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Thermostable Nucleoid Protein Cren7 Slides Along DNA and Rapidly Dissociates From DNA While Not Inhibiting the Sliding of Other DNA-binding Protein 耐热核糖体蛋白 Cren7 可沿 DNA 滑动,并迅速与 DNA 分离,同时不会抑制其他 DNA 结合蛋白的滑动。
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-24 DOI: 10.1016/j.jmb.2024.168803
Trishit Banerjee , K. Geethika , Saori Kanbayashi , Satoshi Takahashi , Soumit S. Mandal , Kiyoto Kamagata
A nucleoid protein Cren7 compacts DNA, contributing to the living of Crenarchaeum in high temperature environment. In this study, we investigated the dynamic behavior of Cren7 on DNA and its functional relation using single-molecule fluorescence microscopy. We found two mobility modes of Cren7, sliding along DNA and pausing on it, and the rapid dissociation kinetics from DNA. The salt dependence analysis suggests a sliding with continuous contact to DNA, rather than hopping/jumping. The mutational analysis demonstrates that Cren7 slides along DNA while Trp (W26) residue interacts with the DNA. Furthermore, Cren7 does not impede the target search by a model transcription factor p53, implying no significant interference to other DNA-binding proteins on DNA. At high concentration of Cren7, the molecules form large clusters on DNA via bridging, which compacts DNA. We discuss how the dynamic behavior of Cren7 on DNA enables DNA-compaction and protein-bypass functions.
一种核壳蛋白 Cren7 可压实 DNA,从而促进了 Crenarchaeum 在高温环境中的生存。本研究利用单分子荧光显微镜研究了 Cren7 在 DNA 上的动态行为及其功能关系。我们发现了 Cren7 在 DNA 上的两种移动模式:沿 DNA 滑动和在 DNA 上停顿,以及与 DNA 快速解离的动力学。盐依赖性分析表明,Cren7 是一种与 DNA 持续接触的滑动模式,而不是跳跃模式。突变分析表明,当 Trp(W26)残基与 DNA 相互作用时,Cren7 沿着 DNA 滑动。此外,Cren7 并不妨碍模型转录因子 p53 的目标搜索,这意味着对 DNA 上的其他 DNA 结合蛋白没有明显干扰。在高浓度 Cren7 的情况下,分子通过桥接作用在 DNA 上形成大的簇,从而压实 DNA。我们讨论了 Cren7 在 DNA 上的动态行为是如何实现 DNA 压实和蛋白质旁路功能的。
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引用次数: 0
Context-Dependent and Locus-Specific Role of H3K36 Methylation in Transcriptional Regulation. H3K36 甲基化在转录调控中的上下文依赖性和基因座特异性作用
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-19 DOI: 10.1016/j.jmb.2024.168796
Min Kyung Lee, Na Hyun Park, Soo Young Lee, TaeSoo Kim

H3K36 methylation is a critical histone modification involved in transcription regulation. It involves the mono (H3K36me1), di (H3K36me2), and/or tri-methylation (H3K36me3) of lysine 36 on histone H3 by methyltransferases. In yeast, Set2 catalyzes all three methylation states. By contrast, in higher eukaryotes, at least eight methyltransferases catalyze different methylation states, including SETD2 for H3K36me3 and the NSD family for H3K36me2 in vivo. Both Set2 and SETD2 interact with the phosphorylated CTD of RNA Pol II, which links H3K36 methylation to transcription. In yeast, H3K36me3 and H3K36me2 peak at the 3' ends of genes. In higher eukaryotes, this is also true for H3K36me3 but not for H3K36me2, which is enriched at the 5' ends of genes and intergenic regions, suggesting that H3K36me2 and H3K36me3 may play different regulatory roles. Whether H3K36me1 demonstrates preferential distribution remains unclear. H3K36me3 is essential for inhibiting transcription elongation. It also suppresses cryptic transcription by promoting histone deacetylation by the histone deacetylases Rpd3S (yeast) and variant NuRD (higher eukaryotes). H3K36me3 also facilitates DNA methylation by DNMT3B, thereby preventing spurious transcription initiation. H3K36me3 not only represses transcription since it promotes the activation of mRNA and cryptic promoters in response to environmental changes by targeting the histone acetyltransferase NuA3 in yeast. Further research is needed to elucidate the methylation state- and locus-specific functions of H3K36me1 and the mechanisms that regulate it.

H3K36 甲基化是参与转录调控的一种关键组蛋白修饰。它包括通过甲基转移酶对组蛋白 H3 上的赖氨酸 36 进行单甲基化(H3K36me1)、双甲基化(H3K36me2)和/或三甲基化(H3K36me3)。在酵母中,Set2 催化所有三种甲基化状态。相比之下,在高等真核生物中,至少有八种甲基转移酶能催化不同的甲基化状态,其中包括在体内催化 H3K36me3 的 SETD2 和催化 H3K36me2 的 NSD 家族。Set2 和 SETD2 都与 RNA Pol II 的磷酸化 CTD 相互作用,从而将 H3K36 甲基化与转录联系起来。在酵母中,H3K36me3 和 H3K36me2 在基因的 3' 端达到峰值。在高等真核生物中,H3K36me3 的情况也是如此,但 H3K36me2 的情况却并非如此,H3K36me2 富集在基因的 5'末端和基因间区域,这表明 H3K36me2 和 H3K36me3 可能发挥着不同的调控作用。H3K36me1是否会优先分布仍不清楚。H3K36me3 对抑制转录延伸至关重要。它还通过促进组蛋白去乙酰化酶 Rpd3S(酵母)和变体 NuRD(高等真核生物)的组蛋白去乙酰化作用来抑制隐性转录。H3K36me3 还有助于 DNMT3B 进行 DNA 甲基化,从而防止错误的转录启动。H3K36me3 不仅能抑制转录,还能通过靶向酵母中的组蛋白乙酰转移酶 NuA3 促进 mRNA 和隐性启动子的激活,以应对环境变化。要阐明H3K36me1的甲基化状态和位点特异性功能及其调控机制,还需要进一步的研究。
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引用次数: 0
GpsB Coordinates StkP Signaling as a PASTA Kinase Adaptor in Streptococcus pneumoniae Cell Division 在肺炎链球菌细胞分裂过程中,GpsB 作为 PASTA 激酶适配器协调 StkP 信号传导。
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-19 DOI: 10.1016/j.jmb.2024.168797
Václava Stauberová , Bohumil Kubeša , Merrin Joseph , Mattia Benedet , Berenice Furlan , Karolína Buriánková , Aleš Ulrych , Rudolf Kupčík , Tomáš Vomastek , Orietta Massidda , Ho-Ching T. Tsui , Malcolm E. Winkler , Pavel Branny , Linda Doubravová
StkP, the Ser/Thr protein kinase of the major human pathogen Streptococcus pneumoniae, monitors cell wall signals and regulates growth and division in response. In vivo, StkP interacts with GpsB, a cell division protein required for septal ring formation and closure, that affects StkP-dependent phosphorylation. Here, we report that although StkP has basal intrinsic kinase activity, GpsB promotes efficient autophosphorylation of StkP and phosphorylation of StkP substrates. Phosphoproteomic analyzes showed that GpsB is phosphorylated at several Ser and Thr residues. We confirmed that StkP directly phosphorylates GpsB in vitro and in vivo, with T79 and T83 being the major phosphorylation sites. In vitro, phosphoablative GpsB substitutions had a lower potential to stimulate StkP activity, whereas phosphomimetic substitutions were functional in terms of StkP activation. In vivo, substitutions of GpsB phosphoacceptor residues, either phosphoablative or mimetic, had a negative effect on GpsB function, resulting in reduced StkP-dependent phosphorylation and impaired cell division. The bacterial two-hybrid assay and co-immunoprecipitation of GpsB from cells with differentially active StkP indicated that increased phosphorylation of GpsB resulted in a more efficient interaction of GpsB with StkP. Our data suggest that GpsB acts as an adaptor that directly promotes StkP activity by mediating interactions within the StkP signaling hub, ensuring StkP recruitment into the complex and substrate specificity. We present a model that interaction of StkP with GpsB and its phosphorylation and dephosphorylation dynamically modulate kinase activity during exponential growth and under cell wall stress of S. pneumoniae, ensuring the proper functioning of the StkP signaling pathway.
StkP是人类主要病原体肺炎链球菌的Ser/Thr蛋白激酶,它能监测细胞壁信号并调节生长和分裂。在体内,StkP 与中隔环形成和闭合所需的细胞分裂蛋白 GpsB 相互作用,从而影响 StkP 依赖性磷酸化。在这里,我们报告说,虽然 StkP 具有基础固有激酶活性,但 GpsB 能促进 StkP 的有效自磷酸化和 StkP 底物的磷酸化。磷酸化蛋白组分析表明,GpsB 在多个 Ser 和 Thr 残基上被磷酸化。我们证实,StkP 在体外和体内直接磷酸化 GpsB,其中 T79 和 T83 是主要的磷酸化位点。在体外,GpsB的磷酸化取代对StkP活性的刺激潜力较低,而磷酸拟态取代对StkP的激活具有功能性。在体内,GpsB 磷酸化受体残基的置换,无论是磷酸化还是拟态,都会对 GpsB 的功能产生负面影响,导致 StkP 依赖性磷酸化减少和细胞分裂受损。细菌双杂交试验以及从具有不同活性 StkP 的细胞中对 GpsB 的共免疫沉淀表明,GpsB 磷酸化的增加导致 GpsB 与 StkP 的相互作用更有效。我们的数据表明,GpsB 是一种适配体,它通过介导 StkP 信号转导中枢内的相互作用直接促进 StkP 的活性,确保 StkP 被招募到复合物中并具有底物特异性。我们提出了一个模型,即 StkP 与 GpsB 的相互作用及其磷酸化和去磷酸化可动态调节肺炎双球菌在指数生长过程中和细胞壁应激状态下的激酶活性,从而确保 StkP 信号通路的正常运作。
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引用次数: 0
Shigella Senses the Environmental Cue Leucine to Promote its Virulence Gene Expression in the Colon 志贺氏菌感知环境线索亮氨酸,促进其毒力基因在结肠中的表达。
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-19 DOI: 10.1016/j.jmb.2024.168798
Huiying Li , Yongyao Lv , Zhiqi Teng , Rui Guo , Lingyan Jiang
Shigella is a foodborne enteropathogenic bacteria that causes severe bacillary dysentery in humans. Shigella primarily colonizes the human colon and causes disease via invasion of colon epithelial cells. However, the signal regulatory mechanisms associated with its colonization and pathogenesis in the colon remain poorly defined. Here, we report a leucine-mediated regulatory mechanism that promotes Shigella virulence gene expression and invasion of colon epithelial cells. Shigella in response to leucine, which is highly abundant in the colon, via the leucine-responsive regulator Lrp and the binding of Lrp with leucine induces the expression of a newly identified small RNA SsrV. SsrV then activates the expression of virF and downstream invasion-related virulence genes by increasing the protein level of the LysR-type transcription regulator LrhA, therefore enabling Shigella invasion of colon epithelial cells. Shigella lacking ssrV displays impaired invasion ability. Collectively, these findings suggest that Shigella employs a leucine-responsive environmental activation mechanism to establish colonization and pathogenicity.
志贺氏菌是一种食源性肠道致病菌,可导致人类严重的菌痢。志贺氏菌主要在人类结肠中定植,通过入侵结肠上皮细胞致病。然而,与志贺氏杆菌在结肠中的定植和致病相关的信号调控机制仍不十分明确。在这里,我们报告了一种由亮氨酸介导的调控机制,它能促进志贺氏菌毒力基因的表达和对结肠上皮细胞的侵袭。志贺氏菌通过亮氨酸反应调节因子 Lrp 对结肠中含量丰富的亮氨酸做出反应,Lrp 与亮氨酸的结合诱导了一种新发现的小 RNA SsrV 的表达。然后,SsrV 通过提高 LysR 型转录调节因子 LrhA 的蛋白水平,激活 virF 和下游入侵相关毒力基因的表达,从而使志贺氏杆菌入侵结肠上皮细胞。缺乏ssrV的志贺氏杆菌侵袭能力受损。总之,这些研究结果表明,志贺氏菌采用了一种亮氨酸反应性环境激活机制来建立定植和致病性。
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引用次数: 0
Ligand Recognition and Activation Mechanism of the Alicarboxylic Acid Receptors 羧酸受体的配体识别和激活机制。
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-19 DOI: 10.1016/j.jmb.2024.168795
Yanru Liu , Ziwei Zhou , Fenghui Guan , Zhen Han , Cheng Zhu , Sheng Ye , Xuekui Yu , Anna Qiao
Endogenous ligands for alicarboxylic acid receptors are important metabolic intermediates that play a significant role in regulating body energy and maintaining homeostasis. However, the molecular mechanism of alicarboxylate ligand-mediated counterpart receptors is currently unclear. We resolve the active state structure of HCA2-niacin, and the structural analysis explains the mechanism of niacin selectivity in the alicarboxylic acid receptors family. Homology modeling, molecular dynamics simulation and mutagenesis experiments reveal different ligand recognition modes and activation mechanisms of the alicarboxylic acid receptors, analyze the flexibility of the binding pocket and elucidate the important role of disulfide bonds on receptor activation and ligand binding. These more detailed molecular mechanisms further elucidate the relevant mechanisms of human metabolism and provide key clues for subsequent drug development of alicarboxylic acid receptors.
羧酸受体的内源配体是重要的代谢中间产物,在调节机体能量和维持体内平衡方面发挥着重要作用。然而,羧酸配体介导对应受体的分子机制目前尚不清楚。我们解析了 HCA2-烟酸的活性状态结构,并通过结构分析解释了烟酸受体家族中烟酸选择性的机制。同源建模、分子动力学模拟和诱变实验揭示了羧酸受体的不同配体识别模式和激活机制,分析了结合口袋的灵活性,阐明了二硫键对受体激活和配体结合的重要作用。这些更详细的分子机制进一步阐明了人体代谢的相关机制,为后续的羧酸受体药物开发提供了关键线索。
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引用次数: 0
Inventing Novel Protein Folds 发明新的蛋白质折叠
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-12 DOI: 10.1016/j.jmb.2024.168791
Nobuyasu Koga , Rie Tatsumi-Koga
The vastness of unexplored protein fold universe remains a significant question. Through systematic de novo design of proteins with novel αβ-folds, we demonstrated that nature has only explored a tiny portion of the possible folds. Numerous possible protein folds are still untouched by nature. This review outlines this study and discusses the prospects for design of functional proteins with novel folds.
尚未探索的蛋白质折叠宇宙的广阔性仍然是一个重要问题。通过系统地从头设计具有新型αβ折叠的蛋白质,我们证明自然界只探索了可能折叠的极小部分。许多可能的蛋白质折叠仍未被自然界触及。这篇综述概述了这项研究,并讨论了设计具有新折叠的功能蛋白质的前景。
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引用次数: 0
Insights into Ligand-Mediated Activation of an Oligomeric Ring-Shaped Gene-Regulatory Protein from Solution- and Solid-State NMR 从溶液和固态核磁共振深入了解配体介导的寡聚环形基因调控蛋白的活化过程
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-11 DOI: 10.1016/j.jmb.2024.168792
Rodrigo Muzquiz , Cameron Jamshidi , Daniel W. Conroy , Christopher P. Jaroniec , Mark P. Foster
The 91 kDa oligomeric ring-shaped ligand binding protein TRAP (trp RNA binding attenuation protein) regulates the expression of a series of genes involved in tryptophan (Trp) biosynthesis in bacilli. When cellular Trp levels rise, the free amino acid binds to sites buried in the interfaces between each of the 11 (or 12, depending on the species) protomers in the ring. Crystal structures of Trp-bound TRAP show the Trp ligands are sequestered from solvent by a pair of loops from adjacent protomers that bury the bound ligand via polar contacts to several threonine residues. Binding of the Trp ligands occurs cooperatively, such that successive binding events occur with higher apparent affinity but the structural basis for this cooperativity is poorly understood. We used solution methyl-TROSY NMR relaxation experiments focused on threonine and isoleucine sidechains, as well as magic angle spinning solid-state NMR 13C–13C and 15N-13C chemical shift correlation spectra on uniformly labeled samples recorded at 800 and 1200 MHz, to characterize the structure and dynamics of the protein. Methyl 13C relaxation dispersion experiments on ligand-free apo TRAP revealed concerted exchange dynamics on the µs-ms time scale, consistent with transient sampling of conformations that could allow ligand binding. Cross-correlated relaxation experiments revealed widespread disorder on fast timescales. Chemical shifts for methyl-bearing side chains in apo- and Trp-bound TRAP revealed subtle changes in the distribution of sampled sidechain rotameric states. These observations reveal a pathway and mechanism for induced conformational changes to generate homotropic Trp-Trp binding cooperativity.
91 kDa 的寡聚环形配体结合蛋白 TRAP(trp RNA 结合衰减蛋白)调节着一系列参与色氨酸(Trp)在芽孢杆菌中生物合成的基因的表达。当细胞中的 Trp 水平升高时,游离氨基酸就会与埋藏在环中 11 个(或 12 个,视物种而定)原生体之间界面上的位点结合。与 Trp 结合的 TRAP 的晶体结构显示,Trp 配体通过相邻原体的一对环路与溶剂隔离,这对环路通过与几个苏氨酸残基的极性接触将结合的配体埋藏起来。Trp 配体的结合是协同进行的,因此连续的结合事件会以更高的表观亲和力发生,但这种协同性的结构基础却鲜为人知。我们利用以苏氨酸和异亮氨酸侧链为重点的溶液甲基-TROSY NMR 驰豫实验,以及以 800 和 1200 MHz 频率记录的均匀标记样品的魔角旋转固态 NMR 13C-13C 和 15N-13C 化学位移相关光谱,来描述该蛋白质的结构和动力学特征。对不含配体的 apo TRAP 进行的甲基 13C 驰豫弥散实验显示,在 µs-ms 时间尺度上存在一致的交换动力学,这与瞬时取样构象可能允许配体结合是一致的。交叉相关弛豫实验揭示了快速时间尺度上的广泛无序性。在载脂蛋白和 Trp 结合的 TRAP 中,含甲基侧链的化学位移显示了取样侧链旋转态分布的微妙变化。这些观察结果揭示了诱导构象变化以产生同向 Trp-Trp 结合合作性的途径和机制。
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引用次数: 0
Species-specific model based on sequence and structural information for ubiquitination sites prediction 基于序列和结构信息的泛素化位点预测物种特异性模型。
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-07 DOI: 10.1016/j.jmb.2024.168781
Weimin Li , Nan Chen , Jie Wang , Yin Luo , Huazhong Liu , Jihong Ding , Qun Jin

Ubiquitination is a common post-translational modification of proteins in eukaryotic cells, and it is also a significant method of regulating protein biological function. Computational methods for predicting ubiquitination sites can serve as a cost-effective and time-saving alternative to experimental methods. Existing computational methods often build classifiers based on protein sequence information, physical and chemical properties of amino acids, evolutionary information, and structural parameters. However, structural information about most proteins cannot be found in existing databases directly. The features of proteins differ among species, and some species have small amounts of ubiquitinated proteins. Therefore, it is necessary to develop species-specific models that can be applied to datasets with small sample sizes. To solve these problems, we propose a species-specific model (SSUbi) based on a capsule network, which integrates proteins’ sequence and structural information. In this model, the feature extraction module is composed of two sub-modules that extract multi-dimensional features from sequence and structural information respectively. In the submodule, the convolution operation is used to extract encoding dimension features, and the channel attention mechanism is used to extract feature map dimension features. After integrating the multi-dimensional features from both types of information, the species-specific capsule network further converts the features into capsule vectors and classifies species-specific ubiquitination sites. The experimental results show that SSUbi can effectively improve the prediction performance of species with small sample sizes and outperform other models.

泛素化是真核细胞中常见的蛋白质翻译后修饰,也是调节蛋白质生物功能的重要方法。使用计算方法预测泛素化位点可以取代昂贵而耗时的实验方法。现有的计算方法通常根据蛋白质序列信息、氨基酸的物理和化学特性、进化信息和结构参数建立分类器。然而,大多数蛋白质的结构信息无法直接从现有数据库中找到。不同物种的蛋白质特征各不相同,有些物种的泛素化蛋白质数量较少。因此,有必要开发可应用于样本量较小的数据集的物种特异性模型。为了解决这些问题,我们提出了一种基于胶囊网络的物种特异性模型(SSUbi),它整合了蛋白质的序列和结构信息。在该模型中,特征提取模块由两个子模块组成,分别从序列和结构信息中提取多维特征。在子模块中,利用卷积运算提取编码维度特征,利用通道注意机制提取特征图维度特征。整合两种信息的多维特征后,物种特异性胶囊网络进一步将特征转换为胶囊向量,并对物种特异性泛素化位点进行分类。实验结果表明,SSUbi 能有效提高小样本量物种的预测性能,并优于其他模型。
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引用次数: 0
Structural Basis of Main Proteases of Coronavirus Bound to Bofutrelvir 冠状病毒主要蛋白酶与 Bofutrelvir 结合的结构基础。
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-06 DOI: 10.1016/j.jmb.2024.168784
Wei-wei Wang , Pei Zeng , Tongchao Liu , Xue-lan Zhou , Cheng Lin , Li Guo , Qi-sheng Wang , Jian Li

Globally, the continuous spread and evolution of SARS-CoV-2, along with its variants, profoundly impact human well-being, health, security, and the growth of socio-economic. In the field of development of drugs against COVID-19, the main protease (Mpro) is a critical target as it plays a core role in the lifecycle of SARS-CoV-2. Bofutrelvir acts as a potent inhibitor of SARS-CoV-2 Mpro, demonstrating high efficacy and broad-spectrum antiviral activity. Compared to therapies that require pharmacokinetic boosters, such as ritonavir, the monotherapy approach of Bofutrelvir reduces the risk of potential drug interactions, making it suitable for a wider patient population. However, further studies on the potency and mechanism of inhibition of Bofutrelvir against the Mpro of COVID-19 and its variants, together with other coronaviruses, are needed to prepare for the possibility of a possible re-emerging threat from an analogous virus in the future. Here, we reveal the effective inhibition of Bofutrelvir against the Mpro of SARS-CoV-2, SARS-CoV, and HCoV-229E through FRET and crystallographic analysis. Furthermore, the inhibitory mechanisms of Bofutrelvir against two SARS-CoV-2 Mpro mutants (G15S and K90R) were also elucidated through FRET and crystallographic studies. Through detailed analysis and comparison of these crystal structures, we identified crucial structural determinants of inhibition and elucidated the binding mode of Bofutrelvir to Mpros from different coronaviruses. These findings are hopeful to accelerate the development of safer and more potent inhibitors against the Mpro of coronavirus, and to provide important references for the prevention and treatment of similar viruses that may emerge in the future.

在全球范围内,SARS-CoV-2 及其变种的持续传播和演变对人类的福祉、健康、安全和社会经济的发展产生了深远的影响。在针对 COVID-19 的药物开发领域,主要蛋白酶(Mpro)是一个关键靶点,因为它在 SARS-CoV-2 的生命周期中发挥着核心作用。Bofutrelvir 是 SARS-CoV-2 Mpro 的强效抑制剂,具有高效、广谱的抗病毒活性。与需要药代动力学促进剂的疗法(如利托那韦)相比,Bofutrelvir 的单药治疗方法降低了潜在药物相互作用的风险,使其适用于更广泛的患者人群。不过,我们还需要进一步研究 Bofutrelvir 对 COVID-19 及其变种以及其他冠状病毒的 Mpro 的抑制效力和机制,以应对未来可能再次出现的类似病毒威胁。在这里,我们通过 FRET 和晶体学分析揭示了 Bofutrelvir 对 SARS-CoV-2、SARS-CoV 和 HCoV-229E 的 Mpro 的有效抑制作用。此外,还通过 FRET 和晶体学研究阐明了 Bofutrelvir 对两种 SARS-CoV-2 Mpro 突变体(G15S 和 K90R)的抑制机制。通过对这些晶体结构的详细分析和比较,我们确定了抑制作用的关键结构决定因素,并阐明了 Bofutrelvir 与不同冠状病毒 Mpro 的结合模式。这些发现有望加速开发更安全、更有效的冠状病毒 Mpro 抑制剂,并为预防和治疗未来可能出现的类似病毒提供重要参考。
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引用次数: 0
Accurate Identification of Periplasmic Urea-binding Proteins by Structure- and Genome Context-assisted Functional Analysis 通过结构和基因组上下文辅助功能分析,准确鉴定外质尿素结合蛋白。
IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-04 DOI: 10.1016/j.jmb.2024.168780
Malin J. Allert , Shivesh Kumar , You Wang , Lorena S. Beese , Homme W. Hellinga

ABC transporters are ancient and ubiquitous nutrient transport systems in bacteria and play a central role in defining lifestyles. Periplasmic solute-binding proteins (SBPs) are components that deliver ligands to their translocation machinery. SBPs have diversified to bind a wide range of ligands with high specificity and affinity. However, accurate assignment of cognate ligands remains a challenging problem in SBPs. Urea metabolism plays an important role in the nitrogen cycle; anthropogenic sources account for more than half of global nitrogen fertilizer. We report identification of urea-binding proteins within a large SBP sequence family that encodes diverse functions. By combining genetic linkage between SBPs, ABC transporter components, enzymes or transcription factors, we accurately identified cognate ligands, as we verified experimentally by biophysical characterization of ligand binding and crystallographic determination of the urea complex of a thermostable urea-binding homolog. Using three-dimensional structure information, these functional assignments were extrapolated to other members in the sequence family lacking genetic linkage information, which revealed that only a fraction bind urea. Using the same combined approaches, we also inferred that other family members bind various short-chain amides, aliphatic amino acids (leucine, isoleucine, valine), γ-aminobutyrate, and as yet unknown ligands. Comparative structural analysis revealed structural adaptations that encode diversification in these SBPs. Systematic assignment of ligands to SBP sequence families is key to understanding bacterial lifestyles, and also provides a rich source of biosensors for clinical and environmental analysis, such as the thermostable urea-binding protein identified here.

ABC 转运体是细菌中古老而无处不在的营养物质转运系统,在确定生命方式方面发挥着核心作用。外质溶质结合蛋白(SBPs)是将配体输送到转运机制的元件。SBPs 种类繁多,能以高特异性和高亲和力结合多种配体。然而,准确分配配体仍然是 SBPs 面临的一个挑战性问题。尿素代谢在氮循环中发挥着重要作用;人为来源占全球氮肥的一半以上。我们报告了在一个编码多种功能的大型 SBP 序列家族中鉴定出的尿素结合蛋白。通过将 SBPs、ABC 转运体成分、酶或转录因子之间的遗传联系结合起来,我们准确地鉴定出了同源配体,并通过配体结合的生物物理表征和恒温尿素结合同源物尿素复合物的晶体学测定进行了实验验证。利用三维结构信息,我们将这些功能分配推断到序列家族中缺乏遗传联系信息的其他成员,结果发现只有一部分能与尿素结合。利用同样的综合方法,我们还推断出其他家族成员结合了各种短链酰胺、脂肪族氨基酸(亮氨酸、异亮氨酸、缬氨酸)、γ -氨基丁酸盐以及尚未知晓的配体。结构比较分析表明,这些 SBPs 的结构适应性编码了多样性。将配体系统地分配到 SBP 序列家族是了解细菌生活方式的关键,同时也为临床和环境分析提供了丰富的生物传感器来源,例如这里发现的恒温尿素结合蛋白。
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引用次数: 0
期刊
Journal of Molecular Biology
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