Journal of Molecular Biology最新文献_第8页

Structural and Biochemical Insights into the Broad-Spectrum TET Enzyme From Methanocaldococcus jannaschii Reveal the Basis of Substrate Specificity in M42 Aminopeptidases jannaschii甲醇钙球菌广谱TET酶的结构和生化分析揭示了M42氨基肽酶底物特异性的基础。

IF 4.5 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2025-12-17 DOI: 10.1016/j.jmb.2025.169596

Joaquin Atalah , Hind Basbous , Gregory Effantin , Sylvie Kieffer-Jaquinod , Guy Schoehn , Eric Girard , Bruno Franzetti

TET peptidases of the M42 family are ∼500 kDa hollow dodecameric complexes ubiquitous in prokaryotes. These enzymes act as strict aminopeptidases, catalyzing the removal of N-terminal amino acids from peptides. A common feature of M42 TET aminopeptidases characterized to date is their marked substrate preference for a limited subset of amino acids. Unlike other hyperthermophilic archaea studied so far, the autotrophic archaeon Methanocaldococcus jannaschii possesses only a single gene encoding an M42 peptidase. This enzyme, named MjTET, is the first reported M42 peptidase to exhibit broad amino acid specificity, including activity on aromatic residues. To assess their peptide degradation efficiencies, the catalytic constants of MjTET were compared to those of its close analogs from Pyrococcus horikoshii. The specialized TETs from P. horikoshii displayed higher catalytic efficiencies than the generalist MjTET, likely reflecting the reliance of Thermococcales on peptide fermentation for energy. Additionally, the structure of MjTET was resolved to 3 Å using cryo-EM and compared with the available models of the four P. horikoshii TETs to identify features underlying substrate specificity. This analysis, combined with mutagenesis studies, revealed a previously uncharacterized loop in the catalytic domain that contributes to substrate discrimination. Collectively, these findings show that substrate specificity in TET enzymes arises from a complex interplay of tertiary structure, oligomeric assembly, and electrostatic surface potential.

Importance

This study first reported a novel TET peptidase from Methanogenic hyperthermophilic archaea. Its enzymatic properties compared to the specialized TET enzyme characterized so far from heterotrophic archaea suggest a link with autotrophy. It also represents an important step in explaining the structural features guiding substrate specificity.

M42家族的TET肽酶是在原核生物中普遍存在的约500 kDa的空心十二聚体复合物。这些酶作为严格的氨基肽酶，催化从肽中去除n端氨基酸。迄今为止表征的M42 TET氨基肽酶的一个共同特征是它们对有限子集氨基酸的显着底物偏好。与迄今为止研究的其他超嗜热古细菌不同，自养古细菌jannaschii甲烷钙球菌只有一个编码M42肽酶的基因。这种酶被命名为MjTET，是第一个报道的具有广泛氨基酸特异性的M42肽酶，包括对芳香残基的活性。为了评估它们的肽降解效率，将MjTET的催化常数与其同源同源物堀井焦球菌的催化常数进行了比较。来自P. horikoshii的特殊tet表现出比通用的MjTET更高的催化效率，可能反映了热球菌对肽发酵能量的依赖。此外，使用冷冻电镜将MjTET的结构分解为3 Å，并将其与四种P. horikoshii tet的现有模型进行比较，以确定底物特异性的特征。该分析与诱变研究相结合，揭示了催化域中以前未表征的环，有助于底物区分。总的来说，这些发现表明TET酶的底物特异性来自三级结构、寡聚物组装和静电表面电位的复杂相互作用。本研究首次报道了从产甲烷的嗜热古菌中提取的一种新的TET肽酶。与迄今为止从异养古菌中鉴定出的特异性TET酶相比，它的酶特性表明它与自养有关。它也代表了解释指导底物特异性的结构特征的重要一步。

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引用次数: 0

Missense3D-PTMdb: A Web Tool for Visualising and Exploring Human Genetic Variants and Post-translational Modification Sites Using AlphaFold Models. Missense3D-PTMdb：一个使用AlphaFold模型可视化和探索人类遗传变异和翻译后修饰位点的网络工具。

IF 4.5 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2025-12-16 DOI: 10.1016/j.jmb.2025.169595

Haotian Zhao, Ryan Pye, Grace Walker, Wendy Tran, Olivia Simmonds, Ifigenia Tsitsa, Suhail Islam, Gordon Hanna, Alessia David

Only a fraction of the >11 million missense variants identified in humans has a known clinical significance. Post-translational modifications (PTMs), such as phosphorylation, glycosylation and ubiquitination, are key regulators of protein function and structure. PTMs depend on correct protein folding and the recognition and binding of enzymes to specific amino acid motifs near modification sites. AlphaFold models provide an unprecedented opportunity to explore variants on 3D structures, enabling systematic identification of amino acid substitutions that could affect PTMs and should be further investigated experimentally. We present Missense3D-PTMdb, a "one-stop-shop" interactive web tool that provides a user-friendly sequence-structure mapping of 20,235 human proteins, 11,5 million naturally occurring human missense variants, >60 PTM types and 203,775 PTM residues and their neighbours in sequence and 3D structure space using AlphaFold models of the human proteome. The resource also supports visualisation of novel variants not in the database. Missense3D-PTMdb is freely available at https://missense3d.bc.ic.ac.uk/ptmdb.

在人类中发现的1,1100万个错义变异中，只有一小部分具有已知的临床意义。翻译后修饰（PTMs），如磷酸化、糖基化和泛素化，是蛋白质功能和结构的关键调节因子。PTMs依赖于正确的蛋白质折叠和酶对修饰位点附近特定氨基酸基序的识别和结合。AlphaFold模型为探索3D结构的变异提供了前所未有的机会，能够系统地识别可能影响PTMs的氨基酸取代，并应进一步进行实验研究。我们提出了Missense3D-PTMdb，一个“一站式”交互式网络工具，使用人类蛋白质组的AlphaFold模型，提供了20,235个人类蛋白质，11,150万个自然存在的人类错义变体，bbbb60个PTM类型和203,775个PTM残基及其邻近序列和3D结构空间的用户友好的序列结构映射。该资源还支持数据库中没有的新变体的可视化。Missense3D-PTMdb可在https://missense3d.bc.ic.ac.uk/ptmdb免费获得。

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引用次数: 0

MASI2.0: Insights of Microbiota Metabolic Potential from Incorporating Genomic Information on Microbiota-Active Substance Interactions MASI2.0：结合微生物群-活性物质相互作用基因组信息的微生物群代谢潜力的见解。

IF 4.5 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2025-12-16 DOI: 10.1016/j.jmb.2025.169594

Dongyue Hou , Yuchen Liu , Hanbo Lin , Jiajie Gu , Juncheng Qian , Shaofei Wang , Yuzong Chen , Dianwen Ju , Xian Zeng

Extensive interactions between microbiota and active substances are health- and disease-relevant. Mechanistic understanding from genomic perspective of these interactions and potential impacts is important for biomedical and pharmaceutical research. However, current data repositories often lack systematic integration from a genomic perspective. Here we describe an update of the MASI microbiota-active substance interactions database. This update includes new data of (1) genomic-derived 166,766 microbiota-drug interactions and 205,505 microbiota-food interactions linked by 415 biosynthetic gene clusters (BGCs), 59 metabolic gene clusters (MGCs), and 7250 genome-scale metabolic network models (GEMs) of ∼1200 microbiota species, and (2) 1848 microbiota-microbiota interaction records mediated by 39 quorum sensing languages, and (3) 46,717 microbiota-disease associations between 640 species and 59 diseases. Overall, this update provides 44,643 interasctions derived from ∼2000 publications and 380,571 genome-derived interactions, covering 1867 microbe species, 1576 therapeutic substances, 357 dietary substances, which is freely accessible at https://www.aiddlab.com/MASI2025/index.html.

微生物群和活性物质之间的广泛相互作用与健康和疾病有关。从基因组的角度理解这些相互作用和潜在影响的机制对生物医学和药物研究具有重要意义。然而，从基因组的角度来看，目前的数据存储库往往缺乏系统的集成。在这里，我们描述了MASI微生物-活性物质相互作用数据库的更新。此次更新包括以下新数据：1)基因组衍生的166766种微生物-药物相互作用和205505种微生物-食物相互作用，这些相互作用由415个生物合成基因簇（bgc）、59个代谢基因簇（MGCs）和7250个基因组尺度代谢网络模型（GEMs）连接；2)由39种群体感应语言介导的1848种微生物-微生物相互作用记录；3)640种物种和59种疾病之间的46717种微生物-疾病关联。总的来说，本次更新提供了44643种相互作用，这些相互作用来自于2000篇出版物和380571种基因组相互作用，涵盖了1867种微生物，1576种治疗物质，357种膳食物质，可在https://www.aiddlab.com/MASI2025/index.html上免费获取。

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引用次数: 0

Rising Star: A Unified Mechanism of Plant NLR Immune Signaling Rising Star：植物NLR免疫信号的统一机制。

IF 4.5 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2025-12-12 DOI: 10.1016/j.jmb.2025.169592

Jijie Chai

Jijie Chai earned his Ph.D. in analytical chemistry from the Institute of Materia Medica, Chinese Academy of Medical Sciences, and conducted postdoctoral research at Princeton University. In 2004, he established his independent research group at National Institute of Biological Science (China), focusing on plant immune receptors, particularly nucleotide-binding leucine-rich repeat (NLR) proteins. NLRs are the largest family immune receptors that enable plants to detect a wide array of pathogen effectors and activate effector-triggered immunity (ETI). Despite their recognition diversity, they elicit remarkably conserved immune responses, a mechanistic puzzle for many years. Chai’s team achieved a landmark breakthrough by reconstituting and solving the cryo-EM structure of activated Arabidopsis coiled-coil NLR (CNL) ZAR1, revealing its assembly into a pentameric resistosome. The N-terminal helices of ZAR1 in the resistosome form a funnel-like structure, which was later confirmed as a Ca²⁺-permeable ion channel. These discoveries directly link NLR activation to Ca²⁺ influx, a central immune signaling event. Chai’s findings further showed that CNLs form structurally conserved resistosomes with intrinsic Ca2⁺-permeable channel activity. Notably, Chai discovered that TIR-domain NLRs (TNLs) also form resistosomes but function as NADases, producing small-molecule signals that activate downstream helper NLRs. These helper NLRs also assemble into Ca²⁺-conducting resistosomes. Together, these findings establish a unified mechanism in plant immunity, with diverse NLRs converge on resistosome formation that ultimately drives Ca²⁺ influx as a central hub of defense activation. This paradigm shift underscores the functional conservation of NLR-mediated immunity across plant species and has fundamentally reshaped the field of plant innate immunity.

柴继杰博士毕业于中国医学科学院药物研究所分析化学专业，并在普林斯顿大学进行博士后研究。2004年在中国国家生物科学研究所成立独立课题组，重点研究植物免疫受体，特别是核苷酸结合富亮氨酸重复序列（NLR）蛋白。nlr是最大的家族免疫受体，使植物能够检测广泛的病原体效应物并激活效应触发免疫（ETI）。尽管它们的识别多样性，但它们引起了非常保守的免疫反应，这是一个多年来的机制难题。Chai的团队通过重建和解决活化的拟南芥卷曲卷曲NLR (CNL) ZAR1的低温电镜结构，揭示了其组装成五聚体的抗性体，取得了里程碑式的突破。抗性小体中ZAR1的n端螺旋形成一个漏斗状结构，后来证实这是一个Ca2+渗透离子通道。这些发现直接将NLR激活与Ca2+内流联系起来，这是一种中枢免疫信号事件。Chai的研究结果进一步表明，cnl形成结构保守的抵抗体，具有内在的Ca2+渗透性通道活性。值得注意的是，Chai发现tir结构域NLRs （TNLs）也形成抵抗体，但作为nadase起作用，产生激活下游辅助NLRs的小分子信号。这些辅助性nlr也组装成Ca2+传导抵抗体。总之，这些发现在植物免疫中建立了一个统一的机制，不同的nlr聚集在抵抗体形成上，最终驱动Ca2+内流作为防御激活的中心枢纽。这种范式转变强调了nlr介导的免疫在植物物种中的功能保护，并从根本上重塑了植物先天免疫领域。

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引用次数: 0

Integrating Sequence, Structure, and Graph-based Features for Elucidating the Stability of Thermophilic Proteins. 整合序列、结构和基于图的特征来阐明嗜热蛋白的稳定性。

IF 4.5 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2025-12-12 DOI: 10.1016/j.jmb.2025.169593

P Ramakrishna Reddy, Fathima Ridha, M Michael Gromiha

Proteins from thermophilic organisms exhibit remarkable stability under extreme thermal conditions. Understanding the molecular mechanisms underlying thermostability is essential for studying protein evolution and engineering robust enzymes. In this study, we systematically analyzed four sets of mesophilic-thermophilic protein pairs to investigate the molecular basis of thermal adaptation. We have constructed independent datasets of mesophilic-thermophilic protein pairs defined by sequence identity and optimal growth temperature (OGT): (a) >90% identity and 60-80 °C OGT, (b) 50-90% identity and >80 °C OGT, (c) 50-90% identity and 60-80 °C OGT, and (d) 50-90% identity and 40-60 °C OGT. Mutational analysis revealed that thermophilic proteins consistently reduced polar, uncharged residues while enriching charged, hydrophobic, and aromatic residues, particularly in extreme thermophiles (>80 °C). Further, by integrating multiple known protein features into a hierarchical rule-based classifier, we identified the thermostable protein from a pair of sequences and also assessed the relative importance of features across datasets to provide interpretable insights into protein thermostability. The hierarchical rule-based method identified stabilizing residues as the primary distinguishing factor, followed by electrostatic energy, volume, and localized electrical effects, which correctly classified 99% of thermophilic proteins. A bagging model trained on the same features achieved a balanced accuracy of 92% in 5-fold cross-validation and 91% on the 20% hold-out test set. Furthermore, independent validation using multiple mutations in proteins accurately identified 94% of stabilizing and destabilizing mutations. The results obtained in this work provide valuable insights to understand the thermal adaptation of proteins and reliably identify thermostable proteins.

来自嗜热生物的蛋白质在极端热条件下表现出显著的稳定性。了解热稳定性的分子机制对于研究蛋白质进化和构建健壮酶至关重要。在这项研究中，我们系统地分析了四组中温-嗜热蛋白对，以探讨热适应的分子基础。我们构建了由序列一致性和最佳生长温度（OGT）定义的中温-嗜热蛋白对的独立数据集：(a) >90%一致性和60-80°C OGT， (b) 50-90%一致性和>80°C OGT, (C) 50-90%一致性和60-80°C OGT， (d) 50-90%一致性和40-60°C OGT。突变分析显示，在极端嗜热菌（bb0 ~ 80°C）中，嗜热蛋白持续减少极性、不带电的残基，同时富集带电、疏水和芳香残基。此外，通过将多个已知的蛋白质特征整合到基于分层规则的分类器中，我们从一对序列中确定了热稳定性蛋白质，并评估了数据集中特征的相对重要性，以提供对蛋白质热稳定性的可解释性见解。基于分层规则的方法将稳定残基作为主要区分因素，其次是静电能、体积和局部电效应，正确分类了99%的嗜热蛋白。在相同特征上训练的装袋模型在5倍交叉验证中实现了92%的平衡精度，在20%的保留测试集上实现了91%的平衡精度。此外，使用蛋白质中多个突变的独立验证准确地识别了94%的稳定和不稳定突变。本研究结果为了解蛋白质的热适应性和可靠地鉴定热稳定性蛋白质提供了有价值的见解。

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引用次数: 0

Cryo-EM of Cardiac AL-224L Amyloid Reveals Shared Structural Motifs and Mutation-induced Differences in λ6 Light Chain Fibrils 心脏AL-224L淀粉样蛋白的冷冻电镜显示了共同的结构基序和突变诱导的λ6轻链原纤维的差异。

IF 4.5 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2025-12-11 DOI: 10.1016/j.jmb.2025.169591

Chad W. Hicks , Tatiana Prokaeva , Brian Spencer , Shobini Jayaraman , Noorul Huda , Sherry Wong , Hui Chen , Vaishali Sanchorawala , Francesca Lavatelli , Olga Gursky

In light chain amyloidosis (AL), aberrant monoclonal antibody light chains (LCs) deposit in vital organs causing organ damage. Each AL patient features a unique LC; previous cryogenic electron microscopy (cryo-EM) studies revealed different amyloid structures in different AL patients. How LC mutations influence amyloid structures remains unclear. We report a cryo-EM structure of cardiac AL-224L amyloid (2.92 Å resolution) from λ6-LC family, which is overrepresented in AL amyloidosis. Comparison with λ6-LC structures from two other patients reveals similarities in amyloid folds, along with major differences caused by specific mutations. Differences in AL-224L include altered C-terminal conformation with an exposed surface forming an apparent ligand-binding site; an enlarged hydrophilic pore with orphan density; and altered steric zipper registry with backbone flipping, which likely represent general adaptive mechanisms in amyloids. The results reveal shared features in λ6-LC amyloid folds and suggest how mutation-induced structural changes influence amyloid-ligand interactions in a patient-specific manner.

在轻链淀粉样变性（AL）中，异常单克隆抗体轻链（LCs）沉积在重要器官中导致器官损伤。每个AL患者都有一个独特的LC；先前的低温电子显微镜（cryo-EM）研究显示不同AL患者的淀粉样蛋白结构不同。LC突变如何影响淀粉样蛋白结构尚不清楚。我们报道了来自λ6-LC家族的心脏AL- 224l淀粉样蛋白（2.92Å分辨率）的冷冻电镜结构，该淀粉样蛋白在AL淀粉样变性中过度代表。与其他两名患者的λ6-LC结构比较，发现淀粉样蛋白折叠相似，但特异性突变导致的主要差异。AL-224L的差异包括c端构象的改变，暴露的表面形成明显的配体结合位点；增大的亲水孔，具有孤儿密度；通过脊柱翻转改变了立体拉链注册，这可能代表了淀粉样蛋白的一般适应机制。结果揭示了λ6-LC淀粉样蛋白折叠的共同特征，并提示突变诱导的结构变化如何以患者特异性的方式影响淀粉样蛋白与配体的相互作用。

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引用次数: 0

Transcriptome-wide RNA Stability Across Cancers Reveals Therapeutic Vulnerabilities 转录组范围内的RNA稳定性图谱揭示了癌症的转录后控制和治疗脆弱性。

IF 4.5 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2025-12-08 DOI: 10.1016/j.jmb.2025.169590

Yang Tong , Yuting Wang , Gerui Liu , Yihu Wei , Xiaoxiao Yang , Jiapei Yuan , Yang Yang , Qiang Zhang

The steady-state abundance of mRNA is governed by the interplay between transcription and degradation, yet the contribution of RNA stability to cancer biology remains incompletely understood. Here, we systematically investigate RNA decay dynamics across 22 cancer types using RNA-seq data from the Cancer Cell Line Encyclopedia. By inferring transcriptome-wide RNA stability profiles, we identify distinct molecular subtypes defined by post-transcriptional regulation. Integrative analyses reveal that RNA-binding proteins (RBPs) and microRNAs (miRNAs), including SNRPA and RBMX, act as key modulators of RNA stability and are essential for cancer cell proliferation and survival. Somatic mutations, particularly those affecting miRNA binding sites, were found to significantly perturb RNA decay, implicating dysregulation of pathways such as nonsense-mediated decay. Furthermore, machine learning models demonstrate that RNA stability profiles predict sensitivity to 24 anticancer drugs, nominating specific RBPs as candidate biomarkers for therapeutic response. Collectively, our findings establish RNA stability as a pivotal layer of gene regulation in cancer, with broad implications for molecular stratification and precision oncology.

mRNA的稳定丰度是由转录和降解之间的相互作用决定的，但RNA稳定性对癌症生物学的贡献仍然不完全清楚。在这里，我们使用来自癌细胞系百科全书的RNA-seq数据系统地研究了22种癌症类型的RNA衰变动力学。通过推断转录组范围内的RNA稳定性谱，我们确定了由转录后调控定义的不同分子亚型。综合分析表明，RNA结合蛋白（rbp）和microRNAs (miRNAs)，包括SNRPA和RBMX，是RNA稳定性的关键调节剂，对癌细胞的增殖和生存至关重要。体细胞突变，特别是那些影响miRNA结合位点的突变，被发现会显著扰乱RNA衰变，这意味着无义介导的衰变等途径的失调。此外，机器学习模型表明，RNA稳定性谱可以预测对24种抗癌药物的敏感性，指定特定的rbp作为治疗反应的候选生物标志物。总的来说，我们的研究结果确立了RNA稳定性作为癌症基因调控的关键层，对分子分层和精确肿瘤学具有广泛的意义。

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引用次数: 0

Influence of Methionine Oxidation on Protein Stability and Association Studied by Free Energy Simulations 用自由能模拟研究了蛋氨酸氧化对蛋白质稳定性和结合体的影响。

IF 4.5 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2025-12-05 DOI: 10.1016/j.jmb.2025.169576

Tristan Alexander Mauck, Martin Zacharias

Cellular metabolic systems but also the extracellular environment can generate reactive oxygen species that lead to oxidation of methionine (MET) and interfere with protein folding and protein–protein association. The molecular mechanism of how MET oxidation (MEO) influences conformational stability and binding is not well understood. We employ alchemical free energy simulations to systematically study the influence of MET oxidation on protein–protein binding using the tetramerization domain of the tumor suppression protein p53 as a model system. A single MEO in one tetramerisation domain destabilizes the tetramer by ≈1.1–1.8 kcal/mol depending slightly on the MEO diastereomer. The simulations on double and triple oxidations reveal increased destabilization (≈3–7 kcal/mol) and significant cooperative effects depending on the relative position of the oxidized residues. The MET oxidation effects are of similar magnitude for the change in stability of the human prion protein (HPP) that served as a second model system and also agreed with available experimental data. The calculations predict a significant dependence of stability changes on the position of the MEO and also indicate non-additive effects of multiple oxidations which may play a role to protect proteins from oxidative damage and stress. Analysis of the Molecular Dynamics trajectories allowed us to interpret the oxidation effects in molecular detail. The simulation methodology could also serve as a general protocol to analyze single and multiple MET oxidations in other systems and its influence on protein binding and stability.

细胞代谢系统以及细胞外环境可以产生活性氧，导致蛋氨酸（MET）氧化并干扰蛋白质折叠和蛋白质-蛋白质结合。MET氧化（MEO）影响构象稳定性和结合的分子机制尚不清楚。我们采用炼金术自由能模拟系统地研究MET氧化对蛋白质-蛋白质结合的影响，使用肿瘤抑制蛋白p53的四聚域作为模型系统。在一个四聚域中，单个MEO使四聚体失稳约1.1-1.6 kcal/mol，这取决于MEO非对映体。双氧化和三氧化的模拟结果表明，随着氧化残基的相对位置的增加，不稳定性增加（≈3-7 kcal/mol），并产生显著的协同效应。作为第二个模型系统的人朊蛋白（HHP）的稳定性变化，MET氧化效应的幅度相似，也与现有的实验数据一致。计算预测了稳定性变化对MEO位置的显著依赖，并表明多重氧化的非加性效应可能在保护蛋白质免受氧化损伤和应激方面发挥作用。分子动力学轨迹的分析使我们能够从分子的细节上解释氧化效应。模拟方法也可以作为一般协议来分析其他系统中的单个和多个MET氧化及其对蛋白质结合和稳定性的影响。

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引用次数: 0

The Nuclease Domain of E. coli RecBCD Helicase Regulates DNA Binding and Base Pair Melting 大肠杆菌RecBCD解旋酶核酸酶结构域调控DNA结合和碱基对融化。

IF 4.5 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2025-12-05 DOI: 10.1016/j.jmb.2025.169571

Linxuan Hao, Rui Zhang, Timothy M. Lohman

E. coli RecBCD, a hetero-trimeric helicase and nuclease, functions in double stranded (ds) DNA break repair. RecBCD possesses ATPase motor domains within both RecB (3′–5′) and RecD (5′–3′) and a nuclease domain within RecB (RecB^Nuc). RecBCD binds to double stranded DNA ends and initiates DNA unwinding by first melting several DNA base pairs (bp) using only its binding free energy. The RecB^Nuc domain is docked ∼70 Å from the duplex DNA binding site in RecBCD-DNA structures but appears to be dynamic and able to move from its docked position. Here, we compare DNA binding of RecBCD and a variant, RecB^ΔNucCD, in which the 30 kDa nuclease domain has been deleted. RecBCD binding to a blunt DNA end is enthalpically unfavorable and entropically driven. Deletion of RecB^Nuc results in an increase in DNA binding affinity, suggesting an allosteric effect of RecB^Nuc. RecB^ΔNucCD binding to DNA possessing fully ‘pre-melted’ DNA ends is associated with a large favorable ΔH_obs, but much smaller than observed for RecBCD, suggesting that deletion of RecB^Nuc limits bp melting from a blunt DNA. We also solved cryo-EM structures showing only 4 bp melted upon RecB^ΔNucCD binding to a blunt ended DNA duplex, less than the 11 bp melted upon RecBCD binding. Thus, the RecB nuclease domain regulates the extent of bp melting by RecBCD. These results suggest that RecB^Nuc may manifest its long-range allosteric effect on DNA binding and DNA melting via linker-linker interactions between RecB and RecC.

大肠杆菌RecBCD是一种异三聚体解旋酶和核酸酶，在双链DNA断裂修复中起作用。RecBCD在RecB（3‘至5’）和RecD（5‘至3’）中都具有atp酶运动结构域，在RecB （RecBNuc）中具有核酸酶结构域。RecBCD结合到双链DNA末端，并通过仅利用其结合自由能首先熔化几个DNA碱基对（bp）来启动DNA解绕。在RecBCD-DNA结构中，RecBNuc结构域与双链DNA结合位点对接约70 Å，但似乎是动态的，能够从其对接位置移动。在这里，我们比较了RecBCD和一个30 kDa核酸酶结构域被删除的变体RecBΔNucCD的DNA结合。RecBCD与钝DNA末端的结合在焓上是不利的，并且是熵驱动的。RecBNuc的缺失导致DNA结合亲和力增加，表明RecBNuc具有变构作用。RecBΔNucCD与具有完全“预融化”DNA末端的DNA结合与一个大的有利ΔHobs相关，但比RecBCD观察到的要小得多，这表明RecBNuc的缺失限制了从原始DNA中融化的bp。我们还解决了低温电镜结构，显示RecBΔNucCD与钝端DNA双链结合时仅熔化了4 bp，少于RecBCD结合时熔化的11 bp。因此，RecB核酸酶结构域调节了RecBCD对bp的融化程度。这些结果表明，RecBNuc可能通过RecB和RecC之间的连接子相互作用来表现其对DNA结合和DNA融化的远程变构作用。

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引用次数: 0

Structure-based Discovery of a Non-competitive FTO Inhibitor Bound to a Cryptic Site at the Domain Interface 基于结构的非竞争性FTO抑制剂的发现结合在域界面上的一个神秘位点。

IF 4.5 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Molecular Biology

Pub Date : 2025-12-05 DOI: 10.1016/j.jmb.2025.169575

Aayushi Singh , Francesco Pettini , Beatrice Gianibbi , Sayan Das , Donatella Barisani , Jeffrey A. Purslow , Simone Furini , Ottavia Spiga , Vincenzo Venditti

The fat mass and obesity-associated fatso (FTO) protein is a member of the AlkB family of dioxygenases whose overexpression links to several metabolic diseases, including obesity, type 2 diabetes, Alzheimer’s, and various types of cancer. FTO is an important target for pharmaceutical research, and several selective and non-selective competitive inhibitors have been developed against the enzyme. However, given the competitive nature of the available inhibitors, obtaining complete subfamily selectivity still presents an unresolved challenge. Here, we describe the discovery of a molecular scaffold for selective inhibition of FTO, which resulted from high throughput virtual screening targeted at FTO cryptic pockets. Analysis of the FTO-inhibitor interaction by solution NMR, molecular dynamics simulations, and enzyme kinetic assays shows that, differently from the FTO inhibitors developed so far, our molecule binds to a cryptic site between the FTO structural domains, and modulates the enzyme function non-competitively by perturbing the binding pose of the α-ketoglutarate and nucleic acid substrates. Since FTO is the only member of the AlkB family that presents multiple structural domains, we expect further development of this allosteric molecule to result in a new family of highly selective FTO inhibitors that can be used alone or in combination with pre-existing compounds to improve their potency and selectivity.

脂肪量和肥胖相关脂肪（FTO）蛋白是双加氧酶AlkB家族的成员，其过表达与几种代谢性疾病有关，包括肥胖、2型糖尿病、阿尔茨海默氏症和各种类型的癌症。FTO是药物研究的重要靶点，目前已经开发出几种针对FTO酶的选择性和非选择性竞争性抑制剂。然而，鉴于现有抑制剂的竞争性，获得完全的亚家族选择性仍然是一个未解决的挑战。在这里，我们描述了一种选择性抑制FTO的分子支架的发现，这是通过针对FTO隐口袋的高通量虚拟筛选产生的。通过溶液核磁共振、分子动力学模拟和酶动力学分析对FTO抑制剂的相互作用进行分析表明，与目前开发的FTO抑制剂不同，我们的分子结合到FTO结构域之间的一个神秘位点上，并通过干扰α-酮戊二酸酯和核酸底物的结合位来非竞争性地调节酶的功能。由于FTO是AlkB家族中唯一具有多个结构域的成员，我们期望进一步开发这种变构分子，从而产生一个新的高选择性FTO抑制剂家族，可以单独使用或与已有化合物联合使用，以提高其效力和选择性。

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