KHK-C (mKHK-C), which shares 90% sequence identity with human KHK-C. Here, a high-resolution X-ray crystal structure of mKHK-C refined to 1.79 Å resolution is presented. The structure was determined in a complex with both the substrate fructose and the product of catalysis, ADP, providing a view of the Michaelis-like complex of the mouse ortholog. Comparison to unliganded structures suggests that KHK undergoes a conformational change upon binding of substrates that places the enzyme in a catalytically competent form in which the β-sheet domain from one subunit rotates by 16.2°, acting as a lid for the opposing active site. Similar kinetic parameters were calculated for the mouse and human enzymes and indicate that mice may be a suitable animal model for the study of fructose-related diseases. Knowledge of the similarity between the mouse and human enzymes is important for understanding preclinical efforts towards targeting this enzyme, and this ground-state, Michaelis-like complex suggests that a conformational change plays a role in the catalytic function of KHK-C.
{"title":"Michaelis-like complex of mouse ketohexokinase isoform C.","authors":"William C Gasper, Sarah Gardner, Adam Ross, Sarah A Oppelt, Karen N Allen, Dean R Tolan","doi":"10.1107/S2059798324003723","DOIUrl":"10.1107/S2059798324003723","url":null,"abstract":"<p><p>Over the past forty years there has been a drastic increase in fructose-related diseases, including obesity, heart disease and diabetes. Ketohexokinase (KHK), the first enzyme in the liver fructolysis pathway, catalyzes the ATP-dependent phosphorylation of fructose to fructose 1-phosphate. Understanding the role of KHK in disease-related processes is crucial for the management and prevention of this growing epidemic. Molecular insight into the structure-function relationship in ligand binding and catalysis by KHK is needed for the design of therapeutic inhibitory ligands. Ketohexokinase has two isoforms: ketohexokinase A (KHK-A) is produced ubiquitously at low levels, whereas ketohexokinase C (KHK-C) is found at much higher levels, specifically in the liver, kidneys and intestines. Structures of the unliganded and liganded human isoforms KHK-A and KHK-C are known, as well as structures of unliganded and inhibitor-bound mouse KHK-C (mKHK-C), which shares 90% sequence identity with human KHK-C. Here, a high-resolution X-ray crystal structure of mKHK-C refined to 1.79 Å resolution is presented. The structure was determined in a complex with both the substrate fructose and the product of catalysis, ADP, providing a view of the Michaelis-like complex of the mouse ortholog. Comparison to unliganded structures suggests that KHK undergoes a conformational change upon binding of substrates that places the enzyme in a catalytically competent form in which the β-sheet domain from one subunit rotates by 16.2°, acting as a lid for the opposing active site. Similar kinetic parameters were calculated for the mouse and human enzymes and indicate that mice may be a suitable animal model for the study of fructose-related diseases. Knowledge of the similarity between the mouse and human enzymes is important for understanding preclinical efforts towards targeting this enzyme, and this ground-state, Michaelis-like complex suggests that a conformational change plays a role in the catalytic function of KHK-C.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":" ","pages":"377-385"},"PeriodicalIF":2.2,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141157488","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-01Epub Date: 2024-06-04DOI: 10.1107/S2059798324004881
Carol Herre, Alex Ho, Ben Eisenbraun, James Vincent, Thomas Nicholson, Giorgos Boutsioukis, Peter A Meyer, Michelle Ottaviano, Kurt L Krause, Jason Key, Piotr Sliz
The expansive scientific software ecosystem, characterized by millions of titles across various platforms and formats, poses significant challenges in maintaining reproducibility and provenance in scientific research. The diversity of independently developed applications, evolving versions and heterogeneous components highlights the need for rigorous methodologies to navigate these complexities. In response to these challenges, the SBGrid team builds, installs and configures over 530 specialized software applications for use in the on-premises and cloud-based computing environments of SBGrid Consortium members. To address the intricacies of supporting this diverse application collection, the team has developed the Capsule Software Execution Environment, generally referred to as Capsules. Capsules rely on a collection of programmatically generated bash scripts that work together to isolate the runtime environment of one application from all other applications, thereby providing a transparent cross-platform solution without requiring specialized tools or elevated account privileges for researchers. Capsules facilitate modular, secure software distribution while maintaining a centralized, conflict-free environment. The SBGrid platform, which combines Capsules with the SBGrid collection of structural biology applications, aligns with FAIR goals by enhancing the findability, accessibility, interoperability and reusability of scientific software, ensuring seamless functionality across diverse computing environments. Its adaptability enables application beyond structural biology into other scientific fields.
{"title":"Introduction of the Capsules environment to support further growth of the SBGrid structural biology software collection.","authors":"Carol Herre, Alex Ho, Ben Eisenbraun, James Vincent, Thomas Nicholson, Giorgos Boutsioukis, Peter A Meyer, Michelle Ottaviano, Kurt L Krause, Jason Key, Piotr Sliz","doi":"10.1107/S2059798324004881","DOIUrl":"10.1107/S2059798324004881","url":null,"abstract":"<p><p>The expansive scientific software ecosystem, characterized by millions of titles across various platforms and formats, poses significant challenges in maintaining reproducibility and provenance in scientific research. The diversity of independently developed applications, evolving versions and heterogeneous components highlights the need for rigorous methodologies to navigate these complexities. In response to these challenges, the SBGrid team builds, installs and configures over 530 specialized software applications for use in the on-premises and cloud-based computing environments of SBGrid Consortium members. To address the intricacies of supporting this diverse application collection, the team has developed the Capsule Software Execution Environment, generally referred to as Capsules. Capsules rely on a collection of programmatically generated bash scripts that work together to isolate the runtime environment of one application from all other applications, thereby providing a transparent cross-platform solution without requiring specialized tools or elevated account privileges for researchers. Capsules facilitate modular, secure software distribution while maintaining a centralized, conflict-free environment. The SBGrid platform, which combines Capsules with the SBGrid collection of structural biology applications, aligns with FAIR goals by enhancing the findability, accessibility, interoperability and reusability of scientific software, ensuring seamless functionality across diverse computing environments. Its adaptability enables application beyond structural biology into other scientific fields.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":" ","pages":"439-450"},"PeriodicalIF":2.2,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11154594/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141236313","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-01Epub Date: 2024-05-28DOI: 10.1107/S2059798324004303
Valentin J Maurer, Marc Siggel, Jan Kosinski
The detection of specific biological macromolecules in cryogenic electron tomography data is frequently approached by applying cross-correlation-based 3D template matching. To reduce computational cost and noise, high binning is used to aggregate voxels before template matching. This remains a prevalent practice in both practical applications and methods development. Here, the relation between template size, shape and angular sampling is systematically evaluated to identify ribosomes in a ground-truth annotated data set. It is shown that at the commonly used binning, a detailed subtomogram average, a sphere and a heart emoji result in near-identical performance. These findings indicate that with current template-matching practices macromolecules can only be detected with high precision if their shape and size are sufficiently different from the background. Using theoretical considerations, the experimental results are rationalized and it is discussed why primarily low-frequency information remains at high binning and that template matching fails to be accurate because similarly shaped and sized macromolecules have similar low-frequency spectra. These challenges are discussed and potential enhancements for future template-matching methodologies are proposed.
{"title":"What shapes template-matching performance in cryogenic electron tomography in situ?","authors":"Valentin J Maurer, Marc Siggel, Jan Kosinski","doi":"10.1107/S2059798324004303","DOIUrl":"10.1107/S2059798324004303","url":null,"abstract":"<p><p>The detection of specific biological macromolecules in cryogenic electron tomography data is frequently approached by applying cross-correlation-based 3D template matching. To reduce computational cost and noise, high binning is used to aggregate voxels before template matching. This remains a prevalent practice in both practical applications and methods development. Here, the relation between template size, shape and angular sampling is systematically evaluated to identify ribosomes in a ground-truth annotated data set. It is shown that at the commonly used binning, a detailed subtomogram average, a sphere and a heart emoji result in near-identical performance. These findings indicate that with current template-matching practices macromolecules can only be detected with high precision if their shape and size are sufficiently different from the background. Using theoretical considerations, the experimental results are rationalized and it is discussed why primarily low-frequency information remains at high binning and that template matching fails to be accurate because similarly shaped and sized macromolecules have similar low-frequency spectra. These challenges are discussed and potential enhancements for future template-matching methodologies are proposed.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":" ","pages":"410-420"},"PeriodicalIF":2.6,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11154592/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141157423","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-01Epub Date: 2024-05-28DOI: 10.1107/S2059798324004248
Fernando Ceballos-Zúñiga, Margarita Menéndez, Inmaculada Pérez-Dorado
Type VII secretion (T7S) systems, also referred to as ESAT-6 secretion (ESX) systems, are molecular machines that have gained great attention due to their implications in cell homeostasis and in host-pathogen interactions in mycobacteria. The latter include important human pathogens such as Mycobacterium tuberculosis (Mtb), the etiological cause of human tuberculosis, which constitutes a pandemic accounting for more than one million deaths every year. The ESX-5 system is exclusively found in slow-growing pathogenic mycobacteria, where it mediates the secretion of a large family of virulence factors: the PE and PPE proteins. The secretion driving force is provided by EccC5, a multidomain ATPase that operates using four globular cytosolic domains: an N-terminal domain of unknown function (EccC5DUF) and three FtsK/SpoIIIE ATPase domains. Recent structural and functional studies of ESX-3 and ESX-5 systems have revealed EccCDUF to be an ATPase-like fold domain with potential ATPase activity, the functionality of which is essential for secretion. Here, the crystal structure of the MtbEccC5DUF domain is reported at 2.05 Å resolution, which reveals a nucleotide-free structure with degenerated cis-acting and trans-acting elements involved in ATP binding and hydrolysis. This crystallographic study, together with a biophysical assessment of the interaction of MtbEccC5DUF with ATP/Mg2+, supports the absence of ATPase activity proposed for this domain. It is shown that this degeneration is also present in DUF domains from other ESX and ESX-like systems, which are likely to exhibit poor or null ATPase activity. Moreover, based on an in silico model of the N-terminal region of MtbEccC5DUF, it is hypothesized that MtbEccC5DUF is a degenerated ATPase domain that may have retained the ability to hexamerize. These observations draw attention to DUF domains as structural elements with potential implications in the opening and closure of the membrane pore during the secretion process via their involvement in inter-protomer interactions.
Ⅶ型分泌(T7S)系统,也称为 ESAT-6 分泌(ESX)系统,是一种分子机器,由于其在细胞稳态和分枝杆菌中宿主-病原体相互作用中的作用而备受关注。后者包括重要的人类病原体,如结核分枝杆菌(Mtb),它是人类结核病的病原体,每年造成一百多万人死亡。ESX-5 系统只存在于生长缓慢的致病分枝杆菌中,它介导着一大批毒力因子的分泌:PE 和 PPE 蛋白。分泌驱动力由 EccC5 提供,它是一种多域 ATP 酶,利用四个球状细胞膜结构域运行:一个功能未知的 N 端结构域(EccC5DUF)和三个 FtsK/SpoIIIE ATP 酶结构域。最近对 ESX-3 和 ESX-5 系统的结构和功能研究发现,EccCDUF 是一个具有潜在 ATPase 活性的 ATPase 样折叠结构域,其功能对于分泌至关重要。本文以 2.05 Å 的分辨率报告了 MtbEccC5DUF 结构域的晶体结构,它揭示了一种无核苷酸结构,其中有退化的顺式作用和反式作用元件,参与 ATP 结合和水解。这项晶体学研究以及对 MtbEccC5DUF 与 ATP/Mg2+ 相互作用的生物物理评估,支持了该结构域不具有 ATP 酶活性的观点。研究表明,其他 ESX 和类 ESX 系统的 DUF 结构域也存在这种退化现象,它们很可能表现出较低或无效的 ATPase 活性。此外,根据对 MtbEccC5DUF N 端区域的硅学模型推测,MtbEccC5DUF 是一个退化的 ATPase 结构域,可能保留了六聚化的能力。这些观察结果使人们注意到 DUF 结构域在分泌过程中通过参与原体间的相互作用而对膜孔的打开和关闭具有潜在影响。
{"title":"New insights into the domain of unknown function (DUF) of EccC<sub>5</sub>, the pivotal ATPase providing the secretion driving force to the ESX-5 secretion system.","authors":"Fernando Ceballos-Zúñiga, Margarita Menéndez, Inmaculada Pérez-Dorado","doi":"10.1107/S2059798324004248","DOIUrl":"10.1107/S2059798324004248","url":null,"abstract":"<p><p>Type VII secretion (T7S) systems, also referred to as ESAT-6 secretion (ESX) systems, are molecular machines that have gained great attention due to their implications in cell homeostasis and in host-pathogen interactions in mycobacteria. The latter include important human pathogens such as Mycobacterium tuberculosis (Mtb), the etiological cause of human tuberculosis, which constitutes a pandemic accounting for more than one million deaths every year. The ESX-5 system is exclusively found in slow-growing pathogenic mycobacteria, where it mediates the secretion of a large family of virulence factors: the PE and PPE proteins. The secretion driving force is provided by EccC<sub>5</sub>, a multidomain ATPase that operates using four globular cytosolic domains: an N-terminal domain of unknown function (EccC<sub>5</sub><sup>DUF</sup>) and three FtsK/SpoIIIE ATPase domains. Recent structural and functional studies of ESX-3 and ESX-5 systems have revealed EccC<sup>DUF</sup> to be an ATPase-like fold domain with potential ATPase activity, the functionality of which is essential for secretion. Here, the crystal structure of the MtbEccC<sub>5</sub><sup>DUF</sup> domain is reported at 2.05 Å resolution, which reveals a nucleotide-free structure with degenerated cis-acting and trans-acting elements involved in ATP binding and hydrolysis. This crystallographic study, together with a biophysical assessment of the interaction of MtbEccC<sub>5</sub><sup>DUF</sup> with ATP/Mg<sup>2+</sup>, supports the absence of ATPase activity proposed for this domain. It is shown that this degeneration is also present in DUF domains from other ESX and ESX-like systems, which are likely to exhibit poor or null ATPase activity. Moreover, based on an in silico model of the N-terminal region of MtbEccC<sub>5</sub><sup>DUF</sup>, it is hypothesized that MtbEccC<sub>5</sub><sup>DUF</sup> is a degenerated ATPase domain that may have retained the ability to hexamerize. These observations draw attention to DUF domains as structural elements with potential implications in the opening and closure of the membrane pore during the secretion process via their involvement in inter-protomer interactions.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":" ","pages":"397-409"},"PeriodicalIF":2.2,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11154593/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141157461","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01Epub Date: 2024-04-29DOI: 10.1107/S205979832400336X
Piotr Neumann, Jana L Heidemann, Jan Wollenhaupt, Achim Dickmanns, Michael Agthe, Manfred S Weiss, Ralf Ficner
CdaA is the most widespread diadenylate cyclase in many bacterial species, including several multidrug-resistant human pathogens. The enzymatic product of CdaA, cyclic di-AMP, is a secondary messenger that is essential for the viability of many bacteria. Its absence in humans makes CdaA a very promising and attractive target for the development of new antibiotics. Here, the structural results are presented of a crystallographic fragment screen against CdaA from Listeria monocytogenes, a saprophytic Gram-positive bacterium and an opportunistic food-borne pathogen that can cause listeriosis in humans and animals. Two of the eight fragment molecules reported here were localized in the highly conserved ATP-binding site. These fragments could serve as potential starting points for the development of antibiotics against several CdaA-dependent bacterial species.
{"title":"A small step towards an important goal: fragment screen of the c-di-AMP-synthesizing enzyme CdaA.","authors":"Piotr Neumann, Jana L Heidemann, Jan Wollenhaupt, Achim Dickmanns, Michael Agthe, Manfred S Weiss, Ralf Ficner","doi":"10.1107/S205979832400336X","DOIUrl":"10.1107/S205979832400336X","url":null,"abstract":"<p><p>CdaA is the most widespread diadenylate cyclase in many bacterial species, including several multidrug-resistant human pathogens. The enzymatic product of CdaA, cyclic di-AMP, is a secondary messenger that is essential for the viability of many bacteria. Its absence in humans makes CdaA a very promising and attractive target for the development of new antibiotics. Here, the structural results are presented of a crystallographic fragment screen against CdaA from Listeria monocytogenes, a saprophytic Gram-positive bacterium and an opportunistic food-borne pathogen that can cause listeriosis in humans and animals. Two of the eight fragment molecules reported here were localized in the highly conserved ATP-binding site. These fragments could serve as potential starting points for the development of antibiotics against several CdaA-dependent bacterial species.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":"80 Pt 5","pages":"350-361"},"PeriodicalIF":2.2,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11066881/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140848269","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01Epub Date: 2024-04-30DOI: 10.1107/S2059798324003243
Kathryn L Shelley, Elspeth F Garman
Radiation damage remains one of the major impediments to accurate structure solution in macromolecular crystallography. The artefacts of radiation damage can manifest as structural changes that result in incorrect biological interpretations being drawn from a model, they can reduce the resolution to which data can be collected and they can even prevent structure solution entirely. In this article, we discuss how to identify and mitigate against the effects of radiation damage at each stage in the macromolecular crystal structure-solution pipeline.
{"title":"Identifying and avoiding radiation damage in macromolecular crystallography.","authors":"Kathryn L Shelley, Elspeth F Garman","doi":"10.1107/S2059798324003243","DOIUrl":"https://doi.org/10.1107/S2059798324003243","url":null,"abstract":"<p><p>Radiation damage remains one of the major impediments to accurate structure solution in macromolecular crystallography. The artefacts of radiation damage can manifest as structural changes that result in incorrect biological interpretations being drawn from a model, they can reduce the resolution to which data can be collected and they can even prevent structure solution entirely. In this article, we discuss how to identify and mitigate against the effects of radiation damage at each stage in the macromolecular crystal structure-solution pipeline.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":"80 Pt 5","pages":"314-327"},"PeriodicalIF":2.2,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11066884/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140849877","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01Epub Date: 2024-04-29DOI: 10.1107/S2059798324003152
Milana Bazayeva, Claudia Andreini, Antonio Rosato
Metalloproteins are ubiquitous in all living organisms and take part in a very wide range of biological processes. For this reason, their experimental characterization is crucial to obtain improved knowledge of their structure and biological functions. The three-dimensional structure represents highly relevant information since it provides insight into the interaction between the metal ion(s) and the protein fold. Such interactions determine the chemical reactivity of the bound metal. The available PDB structures can contain errors due to experimental factors such as poor resolution and radiation damage. A lack of use of distance restraints during the refinement and validation process also impacts the structure quality. Here, the aim was to obtain a thorough overview of the distribution of the distances between metal ions and their donor atoms through the statistical analysis of a data set based on more than 115 000 metal-binding sites in proteins. This analysis not only produced reference data that can be used by experimentalists to support the structure-determination process, for example as refinement restraints, but also resulted in an improved insight into how protein coordination occurs for different metals and the nature of their binding interactions. In particular, the features of carboxylate coordination were inspected, which is the only type of interaction that is commonly present for nearly all metals.
{"title":"A database overview of metal-coordination distances in metalloproteins.","authors":"Milana Bazayeva, Claudia Andreini, Antonio Rosato","doi":"10.1107/S2059798324003152","DOIUrl":"https://doi.org/10.1107/S2059798324003152","url":null,"abstract":"<p><p>Metalloproteins are ubiquitous in all living organisms and take part in a very wide range of biological processes. For this reason, their experimental characterization is crucial to obtain improved knowledge of their structure and biological functions. The three-dimensional structure represents highly relevant information since it provides insight into the interaction between the metal ion(s) and the protein fold. Such interactions determine the chemical reactivity of the bound metal. The available PDB structures can contain errors due to experimental factors such as poor resolution and radiation damage. A lack of use of distance restraints during the refinement and validation process also impacts the structure quality. Here, the aim was to obtain a thorough overview of the distribution of the distances between metal ions and their donor atoms through the statistical analysis of a data set based on more than 115 000 metal-binding sites in proteins. This analysis not only produced reference data that can be used by experimentalists to support the structure-determination process, for example as refinement restraints, but also resulted in an improved insight into how protein coordination occurs for different metals and the nature of their binding interactions. In particular, the features of carboxylate coordination were inspected, which is the only type of interaction that is commonly present for nearly all metals.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":"80 Pt 5","pages":"362-376"},"PeriodicalIF":2.2,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11066882/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140848506","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-01Epub Date: 2024-03-21DOI: 10.1107/S2059798324002316
Callum W Breeze, Yuji Nakano, Eleanor C Campbell, Rebecca L Frkic, David W Lupton, Colin J Jackson
Lanthanide ions have ideal chemical properties for catalysis, such as hard Lewis acidity, fast ligand-exchange kinetics, high coordination-number preferences and low geometric requirements for coordination. As a result, many small-molecule lanthanide catalysts have been described in the literature. Yet, despite the ability of enzymes to catalyse highly stereoselective reactions under gentle conditions, very few lanthanoenzymes have been investigated. In this work, the mononuclear binding of europium(III) and gadolinium(III) to the active site of a mutant of the model enzyme phosphotriesterase are described using X-ray crystallography at 1.78 and 1.61 Å resolution, respectively. It is also shown that despite coordinating a single non-natural metal cation, the PTE-R18 mutant is still able to maintain esterase activity.
镧系离子具有理想的催化化学特性,如硬路易斯酸性、快速配体交换动力学、高配位数偏好和低几何配位要求。因此,文献中描述了许多小分子镧系催化剂。然而,尽管酶能够在温和的条件下催化高度立体选择性的反应,但很少有人研究过镧系酶。在这项工作中,利用分辨率分别为 1.78 和 1.61 Å 的 X 射线晶体学,描述了铕(III)和钆(III)与磷酸三酯酶模型突变体活性位点的单核结合。研究还表明,尽管配位了单个非天然金属阳离子,PTE-R18 突变体仍能保持酯酶活性。
{"title":"Mononuclear binding and catalytic activity of europium(III) and gadolinium(III) at the active site of the model metalloenzyme phosphotriesterase.","authors":"Callum W Breeze, Yuji Nakano, Eleanor C Campbell, Rebecca L Frkic, David W Lupton, Colin J Jackson","doi":"10.1107/S2059798324002316","DOIUrl":"10.1107/S2059798324002316","url":null,"abstract":"<p><p>Lanthanide ions have ideal chemical properties for catalysis, such as hard Lewis acidity, fast ligand-exchange kinetics, high coordination-number preferences and low geometric requirements for coordination. As a result, many small-molecule lanthanide catalysts have been described in the literature. Yet, despite the ability of enzymes to catalyse highly stereoselective reactions under gentle conditions, very few lanthanoenzymes have been investigated. In this work, the mononuclear binding of europium(III) and gadolinium(III) to the active site of a mutant of the model enzyme phosphotriesterase are described using X-ray crystallography at 1.78 and 1.61 Å resolution, respectively. It is also shown that despite coordinating a single non-natural metal cation, the PTE-R18 mutant is still able to maintain esterase activity.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":" ","pages":"289-298"},"PeriodicalIF":4.6,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10994177/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140178910","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}