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Proton-Coupled Electron Transfer upon Oxidation of Tyrosine in a De Novo Protein: Analysis of Proton Acceptor Candidates. 新蛋白中酪氨酸氧化时的质子耦合电子转移:质子受体候选物分析。
IF 2.9 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-18 DOI: 10.1021/acs.biochem.4c00211
Qiwen Zhu, Alexander V Soudackov, Cecilia Tommos, Sharon Hammes-Schiffer

Redox-active residues, such as tyrosine and tryptophan, play important roles in a wide range of biological processes. The α3Y de novo protein, which is composed of three α helices and a tyrosine residue Y32, provides a platform for investigating the redox properties of tyrosine in a well-defined protein environment. Herein, the proton-coupled electron transfer (PCET) reaction that occurs upon oxidation of tyrosine in this model protein by a ruthenium photosensitizer is studied by using a vibronically nonadiabatic PCET theory that includes hydrogen tunneling and excited vibronic states. The input quantities to the analytical nonadiabatic rate constant expression, such as the diabatic proton potential energy curves and associated proton vibrational wave functions, reorganization energy, and proton donor-acceptor distribution functions, are obtained from density functional theory calculations on model systems and molecular dynamics simulations of the solvated α3Y protein. Two possible proton acceptors, namely, water or a glutamate residue in the protein scaffold, are explored. The PCET rate constant is greater when glutamate is the proton acceptor, mainly due to the more favorable driving force and shorter equilibrium proton donor-acceptor distance, although contributions from excited vibronic states mitigate these effects. Nevertheless, water could be the dominant proton acceptor if its equilibrium constant associated with hydrogen bond formation is significantly greater than that for glutamate. Although these calculations do not definitively identify the proton acceptor for this PCET reaction, they elucidate the conditions under which each proton acceptor can be favored. These insights have implications for tyrosine-based PCET in a wide variety of biochemical processes.

酪氨酸和色氨酸等氧化还原活性残基在多种生物过程中发挥着重要作用。由三个 α 螺旋和一个酪氨酸残基 Y32 组成的 α3Y 新生蛋白质为研究酪氨酸在明确定义的蛋白质环境中的氧化还原特性提供了一个平台。在本文中,利用包括氢隧道和激发振子态在内的振子非绝热 PCET 理论,研究了该模型蛋白质中酪氨酸被钌光敏剂氧化时发生的质子耦合电子转移(PCET)反应。分析性非绝热速率常数表达式的输入量,如二绝热质子势能曲线和相关质子振动波函数、重组能和质子供体-受体分布函数,都是从模型系统的密度泛函理论计算和溶解的 α3Y 蛋白质的分子动力学模拟中获得的。探讨了两种可能的质子接受者,即水或蛋白质支架中的谷氨酸残基。谷氨酸作为质子受体时,PCET 的速率常数更大,这主要是由于更有利的驱动力和更短的平衡质子供体-受体距离,尽管激发振动态的贡献减轻了这些影响。不过,如果水与氢键形成相关的平衡常数明显大于谷氨酸的平衡常数,那么水可能是主要的质子接受者。虽然这些计算并没有明确确定 PCET 反应的质子接受体,但它们阐明了每种质子接受体可能被偏爱的条件。这些见解对基于酪氨酸的 PCET 在各种生化过程中的应用具有重要意义。
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引用次数: 0
A Noble Metal Substitution Leads to B12 Cofactor Mimicry by a Rhodibalamin. 贵金属置换导致罗地巴拉明模拟 B12 辅因子
IF 2.9 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-16 DOI: 10.1021/acs.biochem.4c00216
Markus Ruetz, Romila Mascarenhas, Florian Widner, Christoph Kieninger, Markos Koutmos, Bernhard Kräutler, Ruma Banerjee

In mammals, cobalamin is an essential cofactor that is delivered by a multitude of chaperones in an elaborate trafficking pathway to two client enzymes, methionine synthase and methylmalonyl-CoA mutase (MMUT). Rhodibalamins, the rhodium analogs of cobalamins, have been described as antimetabolites due to their ability to inhibit bacterial growth. In this study, we have examined the reactivity of adenosylrhodibalamin (AdoRhbl) with two key human chaperones, MMACHC (also known as CblC) and adenosyltransferase (MMAB, also known as ATR), and with the human and Mycobacterium tuberculosis MMUT. We demonstrate that while AdoRhbl binds tightly to all four proteins, the Rh-carbon bond is resistant to homolytic (on MMAB and MMUT) as well as heterolytic (on MMACHC) rupture. On the other hand, MMAB catalyzes Rh-carbon bond formation, converting rhodi(I)balamin in the presence of ATP to AdoRhbl. We report the first crystal structure of a rhodibalamin (AdoRhbl) bound to a B12 protein, i.e., MMAB, in the presence of triphosphate, which shows a weakened but intact Rh-carbon bond. The structure provides insights into how MMAB cleaves the corresponding Co-carbon bond in a sacrificial homolytic reaction that purportedly functions as a cofactor sequestration strategy. Collectively, the study demonstrates that while the noble metal substitution of cobalt by rhodium sets up structural mimicry, it compromises chemistry, which could be exploited for targeting human and bacterial B12 chaperones and enzymes.

在哺乳动物体内,钴胺素是一种重要的辅助因子,由多种伴侣通过复杂的运输途径输送到两种客户酶,即蛋氨酸合成酶和甲基丙二酰-CoA 突变酶(MMUT)。钴胺的铑类似物 Rhodibalamins 因其抑制细菌生长的能力而被描述为抗代谢物。在这项研究中,我们研究了腺苷罗地巴铵(AdoRhbl)与两种关键的人类伴侣蛋白 MMACHC(又称 CblC)和腺苷转移酶(MMAB,又称 ATR)的反应性,以及与人类和结核分枝杆菌 MMUT 的反应性。我们证明,虽然 AdoRhbl 与所有四种蛋白都能紧密结合,但 Rh 碳键对同源分解(MMAB 和 MMUT)和异源分解(MMACHC)断裂都有抵抗力。另一方面,MMAB 可催化 Rh 碳键的形成,在 ATP 的存在下将 rhodi(I)balamin 转化为 AdoRhbl。我们首次报道了罗地巴拉明(AdoRhbl)在三磷酸存在下与 B12 蛋白(即 MMAB)结合的晶体结构,该结构显示了一个减弱但完整的 Rh 碳键。通过该结构,我们可以深入了解 MMAB 如何在牺牲性同源分解反应中裂解相应的 Co 碳键,该反应据称是一种辅助因子螯合策略。总之,这项研究表明,虽然铑取代钴的贵金属会产生结构模拟,但却会损害化学性质,而这种化学性质可用于靶向人类和细菌的 B12 伴侣和酶。
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引用次数: 0
19F Fast Magic-Angle Spinning NMR Spectroscopy on Microcrystalline Complexes of Fluorinated Ligands and the Carbohydrate Recognition Domain of Galectin-3. 19F 快速魔角旋转核磁共振波谱分析含氟配体与 Galectin-3 碳水化合物识别域的微晶复合物。
IF 2.9 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-15 DOI: 10.1021/acs.biochem.4c00232
Roza Kalabekova, Caitlin M Quinn, Kumar Tekwani Movellan, Angela M Gronenborn, Mikael Akke, Tatyana Polenova

Structural characterization of protein-ligand binding interfaces at atomic resolution is essential for improving the design of specific and potent inhibitors. Herein, we explored fast 19F- and 1H-detected magic angle spinning NMR spectroscopy to investigate the interaction between two fluorinated ligand diastereomers with the microcrystalline galectin-3 carbohydrate recognition domain. The detailed environment around the fluorine atoms was mapped by 2D 13C-19F and 1H-19F dipolar correlation experiments and permitted characterization of the binding interface. Our results demonstrate that 19F MAS NMR is a powerful tool for detailed characterization of protein-ligand interfaces and protein interactions at the atomic level.

原子分辨率的蛋白质配体结合界面结构表征对于改进特异性强效抑制剂的设计至关重要。在此,我们利用快速 19F 和 1H 检测魔角旋转 NMR 光谱来研究两种含氟配体非对映异构体与微晶 galectin-3 碳水化合物识别结构域之间的相互作用。通过二维 13C-19F 和 1H-19F 双极性相关实验绘制了氟原子周围的详细环境图谱,从而确定了结合界面的特征。我们的研究结果表明,19F MAS NMR 是在原子水平上详细描述蛋白质配体界面和蛋白质相互作用的有力工具。
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引用次数: 0
Active-Site Oxygen Accessibility and Catalytic Loop Dynamics of Plant Aromatic Amino Acid Decarboxylases from Molecular Simulations. 从分子模拟看植物芳香族氨基酸脱羧酶的活性位点氧可得性和催化环动力学
IF 2.9 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-15 DOI: 10.1021/acs.biochem.4c00144
Yitao Gou, Tianjie Li, Yi Wang

Aromatic amino acid decarboxylases (AAADs) are pyridoxal-5'-phosphate (PLP)-dependent enzymes that catalyze the decarboxylation of aromatic amino acid l-amino acids. In plants, apart from canonical AAADs that catalyze the straightforward decarboxylation reaction, other members of the AAAD family function as aromatic acetaldehyde synthases (AASs) and catalyze more complex decarboxylation-dependent oxidative deamination. The interconversion between a canonical AAAD and an AAS can be achieved by a single tyrosine-phenylalanine mutation in the large catalytic loop of the enzymes. In this work, we report implicit ligand sampling (ILS) calculations of the canonical l-tyrosine decarboxylase from Papaver somniferum (PsTyDC) that catalyzes l-tyrosine decarboxylation and its Y350F mutant that instead catalyzes the decarboxylation-dependent oxidative deamination of the same substrate. Through comparative analysis of the resulting three-dimensional (3D) O2 free energy profiles, we evaluate the impact of the key tyrosine/phenylalanine mutation on oxygen accessibility to both the wild type and Y350F mutant of PsTyDC. Additionally, using molecular dynamics (MD) simulations of the l-tryptophan decarboxylase from Catharanthus roseus (CrTDC), we further investigate the dynamics of a large catalytic loop known to be indispensable to all AAADs. Results of our ILS and MD calculations shed new light on how key structural elements and loop conformational dynamics underlie the enzymatic functions of different members of the plant AAAD family.

芳香族氨基酸脱羧酶(AAADs)是一种依赖于吡哆醛-5'-磷酸(PLP)的酶,可催化芳香族氨基酸 l-氨基酸的脱羧反应。在植物中,除了典型的 AAADs 能催化直接的脱羧反应外,AAAD 家族的其他成员也能作为芳香族乙醛合成酶(AASs)发挥作用,催化更复杂的脱羧依赖性氧化脱氨反应。典型 AAAD 与 AAS 之间的相互转换可以通过酶的大催化环中的单个酪氨酸-苯丙氨酸突变来实现。在这项工作中,我们报告了隐式配体取样(ILS)计算的结果,计算的对象是催化 l-酪氨酸脱羧的巴布亚新几内亚(Papaver somniferum)典型 l-酪氨酸脱羧酶(PsTyDC)及其 Y350F 突变体,后者转而催化同一底物的脱羧依赖性氧化脱氨。通过对由此产生的三维(3D)氧气自由能曲线进行比较分析,我们评估了关键的酪氨酸/苯丙氨酸突变对 PsTyDC 野生型和 Y350F 突变体氧气可及性的影响。此外,我们还利用分子动力学(MD)模拟了蔷薇色氨酸脱羧酶(CrTDC),进一步研究了已知对所有 AAADs 都不可或缺的大催化环的动力学。我们的 ILS 和 MD 计算结果揭示了植物 AAAD 家族不同成员的酶功能是如何通过关键结构元素和环路构象动力学来实现的。
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引用次数: 0
Transcription Attenuation in Synthetic Promoters in Nonoverlapping Tandem Formation. 非重叠串联合成启动子的转录衰减
IF 2.9 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-12 DOI: 10.1021/acs.biochem.4c00012
Vatsala Chauhan, Ines S C Baptista, Amir M Arsh, Rahul Jagadeesan, Suchintak Dash, Andre S Ribeiro

Closely spaced promoters are ubiquitous in prokaryotic and eukaryotic genomes. How their structure and dynamics relate remains unclear, particularly for tandem formations. To study their transcriptional interference, we engineered two pairs and one trio of synthetic promoters in nonoverlapping, tandem formation, in single-copy plasmids transformed into Escherichia coli cells. From in vivo measurements, we found that these promoters in tandem formation can have attenuated transcription rates. The attenuation strength can be widely fine-tuned by the promoters' positioning, natural regulatory mechanisms, and other factors, including the antibiotic rifampicin, which is known to hamper RNAP promoter escape. From this, and supported by in silico models, we concluded that the attenuation in these constructs emerges from premature terminations generated by collisions between RNAPs elongating from upstream promoters and RNAPs occupying downstream promoters. Moreover, we found that these collisions can cause one or both RNAPs to falloff. Finally, the broad spectrum of possible, externally regulated, attenuation strengths observed in our synthetic tandem promoters suggests that they could become useful as externally controllable regulators of future synthetic circuits.

在原核生物和真核生物基因组中,间隔很近的启动子无处不在。它们的结构和动力学如何相关仍不清楚,尤其是串联形式的启动子。为了研究它们对转录的干扰,我们在转化到大肠杆菌细胞中的单拷贝质粒中设计了两对和三对不重叠、串联的合成启动子。通过活体测量,我们发现这些串联启动子的转录率会减弱。衰减强度可通过启动子的位置、自然调控机制和其他因素(包括已知会阻碍 RNAP 启动子逃逸的抗生素利福平)进行广泛的微调。由此,我们得出结论,这些构建物中的衰减是由于从上游启动子伸长的 RNAP 与占据下游启动子的 RNAP 之间发生碰撞而产生的过早终止。此外,我们还发现,这些碰撞可导致一个或两个 RNAP 脱落。最后,在我们合成的串联启动子中观察到的外部调节衰减强度的广泛范围表明,它们可以成为未来合成电路的外部可控调节器。
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引用次数: 0
Novel DNA-Binding Activity Exhibited by Poly(aspartic acid) Hydrolase-1 Inhibits Poly(aspartic acid) Hydrolase Activity. 聚(天冬氨酸)水解酶-1 显示的新型 DNA 结合活性抑制了聚(天冬氨酸)水解酶的活性。
IF 2.9 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-12 DOI: 10.1021/acs.biochem.4c00127
Joshua Couch, Justin D Marsee, Waylan W Callaway, Thi Ho, Kathryn E Glorioso, Michael Mercante, Britney Williams, Connor Coughran, Mitch H Weiland, Justin M Miller

Significant attention has been shifted toward the use and development of biodegradable polymeric materials to mitigate environmental accumulation and potential health impacts. One such material, poly(aspartic acid) (PAA), is a biodegradable alternative to superabsorbent poly(carboxylates), like poly(acrylate). Three enzymes are known to hydrolyze PAA: PahZ1KT-1 and PahZ2KT-1 from Sphingomonas sp. KT-1 and PahZ1KP-2 from Pedobacter sp. KP-2. We previously reported the X-ray crystal structure for PahZ1KT-1, which revealed a homodimer complex with a strongly cationic surface spanning one side of each monomer. Here, we report the first characterization of any polymer hydrolase binding to DNA, where modeling data predict binding of the polyanionic DNA near the cationic substrate binding surface. Our data reveal that PahZ1 homologues from Sphingomonas sp. KT-1 and Pedobacter sp. KP-2 bind ssDNA and dsDNA with nanomolar binding affinities. PahZ1KT-1 binds ssDNA and dsDNA with an apparent dissociation constant, KD,app = 81 ± 14 and 19 ± 1 nM, respectively, and these estimates are similar to the same behaviors exhibited by PahZ1KP-2. Gel permeation chromatography data reveal that dsDNA binding promotes inhibition of PahZ1-catalyzed PAA biodegradation for each homologue. We propose a working model wherein binding of PahZ1 to extracellular biofilm DNA aids in the localization of the hydrolase to the environment in which PAA would first be encountered, thereby providing a mechanism to degrade extracellular PAA and potentially harvest aspartic acid for nutritional uptake.

可生物降解聚合材料的使用和开发已成为人们关注的焦点,以减少环境积累和对健康的潜在影响。聚(天冬氨酸)(PAA)就是这样一种材料,它是超吸收性聚(羧酸盐)(如聚(丙烯酸酯))的一种可生物降解的替代品。目前已知有三种酶可以水解 PAA:KT-1中的PahZ1KT-1和PahZ2KT-1以及KP-2中的PahZ1KP-2。我们以前曾报道过 PahZ1KT-1 的 X 射线晶体结构,该结构揭示了一个同源二聚体复合物,其强阳离子表面横跨每个单体的一侧。在这里,我们首次报道了聚合物水解酶与 DNA 结合的特性,建模数据预测多阴离子 DNA 会在阳离子底物结合表面附近结合。我们的数据显示,来自鞘氨单胞菌 KT-1 和拟杆菌 KP-2 的 PahZ1 同源物能以纳摩尔级的结合亲和力结合 ssDNA 和 dsDNA。PahZ1KT-1 结合 ssDNA 和 dsDNA 的表观解离常数 KD,app 分别为 81 ± 14 和 19 ± 1 nM,这些估计值与 PahZ1KP-2 表现出的相同行为相似。凝胶渗透色谱法数据显示,dsDNA 结合会抑制 PahZ1 催化的每种同源物的 PAA 生物降解。我们提出了一个工作模型,即 PahZ1 与细胞外生物膜 DNA 的结合有助于将水解酶定位到首先遇到 PAA 的环境中,从而提供一种降解细胞外 PAA 的机制,并有可能收获天冬氨酸用于营养吸收。
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引用次数: 0
Theoretical Insights into MutY Glycosylase DNA Repair Mechanism. MutY 糖基化酶 DNA 修复机制的理论启示
IF 2.9 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-11 DOI: 10.1021/acs.biochem.4c00153
Alessio Olivieri, Alessandro Nicola Nardi, Marco D'Abramo

Maintaining the integrity of the genome is fundamental to living organisms. To this end, nature developed several mechanisms to find and promptly repair DNA lesions. Among them, base excision repair (BER) enzymes evolved to efficiently carry out this task. Notably, the mechanisms allowing these proteins to search for, detect, and fix DNA damage on a biologically relevant time scale still remain partially unclear. By taking MutY, a BER enzyme implied in the repair of the 8-oxoguanine-adenine mismatches, as a model system, we shed some light on the repair mechanism through a theoretical-computational approach. First, we estimated the effect of the oxidation state of the MutY iron-sulfur cluster on the protein-DNA binding. Then, the redox thermodynamics of both the protein cluster and DNA nucleobases are calculated. Finally, the charge migration kinetics along the double strand bound to the enzyme has been evaluated. The rationalization of our results indicates that the search for DNA lesions is essentially dictated by the redox chemistry of the species involved, i.e., the iron-sulfur redox cofactor and the DNA bound to the enzyme.

保持基因组的完整性是生物的根本。为此,大自然开发了多种机制来发现并及时修复 DNA 病变。其中,碱基切除修复(BER)酶就是为了高效完成这项任务而进化出来的。值得注意的是,允许这些蛋白质在生物学相关的时间尺度内搜索、检测和修复DNA损伤的机制仍然部分不清楚。MutY是一种参与修复8-氧代鸟嘌呤-腺嘌呤错配的BER酶,我们以MutY为模型系统,通过理论-计算方法揭示了修复机制。首先,我们估算了 MutY 铁硫簇的氧化态对蛋白质-DNA 结合的影响。然后,计算了蛋白质簇和 DNA 核碱基的氧化还原热力学。最后,评估了与酶结合的双链的电荷迁移动力学。我们的研究结果表明,DNA 病变的寻找基本上是由相关物种的氧化还原化学决定的,即铁硫氧化还原辅助因子和与酶结合的 DNA。
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引用次数: 0
A Facile LC-MS Method for Profiling Cholesterol and Cholesteryl Esters in Mammalian Cells and Tissues. 分析哺乳动物细胞和组织中胆固醇和胆固醇酯的简便 LC-MS 方法。
IF 2.9 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-10 DOI: 10.1021/acs.biochem.4c00160
Aakash Chandramouli, Siddhesh S Kamat

Cholesterol is central to mammalian lipid metabolism and serves many critical functions in the regulation of diverse physiological processes. Dysregulation in cholesterol metabolism is causally linked to numerous human diseases, and therefore, in vivo, the concentrations and flux of cholesterol and cholesteryl esters (fatty acid esters of cholesterol) are tightly regulated. While mass spectrometry has been an analytical method of choice for detecting cholesterol and cholesteryl esters in biological samples, the hydrophobicity, chemically inert nature, and poor ionization of these neutral lipids have often proved a challenge in developing lipidomics compatible liquid chromatography-mass spectrometry (LC-MS) methods to study them. To overcome this problem, here, we report a reverse-phase LC-MS method that is compatible with existing high-throughput lipidomics strategies and capable of identifying and quantifying cholesterol and cholesteryl esters from mammalian cells and tissues. Using this sensitive yet robust LC-MS method, we profiled different mammalian cell lines and tissues and provide a comprehensive picture of cholesterol and cholesteryl esters content in them. Specifically, among cholesteryl esters, we find that mammalian cells and tissues largely possess monounsaturated and polyunsaturated variants. Taken together, our lipidomics compatible LC-MS method to study this lipid class opens new avenues in understanding systemic and tissue-level cholesterol metabolism under various physiological conditions.

胆固醇是哺乳动物脂质代谢的核心,在调节各种生理过程中发挥着许多重要功能。胆固醇代谢失调与多种人类疾病有因果关系,因此体内胆固醇和胆固醇酯(胆固醇的脂肪酸酯)的浓度和通量受到严格调控。虽然质谱法一直是检测生物样本中胆固醇和胆固醇酯的首选分析方法,但这些中性脂质的疏水性、化学惰性和电离性差,往往成为开发与脂质组学兼容的液相色谱-质谱(LC-MS)方法来研究它们的挑战。为了克服这一问题,我们在此报告了一种反相液相色谱-质谱方法,该方法与现有的高通量脂质组学策略兼容,能够鉴定和量化哺乳动物细胞和组织中的胆固醇和胆固醇酯。利用这种灵敏而稳健的 LC-MS 方法,我们对不同的哺乳动物细胞系和组织进行了分析,全面了解了它们中胆固醇和胆固醇酯的含量。具体来说,在胆固醇酯中,我们发现哺乳动物细胞和组织大多具有单不饱和和多不饱和变体。总之,我们采用与脂质组学兼容的 LC-MS 方法来研究这类脂质,为了解各种生理条件下全身和组织水平的胆固醇代谢开辟了新途径。
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引用次数: 0
Cryo-EM Structure and Biochemical Analysis of the Human Chemokine Receptor CCR8. 人类趋化因子受体 CCR8 的低温电子显微镜结构和生化分析。
IF 2.9 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-10 DOI: 10.1021/acs.biochem.4c00121
Qi Peng, Haihai Jiang, Xinyu Cheng, Na Wang, Sili Zhou, Yuting Zhang, Tingting Yang, Yixiang Chen, Wei Zhang, Sijia Lv, Weiwei Nan, JianFei Wang, Guo-Huang Fan, Jian Li, Jin Zhang

The C-C motif chemokine receptor 8 (CCR8) is a class A G-protein-coupled receptor that has emerged as a promising therapeutic target in cancer and autoimmune diseases. In the present study, we solved the cryo-electron microscopy (cryo-EM) structure of the human CCR8-Gi complex in the absence of a ligand at 2.58 Å. Structural analysis and comparison revealed that our apo CCR8 structure undergoes some conformational changes and is similar to that in the CCL1-CCR8 complex structure, indicating an active state. In addition, the key residues of CCR8 involved in the recognition of LMD-009, a potent nonpeptide agonist, were investigated by mutating CCR8 and testing the calcium flux induced by LMD-009-CCR8 interaction. Three mutants of CCR8, Y1133.32A, Y1724.64A, and E2867.39A, showed a dramatically decreased ability in mediating calcium mobilization, indicating their key interaction with LMD-009 and key roles in activation. These structural and biochemical analyses enrich molecular insights into the agonism and activation of CCR8 and will facilitate CCR8-targeted therapy.

C-C motif趋化因子受体8(CCR8)是一种A类G蛋白偶联受体,已成为癌症和自身免疫性疾病的治疗靶点。在本研究中,我们在没有配体的情况下,用低温电子显微镜(cryo-EM)解析了人类 CCR8-Gi 复合物的 2.58 Å 结构。结构分析和比较发现,我们的载体 CCR8 结构发生了一些构象变化,与 CCL1-CCR8 复合物结构相似,表明其处于活性状态。此外,我们还通过突变 CCR8 和测试 LMD-009-CCR8 相互作用诱导的钙离子通量,研究了 CCR8 识别强效非肽激动剂 LMD-009 的关键残基。CCR8的三个突变体Y1133.32A、Y1724.64A和E2867.39A在介导钙动员方面的能力急剧下降,这表明它们与LMD-009之间存在关键的相互作用,并在激活过程中发挥关键作用。这些结构和生化分析丰富了对 CCR8 激动和活化的分子认识,将有助于 CCR8 靶向治疗。
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引用次数: 0
Antimalarial Delivery with a Ferritin-Based Protein Cage: A Step toward Developing Smart Therapeutics against Malaria 基于铁蛋白的蛋白笼抗疟输送:向开发抗疟疾智能疗法迈出的一步。
IF 2.9 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-08 DOI: 10.1021/acs.biochem.3c00692
Shruti Bhatt, Subrata Dasgupta, Chiging Tupe, Cherish Prashar, Utpal Adhikari, Kailash C Pandey*, Suman Kundu and Soumyananda Chakraborti*, 

Over the past two decades, the utilization of protein cages has witnessed exponential growth driven by their extensive applications in biotechnology and therapeutics. In the context of the recent Covid-19 pandemic, protein-cage-based scaffolds played a pivotal role in vaccine development. Beyond vaccines, these protein cages have proven valuable in diverse drug delivery applications thanks to their distinctive architecture and structural stability. Among the various types of protein cages, ferritin-based cages have taken the lead in drug delivery applications. This is primarily attributed to their ease of production, exceptional thermal stability, and nontoxic nature. While ferritin-based cages are commonly employed in anticancer drug delivery and contrast agent delivery, their efficacy in malarial drug delivery had not been explored until this study. In this investigation, several antimalarial drugs were encapsulated within horse spleen ferritin, and the binding and loading processes were validated through both experimental and computational techniques. The data unequivocally demonstrate the facile incorporation of antimalarial drugs into ferritin without disrupting its three-dimensional structure. Computational docking and molecular dynamics simulations were employed to pinpoint the precise location of the drug binding site within ferritin. Subsequent efficacy testing on Plasmodium revealed that the developed nanoconjugate, comprising the drug–ferritin conjugate, exhibited significant effectiveness in eradicating the parasite. In conclusion, the findings strongly indicate that ferritin-based carrier systems hold tremendous promise for the future of antimalarial drug delivery, offering high selectivity and limited side effects.

过去二十年来,蛋白质笼在生物技术和治疗领域的广泛应用推动了蛋白质笼利用率的指数级增长。在最近的 Covid-19 大流行中,基于蛋白笼的支架在疫苗开发中发挥了关键作用。除疫苗外,这些蛋白笼凭借其独特的结构和结构稳定性,已被证明在多种药物输送应用中具有重要价值。在各种类型的蛋白笼中,基于铁蛋白的蛋白笼在给药应用中占据领先地位。这主要归功于它们易于生产、具有优异的热稳定性和无毒性。虽然铁蛋白笼通常用于抗癌药物递送和造影剂递送,但在本研究之前,还没有人探索过它们在疟疾药物递送方面的功效。在这项研究中,几种抗疟疾药物被包裹在马脾脏铁蛋白中,并通过实验和计算技术验证了结合和装载过程。这些数据清楚地表明,抗疟药物可以在不破坏铁蛋白三维结构的情况下轻松地与铁蛋白结合。计算对接和分子动力学模拟被用来确定铁蛋白中药物结合位点的精确位置。随后对疟原虫进行的药效测试表明,所开发的由药物-铁蛋白共轭物组成的纳米共轭物在消灭寄生虫方面具有显著效果。总之,研究结果有力地表明,基于铁蛋白的载体系统具有高选择性和有限的副作用,在未来的抗疟药物递送中大有可为。
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引用次数: 0
期刊
Biochemistry Biochemistry
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