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Improved joint X-ray and neutron refinement procedure in Phenix. 改进了Phenix的X射线和中子联合细化程序。
IF 2.2 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-12-01 Epub Date: 2023-11-09 DOI: 10.1107/S2059798323008914
Dorothee Liebschner, Pavel V Afonine, Billy K Poon, Nigel W Moriarty, Paul D Adams

Neutron diffraction is one of the three crystallographic techniques (X-ray, neutron and electron diffraction) used to determine the atomic structures of molecules. Its particular strengths derive from the fact that H (and D) atoms are strong neutron scatterers, meaning that their positions, and thus protonation states, can be derived from crystallographic maps. However, because of technical limitations and experimental obstacles, the quality of neutron diffraction data is typically much poorer (completeness, resolution and signal to noise) than that of X-ray diffraction data for the same sample. Further, refinement is more complex as it usually requires additional parameters to describe the H (and D) atoms. The increase in the number of parameters may be mitigated by using the `riding hydrogen' refinement strategy, in which the positions of H atoms without a rotational degree of freedom are inferred from their neighboring heavy atoms. However, this does not address the issues related to poor data quality. Therefore, neutron structure determination often relies on the presence of an X-ray data set for joint X-ray and neutron (XN) refinement. In this approach, the X-ray data serve to compensate for the deficiencies of the neutron diffraction data by refining one model simultaneously against the X-ray and neutron data sets. To be applicable, it is assumed that both data sets are highly isomorphous, and preferably collected from the same crystals and at the same temperature. However, the approach has a number of limitations that are discussed in this work by comparing four separately re-refined neutron models. To address the limitations, a new method for joint XN refinement is introduced that optimizes two different models against the different data sets. This approach is tested using neutron models and data deposited in the Protein Data Bank. The efficacy of refining models with H atoms as riding or as individual atoms is also investigated.

中子衍射是用于确定分子原子结构的三种晶体学技术(X射线、中子和电子衍射)之一。它的特殊强度源于H(和D)原子是强中子散射体这一事实,这意味着它们的位置以及质子化状态可以从晶体学图中得出。然而,由于技术限制和实验障碍,中子衍射数据的质量通常比同一样品的X射线衍射数据差得多(完整性、分辨率和信噪比)。此外,细化更为复杂,因为它通常需要额外的参数来描述H(和D)原子。参数数量的增加可以通过使用“骑氢”细化策略来缓解,在该策略中,没有旋转自由度的H原子的位置是从它们相邻的重原子推断出来的。然而,这并不能解决与数据质量差有关的问题。因此,中子结构的确定通常依赖于X射线数据集的存在,用于联合X射线和中子(XN)细化。在这种方法中,X射线数据通过对X射线和中子数据集同时细化一个模型来弥补中子衍射数据的不足。为了适用,假设两个数据集都是高度同晶的,并且优选地从相同的晶体和在相同的温度下收集。然而,该方法有许多局限性,本工作通过比较四个单独重新精炼的中子模型来讨论这些局限性。为了解决这些限制,引入了一种新的联合XN精化方法,该方法针对不同的数据集优化两个不同的模型。该方法使用中子模型和蛋白质数据库中存储的数据进行了测试。还研究了以H原子为骑行原子或单个原子的精炼模型的有效性。
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引用次数: 0
Structural and functional characterization of the novel endo-α(1,4)-fucoidanase Mef1 from the marine bacterium Muricauda eckloniae. 海洋细菌Muricauda eckloniae新型内切-α(1,4)-岩藻糖苷酶Mef1的结构和功能表征
IF 2.2 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-11-01 Epub Date: 2023-10-25 DOI: 10.1107/S2059798323008732
Maria Dalgaard Mikkelsen, Vy Ha Nguyen Tran, Sebastian Meier, Thuan Thi Nguyen, Jesper Holck, Hang Thi Thuy Cao, Tran Thi Thanh Van, Pham Duc Thinh, Anne S Meyer, Jens Preben Morth

Fucoidanases (EC 3.2.1.-) catalyze the hydrolysis of glycosidic bonds between fucose residues in fucoidans. Fucoidans are a compositionally and structurally diverse class of fucose-containing sulfated polysaccharides that are primarily found in brown seaweeds. Here, the structural characterization of a novel endo-α(1,4)-fucoidanase, Mef1, from the marine bacterium Muricauda eckloniae is presented, showing sequence similarity to members of glycoside hydrolase family 107. Using carbohydrate polyacrylamide gel electrophoresis and nuclear magnetic resonance analyses, it is shown that the fucoidanase Mef1 catalyzes the cleavage of α(1,4)-linkages between fucose residues sulfated on C2 in the structure [-3)-α-L-Fucp2S-(1,4)-α-L-Fucp2S-(1-]n in fucoidan from Fucus evanescens. Kinetic analysis of Mef1 activity by Fourier transform infrared spectroscopy revealed that the specific Mef1 fucoidanase activity (Uf) on F. evanescens fucoidan was 0.1 × 10-3 Uf µM-1. By crystal structure determination of Mef1 at 1.8 Å resolution, a single-domain organization comprising a (β/α)8-barrel domain was determined. The active site was in an extended, positively charged groove that is likely to be designed to accommodate the binding of the negatively charged, sulfated fucoidan substrate. The active site of Mef1 comprises the amino acids His270 and Asp187, providing acid/base and nucleophile groups, respectively, for the hydrolysis of glycosidic bonds in the fucoidan backbone. Electron densities were identified for two possible Ca2+ ions in the enzyme, one of which is partially exposed to the active-site groove, while the other is very tightly coordinated. A water wire was discovered leading from the exterior of the Mef1 enzyme into the active site, passing the tightly coordinated Ca2+ site.

岩藻糖苷酶(EC 3.2.1.-)催化岩藻糖苷中岩藻糖残基之间糖苷键的水解。岩藻糖胶是一类主要存在于棕色海藻中的含有岩藻糖的硫酸多糖,其组成和结构各不相同。本文介绍了一种新的内源性α(1,4)-岩藻糖苷酶Mef1的结构特征,该酶来自海洋细菌埃科木霉,与糖苷水解酶家族107的成员具有序列相似性。利用碳水化合物聚丙烯酰胺凝胶电泳和核磁共振分析,结果表明,岩藻糖胶酶Mef1催化埃文氏褐藻糖胶中结构[-3)-α-L-Fucp2S-(1,4)-α-L-Fucp2S-(1-]n中C2上硫酸盐化的岩藻糖残基之间的α(1,4 Uf µM-1.在1.8下通过晶体结构测定Mef1 Å分辨率,确定了包含(β/α)8桶结构域的单结构域组织。活性位点位于一个延伸的带正电荷的凹槽中,该凹槽可能被设计为适应带负电荷的硫酸岩藻糖胶底物的结合。Mef1的活性位点包括氨基酸His270和Asp187,分别为岩藻糖胶主链中的糖苷键的水解提供酸/碱和亲核基团。确定了酶中两种可能的Ca2+离子的电子密度,其中一种部分暴露于活性位点凹槽,而另一种则非常紧密地配位。发现一根水管线从Mef1酶的外部进入活性位点,穿过紧密配位的Ca2+位点。
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引用次数: 0
Atypical homodimerization revealed by the structure of the (S)-enantioselective haloalkane dehalogenase DmmarA from Mycobacterium marinum. 海洋分枝杆菌(S)-对映选择性卤代烷烃脱卤酶DmmarA的结构揭示了非典型的同源二聚。
IF 2.6 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-11-01 Epub Date: 2023-10-20 DOI: 10.1107/S2059798323006642
Karolina Snajdarova, Sérgio M Marques, Jiri Damborsky, David Bednar, Martin Marek

Haloalkane dehalogenases (HLDs) are a family of α/β-hydrolase fold enzymes that employ SN2 nucleophilic substitution to cleave the carbon-halogen bond in diverse chemical structures, the biological role of which is still poorly understood. Atomic-level knowledge of both the inner organization and supramolecular complexation of HLDs is thus crucial to understand their catalytic and noncatalytic functions. Here, crystallographic structures of the (S)-enantioselective haloalkane dehalogenase DmmarA from the waterborne pathogenic microbe Mycobacterium marinum were determined at 1.6 and 1.85 Å resolution. The structures show a canonical αβα-sandwich HLD fold with several unusual structural features. Mechanistically, the atypical composition of the proton-relay catalytic triad (aspartate-histidine-aspartate) and uncommon active-site pocket reveal the molecular specificities of a catalytic apparatus that exhibits a rare (S)-enantiopreference. Additionally, the structures reveal a previously unobserved mode of symmetric homodimerization, which is predominantly mediated through unusual L5-to-L5 loop interactions. This homodimeric association in solution is confirmed experimentally by data obtained from small-angle X-ray scattering. Utilizing the newly determined structures of DmmarA, molecular modelling techniques were employed to elucidate the underlying mechanism behind its uncommon enantioselectivity. The (S)-preference can be attributed to the presence of a distinct binding pocket and variance in the activation barrier for nucleophilic substitution.

卤代烷烃脱卤酶(HLDs)是一类α/β-水解酶折叠酶,利用SN2亲核取代来切割不同化学结构中的碳-卤键,其生物学作用仍知之甚少。因此,了解HLD的内部组织和超分子络合的原子水平知识对于理解其催化和非催化功能至关重要。在这里,水性致病微生物海洋分枝杆菌的(S)-对映选择性卤代烷烃脱卤酶DmmarA的晶体结构在1.6和1.85下测定 Å分辨率。该结构显示出典型的αβα三明治HLD折叠,具有一些不寻常的结构特征。从机理上讲,质子中继催化三联体(天冬氨酸组氨酸天冬氨酸)的非典型组成和罕见的活性位点口袋揭示了催化装置的分子特异性,该催化装置表现出罕见的(S)-对映体参比。此外,这些结构揭示了一种以前未观察到的对称同源二聚模式,它主要通过不寻常的L5-L5环相互作用介导。通过小角度X射线散射获得的数据,实验证实了溶液中的这种同二聚体缔合。利用新确定的DmmarA结构,采用分子建模技术来阐明其不同寻常的对映选择性背后的潜在机制。(S)-偏好可归因于存在明显的结合口袋和亲核取代的活化势垒的变化。
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引用次数: 0
Domain structure and cross-linking in a giant adhesin from the Mobiluncus mulieris bacterium. 一种巨大黏着蛋白的结构域结构和交联。
IF 2.2 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-11-01 Epub Date: 2023-10-20 DOI: 10.1107/S2059798323007507
Paul G Young, Jacob M Paynter, Julia K Wardega, Martin J Middleditch, Leo S Payne, Edward N Baker, Christopher J Squire

Cell-surface proteins known as adhesins enable bacteria to colonize particular environments, and in Gram-positive bacteria often contain autocatalytically formed covalent intramolecular cross-links. While investigating the prevalence of such cross-links, a remarkable example was discovered in Mobiluncus mulieris, a pathogen associated with bacterial vaginosis. This organism encodes a putative adhesin of 7651 residues. Crystallography and mass spectrometry of two selected domains, and AlphaFold structure prediction of the remainder of the protein, were used to show that this adhesin belongs to the family of thioester, isopeptide and ester-bond-containing proteins (TIE proteins). It has an N-terminal domain homologous to thioester adhesion domains, followed by 51 immunoglobulin (Ig)-like domains containing ester- or isopeptide-bond cross-links. The energetic cost to the M. mulieris bacterium in retaining such a large adhesin as a single gene or protein construct suggests a critical role in pathogenicity and/or persistence.

被称为粘附素的细胞表面蛋白使细菌能够在特定环境中定植,在革兰氏阳性细菌中,通常含有自催化形成的共价分子内交联。在调查这种交叉连接的流行率时,在与细菌性阴道病相关的病原体穆氏弯孢中发现了一个显著的例子。该生物体编码一个假定的7651个残基的粘附素。使用两个选定结构域的晶体学和质谱分析,以及蛋白质其余部分的AlphaFold结构预测,表明这种粘附素属于硫酯、异肽和含酯键的蛋白质(TIE蛋白质)家族。它有一个与硫酯粘附结构域同源的N端结构域,然后是51个含有酯键或异肽键交联的免疫球蛋白(Ig)样结构域。在保留如此大的粘附素作为单个基因或蛋白质构建体方面,M.mulieris细菌的能量成本表明其在致病性和/或持久性中起着关键作用。
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引用次数: 0
Cocrystallization of ubiquitin-deubiquitinase complexes through disulfide linkage. 泛素-去泛素酶复合物通过二硫键共结晶。
IF 2.6 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-11-01 Epub Date: 2023-10-25 DOI: 10.1107/S2059798323008501
Kristos I Negron Teron, Chittaranjan Das

Structural characterization of the recognition of ubiquitin (Ub) by deubiquitinases (DUBs) has largely relied on covalent complexation of the DUB through its catalytic cysteine with a Ub C-terminal electrophile. The Ub electrophiles are accessed through intein chemistry in conjunction with chemical synthesis. Here, it was asked whether DUB-Ub covalent complexes could instead be accessed by simpler disulfide chemistry using a Ub cysteine mutant in which the last glycine has been replaced with a cysteine. The Ub cysteine mutant displayed a wide variability in disulfide formation across a panel of eukaryotic and prokaryotic DUBs, with some showing no detectable reaction while others robustly produced a disulfide complex. Using this approach, two disulfide-linked ubiquitin-bound complexes were crystallized, one involving the Legionella pneumophila effector SdeA DUB and the other involving the Orientia effector OtDUB. These DUBs had previously been crystallized in Ub-bound forms using the C-terminal electrophile strategy and noncovalent complexation, respectively. While the disulfide-linked SdeA DUB-Ub complex crystallized as expected, in the OtDUB complex the disulfide bond to the Ub mutant involved a cysteine that differed from the catalytic cysteine. Disulfide formation with the SdeA DUB catalytic cysteine was accompanied by local distortion of the helix carrying the active-site cysteine, whereas OtDUB reacted with the Ub mutant using a surface-exposed cysteine.

去泛素酶(DUBs)识别泛素(Ub)的结构表征在很大程度上依赖于DUB通过其催化半胱氨酸与Ub C-末端亲电试剂的共价络合。Ub亲电试剂是通过内部化学结合化学合成获得的。在这里,有人问DUB-Ub共价复合物是否可以通过更简单的二硫化物化学来获得,使用Ub半胱氨酸突变体,其中最后一个甘氨酸被半胱氨酸取代。Ub半胱氨酸突变体在一组真核和原核DUB中显示出二硫化物形成的广泛可变性,其中一些没有显示出可检测的反应,而另一些则强烈产生二硫化物复合物。使用这种方法,结晶了两个二硫键连接的泛素结合复合物,一个涉及嗜肺军团菌效应子SdeA-DUB,另一个涉及Orientia效应子OtDUB。这些DUB之前分别使用C末端亲电策略和非共价络合以Ub结合的形式结晶。虽然二硫键连接的SdeA-DUB-Ub复合物如预期结晶,但在OtDUB复合物中,与Ub突变体的二硫键涉及不同于催化半胱氨酸的半胱氨酸。SdeA-DUB催化半胱氨酸形成的二硫化物伴随着携带活性位点半胱氨酸的螺旋的局部畸变,而OtDUB使用表面暴露的半胱氨酸与Ub突变体反应。
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引用次数: 0
Structural analysis of wild-type and Val120Thr mutant Candida boidinii formate dehydrogenase by X-ray crystallography. 野生型和Val120Thr突变型博伊迪尼念珠菌甲酸脱氢酶的X射线晶体学结构分析。
IF 2.6 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-11-01 Epub Date: 2023-10-20 DOI: 10.1107/S2059798323008070
Mehmet Gul, Busra Yuksel, Huri Bulut, Hasan DeMirci

Candida boidinii NAD+-dependent formate dehydrogenase (CbFDH) has gained significant attention for its potential application in the production of biofuels and various industrial chemicals from inorganic carbon dioxide. The present study reports the atomic X-ray crystal structures of wild-type CbFDH at cryogenic and ambient temperatures, as well as that of the Val120Thr mutant at cryogenic temperature, determined at the Turkish Light Source `Turkish DeLight'. The structures reveal new hydrogen bonds between Thr120 and water molecules in the active site of the mutant CbFDH, suggesting increased stability of the active site and more efficient electron transfer during the reaction. Further experimental data is needed to test these hypotheses. Collectively, these findings provide invaluable insights into future protein-engineering efforts that could potentially enhance the efficiency and effectiveness of CbFDH.

布氏假丝酵母NAD+依赖性甲酸脱氢酶(CbFDH)因其在利用无机二氧化碳生产生物燃料和各种工业化学品方面的潜在应用而备受关注。本研究报道了在土耳其光源“Turkish DeLight”测定的野生型CbFDH在低温和环境温度下的原子X射线晶体结构,以及Val120Thr突变体在低温下的原子X-射线晶体结构。这些结构揭示了突变体CbFDH活性位点中Thr120和水分子之间的新氢键,表明活性位点的稳定性增加,反应过程中电子转移更有效。需要进一步的实验数据来检验这些假设。总之,这些发现为未来的蛋白质工程工作提供了宝贵的见解,有可能提高CbFDH的效率和有效性。
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引用次数: 0
Crystal structures of the DExH-box RNA helicase DHX9. DExH-box RNA解旋酶DHX9。
IF 2.2 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-11-01 Epub Date: 2023-10-20 DOI: 10.1107/S2059798323007611
Young Tae Lee, E Allen Sickmier, Simina Grigoriu, Jennifer Castro, P Ann Boriack-Sjodin

DHX9 is a DExH-box RNA helicase with versatile functions in transcription, translation, RNA processing and regulation of DNA replication. DHX9 has recently emerged as a promising target for oncology, but to date no mammalian structures have been published. Here, crystal structures of human, dog and cat DHX9 bound to ADP are reported. The three mammalian DHX9 structures share identical structural folds. Additionally, the overall architecture and the individual domain structures of DHX9 are highly conserved with those of MLE, the Drosophila orthologue of DHX9 previously solved in complex with RNA and a transition-state analogue of ATP. Due to differences in the bound substrates and global domain orientations, the localized loop conformations and occupancy of dsRNA-binding domain 2 (dsRBD2) differ between the mammalian DHX9 and MLE structures. The combined effects of the structural changes considerably alter the RNA-binding channel, providing an opportunity to compare active and inactive states of the helicase. Finally, the mammalian DHX9 structures provide a potential tool for structure-based drug-design efforts.

DHX9是一种DExH盒RNA解旋酶,在转录、翻译、RNA加工和DNA复制调控方面具有多种功能。DHX9最近成为肿瘤学的一个很有前途的靶点,但迄今为止还没有发表哺乳动物的结构。本文报道了人、狗和猫与ADP结合的DHX9的晶体结构。三种哺乳动物DHX9结构具有相同的结构折叠。此外,DHX9的整体结构和单个结构域结构与MLE的结构域和结构域结构域高度保守,MLE是DHX9以前与RNA和ATP的过渡态类似物在复合物中溶解的果蝇直系同源物。由于结合底物和全局结构域取向的差异,哺乳动物DHX9和MLE结构之间的dsRNA结合结构域2(dsRBD2)的定位环构象和占有率不同。结构变化的综合作用显著改变了RNA结合通道,为比较解旋酶的活性和非活性状态提供了机会。最后,哺乳动物DHX9结构为基于结构的药物设计工作提供了一个潜在的工具。
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引用次数: 0
Anaerobic fixed-target serial crystallography using sandwiched silicon nitride membranes. 使用夹层氮化硅膜的厌氧固定靶系列晶体学。
IF 2.6 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-11-01 DOI: 10.1107/S205979832300880X
Monika Bjelčić, Kajsa G V Sigfridsson Clauss, Oskar Aurelius, Mirko Milas, Jie Nan, Thomas Ursby

In recent years, the emergence of serial crystallography, initially pioneered at X-ray free-electron lasers (XFELs), has sparked a growing interest in collecting macromolecular crystallographic data at room temperature. Various fixed-target serial crystallography techniques have been developed, ranging from commercially available chips to in-house designs implemented at different synchrotron facilities. Nevertheless, there is currently no commercially available chip (known to the authors) specifically designed for the direct handling of oxygen-sensitive samples. This study presents a methodology employing silicon nitride chips arranged in a `sandwich' configuration, enabling reliable room-temperature data collection from oxygen-sensitive samples. The method involves the utilization of a custom-made 3D-printed assembling tool and a MX sample holder. To validate the effectiveness of the proposed method, deoxyhemoglobin and methemoglobin samples were investigated using the BioMAX X-ray macromolecular crystallography beamline, the Balder X-ray absorption spectroscopy beamline and UV-Vis absorption spectroscopy.

近年来,最初在X射线自由电子激光器(XFEL)上开创的系列晶体学的出现,引发了人们对在室温下收集大分子晶体学数据的兴趣。已经开发了各种固定目标系列结晶学技术,从商用芯片到在不同同步加速器设施中实现的内部设计。然而,目前还没有专门设计用于直接处理氧敏感样品的商用芯片(作者已知)。这项研究提出了一种方法,采用“三明治”配置的氮化硅芯片,能够从氧敏感样品中可靠地收集室温数据。该方法包括使用定制的3D打印组装工具和MX样品支架。为了验证所提出方法的有效性,使用BioMAX X射线大分子晶体学光束线、Balder X射线吸收光谱光束线和UV-Vis吸收光谱对脱氧血红蛋白和高铁血红蛋白样品进行了研究。
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引用次数: 0
The impact of molecular variants, crystallization conditions and the space group on ligand-protein complexes: a case study on bacterial phosphotriesterase. 分子变体、结晶条件和空间群对配体-蛋白质复合物的影响:细菌磷酸三酯酶的个案研究。
IF 2.6 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-11-01 Epub Date: 2023-10-20 DOI: 10.1107/S2059798323007672
Orly Dym, Nidhi Aggarwal, Yacov Ashani, Haim Leader, Shira Albeck, Tamar Unger, Shelly Hamer-Rogotner, Israel Silman, Dan S Tawfik, Joel L Sussman

A bacterial phosphotriesterase was employed as an experimental paradigm to examine the effects of multiple factors, such as the molecular constructs, the ligands used during protein expression and purification, the crystallization conditions and the space group, on the visualization of molecular complexes of ligands with a target enzyme. In this case, the ligands used were organophosphates that are fragments of the nerve agents and insecticides on which the enzyme acts as a bioscavenger. 12 crystal structures of various phosphotriesterase constructs obtained by directed evolution were analyzed, with resolutions of up to 1.38 Å. Both apo forms and holo forms, complexed with the organophosphate ligands, were studied. Crystals obtained from three different crystallization conditions, crystallized in four space groups, with and without N-terminal tags, were utilized to investigate the impact of these factors on visualizing the organophosphate complexes of the enzyme. The study revealed that the tags used for protein expression can lodge in the active site and hinder ligand binding. Furthermore, the space group in which the protein crystallizes can significantly impact the visualization of bound ligands. It was also observed that the crystallization precipitants can compete with, and even preclude, ligand binding, leading to false positives or to the incorrect identification of lead drug candidates. One of the co-crystallization conditions enabled the definition of the spaces that accommodate the substituents attached to the P atom of several products of organophosphate substrates after detachment of the leaving group. The crystal structures of the complexes of phosphotriesterase with the organophosphate products reveal similar short interaction distances of the two partially charged O atoms of the P-O bonds with the exposed β-Zn2+ ion and the buried α-Zn2+ ion. This suggests that both Zn2+ ions have a role in stabilizing the transition state for substrate hydrolysis. Overall, this study provides valuable insights into the challenges and considerations involved in studying the crystal structures of ligand-protein complexes, highlighting the importance of careful experimental design and rigorous data analysis in ensuring the accuracy and reliability of the resulting phosphotriesterase-organophosphate structures.

采用细菌磷酸三酯酶作为实验范式,研究多种因素对配体与靶酶的分子复合物可视化的影响,如分子结构、蛋白质表达和纯化过程中使用的配体、结晶条件和空间群。在这种情况下,使用的配体是有机磷酸酯,它是神经毒剂和杀虫剂的片段,酶在其上起到生物洞穴的作用。分析了通过定向进化获得的各种磷酸三酯酶构建体的12个晶体结构,分辨率高达1.38 Å。研究了apo形式和holo形式与有机磷配体的络合。从三种不同的结晶条件中获得的晶体,在四个空间群中结晶,具有和不具有N-末端标签,用于研究这些因素对酶的有机磷复合物的可视化的影响。研究表明,用于蛋白质表达的标签可以滞留在活性位点并阻碍配体结合。此外,蛋白质结晶的空间组可以显著影响结合配体的可视化。还观察到结晶沉淀剂可以与配体结合竞争,甚至阻止配体结合,导致假阳性或导致候选先导药物的错误识别。共结晶条件之一使得能够定义空间,该空间容纳在脱离基团后连接到有机磷酸盐底物的几种产物的P原子上的取代基。磷酸三酯酶与有机磷产物的配合物的晶体结构显示,P-O键的两个部分带电的O原子与暴露的β-Zn2+离子和掩埋的α-Zn2+阳离子的短相互作用距离相似。这表明两种Zn2+离子都在稳定底物水解的过渡态中发挥作用。总的来说,这项研究为研究配体-蛋白质复合物的晶体结构所涉及的挑战和考虑因素提供了有价值的见解,强调了仔细的实验设计和严格的数据分析在确保所得磷酸三酯酶-有机磷结构的准确性和可靠性方面的重要性。
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引用次数: 0
Structural basis for the allosteric pathway of 4-amino-4-deoxychorismate synthase. 4-氨基-4-脱氧氯合酶变构途径的结构基础。
IF 2.2 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-10-01 Epub Date: 2023-09-15 DOI: 10.1107/S2059798323006320
Yusuke Nakamichi, Jyumpei Kobayashi, Koichi Toyoda, Masako Suda, Kazumi Hiraga, Masayuki Inui, Masahiro Watanabe

4-Amino-4-deoxychorismate synthase (ADCS), a chorismate-utilizing enzyme, is composed of two subunits: PabA and PabB. PabA is a glutamine amidotransferase that hydrolyzes glutamine into glutamate and ammonia. PabB is an aminodeoxychorismate synthase that converts chorismate to 4-amino-4-deoxychorismate (ADC) using the ammonia produced by PabA. ADCS functions under allosteric regulation between PabA and PabB. However, the allosteric mechanism remains unresolved because the structure of the PabA-PabB complex has not been determined. Here, the crystal structure and characterization of PapA from Streptomyces venezuelae (SvPapA), a bifunctional enzyme comprising the PabA and PabB domains, is reported. SvPapA forms a unique dimer in which PabA and PabB domains from different monomers complement each other and form an active structure. The chorismate-bound structure revealed that recognition of the C1 carboxyl group by Thr501 and Gly502 of the 498-PIKTG-502 motif in the PabB domain is essential for the catalytic Lys500 to reach the C2 atom, a reaction-initiation site. SvPapA demonstrated ADCS activity in the presence of Mg2+ when glutamate or NH+4 was used as the amino donor. The crystal structure indicated that the Mg2+-binding position changed depending on the binding of chorismate. In addition, significant structural changes were observed in the PabA domain depending on the presence or absence of chorismate. This study provides insights into the structural factors that are involved in the allosteric regulation of ADCS.

4-氨基-4-脱氧合唱酸合成酶(ADCS)是一种利用合唱酸的酶,由两个亚基组成:PabA和PabB。PabA是一种谷氨酰胺酰胺转移酶,可将谷氨酰胺水解为谷氨酸和氨。PabB是一种氨基脱氧氯合酶,利用PabA产生的氨将氯合酶转化为4-氨基-4-脱氧氯合物(ADC)。ADCS在PabA和PabB之间的变构调节下发挥作用。然而,由于PabA-PabB复合物的结构尚未确定,变构机制仍未解决。本文报道了委内瑞拉链霉菌(SvPapA)的PapA的晶体结构和性质,该酶是一种包含PabA和PabB结构域的双功能酶。SvPapA形成一种独特的二聚体,其中来自不同单体的PabA和PabB结构域相互补充并形成活性结构。合唱结合结构表明,Thr501和Gly502对PabB结构域中498-PIKTG-502基序的C1羧基的识别对于催化Lys500到达C2原子(反应起始位点)至关重要。当谷氨酸或NH+4用作氨基供体时,SvPapA在Mg2+存在下表现出ADCS活性。晶体结构表明,Mg2+的结合位置随氯离子的结合而变化。此外,在PabA结构域中观察到显著的结构变化,这取决于是否存在chorismate。这项研究提供了对参与ADCS变构调节的结构因素的见解。
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Acta Crystallographica. Section D, Structural Biology
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