Pub Date : 2025-01-01DOI: 10.1107/s2059798324012518
Marine Le Berre,Thibault Tubiana,Philippa Reuterswärd Waldner,Noureddine Lazar,Ines Li de la Sierra-Gallay,Joana M Santos,Manuel Llinás,Sylvie Nessler
The apicomplexan AP2 (ApiAP2) proteins are the best characterized family of DNA-binding proteins in Plasmodium spp. malaria parasites. Apart from the AP2 DNA-binding domain, there is little sequence similarity between ApiAP2 proteins. However, a conserved AP2-coincident domain mostly at the C-terminus (ACDC domain) is observed in a subset of the ApiAP2 proteins. The structure and function of this domain remain unknown. We report two crystal structures of ACDC domains derived from distinct Plasmodium ApiAP2 proteins, revealing a conserved, unique, noncanonical, four-helix bundle architecture. We used these structures to perform in silico docking calculations against a library of known antimalarial compounds and identified potential small-molecule ligands that bind in a highly conserved hydrophobic pocket that is present in all apicomplexan ACDC domains. These ligands provide a new molecular basis for the future design of ACDC inhibitors.
{"title":"Structural characterization of the ACDC domain from ApiAP2 proteins, a potential molecular target against apicomplexan parasites.","authors":"Marine Le Berre,Thibault Tubiana,Philippa Reuterswärd Waldner,Noureddine Lazar,Ines Li de la Sierra-Gallay,Joana M Santos,Manuel Llinás,Sylvie Nessler","doi":"10.1107/s2059798324012518","DOIUrl":"https://doi.org/10.1107/s2059798324012518","url":null,"abstract":"The apicomplexan AP2 (ApiAP2) proteins are the best characterized family of DNA-binding proteins in Plasmodium spp. malaria parasites. Apart from the AP2 DNA-binding domain, there is little sequence similarity between ApiAP2 proteins. However, a conserved AP2-coincident domain mostly at the C-terminus (ACDC domain) is observed in a subset of the ApiAP2 proteins. The structure and function of this domain remain unknown. We report two crystal structures of ACDC domains derived from distinct Plasmodium ApiAP2 proteins, revealing a conserved, unique, noncanonical, four-helix bundle architecture. We used these structures to perform in silico docking calculations against a library of known antimalarial compounds and identified potential small-molecule ligands that bind in a highly conserved hydrophobic pocket that is present in all apicomplexan ACDC domains. These ligands provide a new molecular basis for the future design of ACDC inhibitors.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"27 1","pages":"38-48"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142989313","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-01DOI: 10.1107/s2059798324009707
Gwendell M Thomas,Stephen Quirk,Raquel L Lieberman
Pollution from plastics is a global problem that threatens the biosphere for a host of reasons, including the time scale that it takes for most plastics to degrade. Biodegradation is an ideal solution for remediating bioplastic waste as it does not require the high temperatures necessary for thermal degradation and does not introduce additional pollutants into the environment. Numerous organisms can scavenge for bioplastics, such as polylactic acid (PLA) or poly-(R)-hydroxybutyrate (PHB), which they can use as an energy source. Recently, a promiscuous PHBase from the thermophilic soil bacterium Lihuaxuella thermophila (LtPHBase) was identified. LtPHBase can accommodate many substrates, including PHB granules and films and PHB block copolymers, as well as the unrelated polymers polylactic acid (PLA) and polycaprolactone (PCL). LtPHBase uses the expected Ser-His-Asp catalytic triad for hydrolysis at an optimal enzyme activity near 70°C. Here, the 1.75 Å resolution crystal structure of apo LtPHBase is presented and its chemical stability is profiled. Knowledge of its substrate preferences was extended to different-sized PHB granules. It is shown that LtPHBase is highly resistant to unfolding, with barriers typical for thermophilic enzymes, and shows a preference for low-molecular-mass PHB granules. These insights have implications for the long-term potential of LtPHBase as an industrial PHB hydrolase and shed light on the evolutionary role that this enzyme plays in bacterial metabolism.
{"title":"Structure and stability of an apo thermophilic esterase that hydrolyzes polyhydroxybutyrate.","authors":"Gwendell M Thomas,Stephen Quirk,Raquel L Lieberman","doi":"10.1107/s2059798324009707","DOIUrl":"https://doi.org/10.1107/s2059798324009707","url":null,"abstract":"Pollution from plastics is a global problem that threatens the biosphere for a host of reasons, including the time scale that it takes for most plastics to degrade. Biodegradation is an ideal solution for remediating bioplastic waste as it does not require the high temperatures necessary for thermal degradation and does not introduce additional pollutants into the environment. Numerous organisms can scavenge for bioplastics, such as polylactic acid (PLA) or poly-(R)-hydroxybutyrate (PHB), which they can use as an energy source. Recently, a promiscuous PHBase from the thermophilic soil bacterium Lihuaxuella thermophila (LtPHBase) was identified. LtPHBase can accommodate many substrates, including PHB granules and films and PHB block copolymers, as well as the unrelated polymers polylactic acid (PLA) and polycaprolactone (PCL). LtPHBase uses the expected Ser-His-Asp catalytic triad for hydrolysis at an optimal enzyme activity near 70°C. Here, the 1.75 Å resolution crystal structure of apo LtPHBase is presented and its chemical stability is profiled. Knowledge of its substrate preferences was extended to different-sized PHB granules. It is shown that LtPHBase is highly resistant to unfolding, with barriers typical for thermophilic enzymes, and shows a preference for low-molecular-mass PHB granules. These insights have implications for the long-term potential of LtPHBase as an industrial PHB hydrolase and shed light on the evolutionary role that this enzyme plays in bacterial metabolism.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"235 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142488228","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-01DOI: 10.1107/s2059798324009902
Michael J Barnett,Rick P Millane,Richard L Kingston
For protein crystals in which more than two thirds of the volume is occupied by solvent, the featureless nature of the solvent region often generates a constraint that is powerful enough to allow direct phasing of X-ray diffraction data. Practical implementation relies on the use of iterative projection algorithms with good global convergence properties to solve the difficult nonconvex phase-retrieval problem. In this paper, some aspects of phase retrieval using iterative projection algorithms are systematically explored, where the diffraction data and density-value distributions in the protein and solvent regions provide the sole constraints. The analysis is based on the addition of random error to the phases of previously determined protein crystal structures, followed by evaluation of the ability to recover the correct phase set as the distance from the solution increases. The properties of the difference-map (DM), relaxed-reflect-reflect (RRR) and relaxed averaged alternating reflectors (RAAR) algorithms are compared. All of these algorithms prove to be effective for crystallographic phase retrieval, and the useful ranges of the adjustable parameter which controls their behavior are established. When these algorithms converge to the solution, the algorithm trajectory becomes stationary; however, the density function continues to fluctuate significantly around its mean position. It is shown that averaging over the algorithm trajectory in the stationary region, following convergence, improves the density estimate, with this procedure outperforming previous approaches for phase or density refinement.
对于三分之二以上体积被溶剂占据的蛋白质晶体来说，溶剂区域的无特征性往往会产生一个强大的约束条件，足以允许对 X 射线衍射数据进行直接相位分析。实际应用依赖于使用具有良好全局收敛特性的迭代投影算法来解决困难的非凸相位检索问题。本文系统地探讨了使用迭代投影算法进行相位检索的一些方面，其中蛋白质和溶剂区域的衍射数据和密度值分布是唯一的约束条件。分析的基础是在先前确定的蛋白质晶体结构相位中加入随机误差，然后评估随着与溶液距离的增加恢复正确相位集的能力。比较了差分图算法（DM）、松弛反射算法（RRR）和松弛平均交替反射算法（RAAR）的特性。所有这些算法都被证明对晶体学相位检索有效，并确定了控制其行为的可调参数的有用范围。当这些算法收敛到解决方案时，算法轨迹会变得静止；然而，密度函数会继续围绕其平均位置大幅波动。研究表明，在算法收敛后，对静止区域内的算法轨迹进行平均，可以改善密度估算，这一过程优于以往的相位或密度细化方法。
{"title":"Analysis of crystallographic phase retrieval using iterative projection algorithms.","authors":"Michael J Barnett,Rick P Millane,Richard L Kingston","doi":"10.1107/s2059798324009902","DOIUrl":"https://doi.org/10.1107/s2059798324009902","url":null,"abstract":"For protein crystals in which more than two thirds of the volume is occupied by solvent, the featureless nature of the solvent region often generates a constraint that is powerful enough to allow direct phasing of X-ray diffraction data. Practical implementation relies on the use of iterative projection algorithms with good global convergence properties to solve the difficult nonconvex phase-retrieval problem. In this paper, some aspects of phase retrieval using iterative projection algorithms are systematically explored, where the diffraction data and density-value distributions in the protein and solvent regions provide the sole constraints. The analysis is based on the addition of random error to the phases of previously determined protein crystal structures, followed by evaluation of the ability to recover the correct phase set as the distance from the solution increases. The properties of the difference-map (DM), relaxed-reflect-reflect (RRR) and relaxed averaged alternating reflectors (RAAR) algorithms are compared. All of these algorithms prove to be effective for crystallographic phase retrieval, and the useful ranges of the adjustable parameter which controls their behavior are established. When these algorithms converge to the solution, the algorithm trajectory becomes stationary; however, the density function continues to fluctuate significantly around its mean position. It is shown that averaging over the algorithm trajectory in the stationary region, following convergence, improves the density estimate, with this procedure outperforming previous approaches for phase or density refinement.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"13 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142488227","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01DOI: 10.1107/s2059798324008659
Kamel El Omari,Ismay Forsyth,Ramona Duman,Christian M Orr,Vitaliy Mykhaylyk,Erika J Mancini,Armin Wagner
AlphaFold2 has revolutionized structural biology by offering unparalleled accuracy in predicting protein structures. Traditional methods for determining protein structures, such as X-ray crystallography and cryo-electron microscopy, are often time-consuming and resource-intensive. AlphaFold2 provides models that are valuable for molecular replacement, aiding in model building and docking into electron density or potential maps. However, despite its capabilities, models from AlphaFold2 do not consistently match the accuracy of experimentally determined structures, need to be validated experimentally and currently miss some crucial information, such as post-translational modifications, ligands and bound ions. In this paper, the advantages are explored of collecting X-ray anomalous data to identify chemical elements, such as metal ions, which are key to understanding certain structures and functions of proteins. This is achieved through methods such as calculating anomalous difference Fourier maps or refining the imaginary component of the anomalous scattering factor f''. Anomalous data can serve as a valuable complement to the information provided by AlphaFold2 models and this is particularly significant in elucidating the roles of metal ions.
AlphaFold2 预测蛋白质结构的准确性无与伦比，彻底改变了结构生物学。确定蛋白质结构的传统方法，如 X 射线晶体学和低温电子显微镜，往往耗费大量时间和资源。AlphaFold2 提供的模型对分子置换、帮助建立模型和对接电子密度图或电位图非常有价值。然而，尽管 AlphaFold2 模型功能强大，但其准确性并不能始终与实验测定的结构相匹配，需要通过实验验证，而且目前还遗漏了一些关键信息，如翻译后修饰、配体和结合离子。本文探讨了收集 X 射线反常数据以识别金属离子等化学元素的优势，这些元素是了解蛋白质某些结构和功能的关键。这是通过计算异常差分傅立叶图或完善异常散射因子 f'' 的虚分量等方法实现的。反常数据可以作为 AlphaFold2 模型所提供信息的宝贵补充，这对于阐明金属离子的作用尤为重要。
{"title":"Utilizing anomalous signals for element identification in macromolecular crystallography.","authors":"Kamel El Omari,Ismay Forsyth,Ramona Duman,Christian M Orr,Vitaliy Mykhaylyk,Erika J Mancini,Armin Wagner","doi":"10.1107/s2059798324008659","DOIUrl":"https://doi.org/10.1107/s2059798324008659","url":null,"abstract":"AlphaFold2 has revolutionized structural biology by offering unparalleled accuracy in predicting protein structures. Traditional methods for determining protein structures, such as X-ray crystallography and cryo-electron microscopy, are often time-consuming and resource-intensive. AlphaFold2 provides models that are valuable for molecular replacement, aiding in model building and docking into electron density or potential maps. However, despite its capabilities, models from AlphaFold2 do not consistently match the accuracy of experimentally determined structures, need to be validated experimentally and currently miss some crucial information, such as post-translational modifications, ligands and bound ions. In this paper, the advantages are explored of collecting X-ray anomalous data to identify chemical elements, such as metal ions, which are key to understanding certain structures and functions of proteins. This is achieved through methods such as calculating anomalous difference Fourier maps or refining the imaginary component of the anomalous scattering factor f''. Anomalous data can serve as a valuable complement to the information provided by AlphaFold2 models and this is particularly significant in elucidating the roles of metal ions.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"27 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142245298","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-04DOI: 10.1107/s2059798324008210
Y Li,L C Pacoste,W Gu,S J Thygesen,K J Stacey,T Ve,B Kobe,H Xu,J D Nanson
Eukaryotic TIR (Toll/interleukin-1 receptor protein) domains signal via TIR-TIR interactions, either by self-association or by interaction with other TIR domains. In mammals, TIR domains are found in Toll-like receptors (TLRs) and cytoplasmic adaptor proteins involved in pro-inflammatory signaling. Previous work revealed that the MAL TIR domain (MALTIR) nucleates the assembly of MyD88TIR into crystalline arrays in vitro. A microcrystal electron diffraction (MicroED) structure of the MyD88TIR assembly has previously been solved, revealing a two-stranded higher-order assembly of TIR domains. In this work, it is demonstrated that the TIR domain of TLR2, which is reported to signal as a heterodimer with either TLR1 or TLR6, induces the formation of crystalline higher-order assemblies of MyD88TIR in vitro, whereas TLR1TIR and TLR6TIR do not. Using an improved data-collection protocol, the MicroED structure of TLR2TIR-induced MyD88TIR microcrystals was determined at a higher resolution (2.85 Å) and with higher completeness (89%) compared with the previous structure of the MALTIR-induced MyD88TIR assembly. Both assemblies exhibit conformational differences in several areas that are important for signaling (for example the BB loop and CD loop) compared with their monomeric structures. These data suggest that TLR2TIR and MALTIR interact with MyD88 in an analogous manner during signaling, nucleating MyD88TIR assemblies unidirectionally.
真核生物 TIR（Toll/白细胞介素-1 受体蛋白）结构域通过 TIR-TIR 相互作用发出信号，这种作用可以是自我结合，也可以是与其他 TIR 结构域相互作用。在哺乳动物中，Toll 样受体（Toll-like receptors，TLRs）和参与促炎信号转导的细胞质适配蛋白中都有 TIR 结构域。以前的研究发现，MAL TIR 结构域（MALTIR）能在体外将 MyD88TIR 组装成晶体阵列。之前已经解决了 MyD88TIR 组装的微晶电子衍射（MicroED）结构，揭示了 TIR 结构域的双链高阶组装。这项研究证明，TLR2 的 TIR 结构域（据报道它与 TLR1 或 TLR6 组成异源二聚体）能在体外诱导 MyD88TIR 晶体高阶组装体的形成，而 TLR1TIR 和 TLR6TIR 则不能。利用改进的数据收集方案，确定了 TLR2TIR 诱导的 MyD88TIR 微晶体的 MicroED 结构，与之前 MALTIR 诱导的 MyD88TIR 组装结构相比，分辨率更高（2.85 Å），完整性更高（89%）。与它们的单体结构相比，这两种组装体在对信号传导很重要的几个区域（如 BB 环和 CD 环）都表现出构象差异。这些数据表明，TLR2TIR 和 MALTIR 在信号传导过程中以类似的方式与 MyD88 相互作用，单向核化 MyD88TIR 组装。
{"title":"Microcrystal electron diffraction structure of Toll-like receptor 2 TIR-domain-nucleated MyD88 TIR-domain higher-order assembly.","authors":"Y Li,L C Pacoste,W Gu,S J Thygesen,K J Stacey,T Ve,B Kobe,H Xu,J D Nanson","doi":"10.1107/s2059798324008210","DOIUrl":"https://doi.org/10.1107/s2059798324008210","url":null,"abstract":"Eukaryotic TIR (Toll/interleukin-1 receptor protein) domains signal via TIR-TIR interactions, either by self-association or by interaction with other TIR domains. In mammals, TIR domains are found in Toll-like receptors (TLRs) and cytoplasmic adaptor proteins involved in pro-inflammatory signaling. Previous work revealed that the MAL TIR domain (MALTIR) nucleates the assembly of MyD88TIR into crystalline arrays in vitro. A microcrystal electron diffraction (MicroED) structure of the MyD88TIR assembly has previously been solved, revealing a two-stranded higher-order assembly of TIR domains. In this work, it is demonstrated that the TIR domain of TLR2, which is reported to signal as a heterodimer with either TLR1 or TLR6, induces the formation of crystalline higher-order assemblies of MyD88TIR in vitro, whereas TLR1TIR and TLR6TIR do not. Using an improved data-collection protocol, the MicroED structure of TLR2TIR-induced MyD88TIR microcrystals was determined at a higher resolution (2.85 Å) and with higher completeness (89%) compared with the previous structure of the MALTIR-induced MyD88TIR assembly. Both assemblies exhibit conformational differences in several areas that are important for signaling (for example the BB loop and CD loop) compared with their monomeric structures. These data suggest that TLR2TIR and MALTIR interact with MyD88 in an analogous manner during signaling, nucleating MyD88TIR assemblies unidirectionally.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"4 1","pages":"699-712"},"PeriodicalIF":0.0,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142233305","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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