Journal of Structural Biology: X最新文献_第2页

Structural characterization and inhibition of carbonic anhydrase from Candida parapsilosis 假丝酵母菌碳酸酐酶的结构特征及抑制作用

IF 5.1 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-12-01 Epub Date: 2025-11-12 DOI: 10.1016/j.yjsbx.2025.100140

Jiří Dostál , Zdeňka Uhrová , Magdalena Škrlová , Stanislav Macháček , Kamila Clarová , Martin Lepšík , Ondřej Bulvas , Milan Vrábel , Olga Heidingsfeld , Iva Pichová

Fungal carbonic anhydrases (CAs) are metalloenzymes that catalyze the reversible hydration of carbon dioxide and play an essential role in fungal adaptation to environments with fluctuating CO₂ concentrations. The opportunistic pathogen Candida parapsilosis expresses a single β-class CA, CpNce103p, which is structurally distinct from human α-class CAs and therefore may represent a promising antifungal target. Here, we report the biochemical and structural characterization of CpNce103p, including its crystal structure in complex with the classical CA inhibitor acetazolamide. Mass photometry and X-ray crystallography revealed that CpNce103p forms a stable homotetramer, similar to its homolog CaNce103p from C. albicans. Inhibition constants (Ki) were measured for a panel of 16 sulfonamide derivatives. One compound attained single-digit micromolar inhibition of CpNce103p, similar to the Ki of acetazolamide. Docking suggested the compound’s binding mode, featuring zinc coordination and accommodation within the tight cavity. Our findings provide a structural basis for rational inhibitor design targeting fungal β-CAs and support CpNce103p as a viable target for the development of antifungal agents with selective activity.

真菌碳酸酐酶（CAs）是一种催化二氧化碳可逆水化的金属酶，在真菌适应二氧化碳浓度波动的环境中起着重要作用。机会致病菌假丝酵母（Candida parapsilosis）表达一个单一的β-class CA CpNce103p，它在结构上不同于人类α-class CA，因此可能是一个有前景的抗真菌靶点。本文报道了CpNce103p的生化和结构表征，包括其与经典CA抑制剂乙酰唑胺配合物的晶体结构。质谱和x射线晶体学显示CpNce103p形成稳定的同四聚体，与白色念珠菌的同源物CaNce103p相似。测定了16种磺胺衍生物的抑制常数（Ki）。其中一种化合物对CpNce103p具有个位数的微摩尔抑制作用，类似于乙酰唑胺的Ki。对接表明了化合物的结合模式，在紧密的腔内具有锌的配位和调节。我们的研究结果为合理设计针对真菌β-CAs的抑制剂提供了结构基础，并支持CpNce103p作为开发具有选择性活性的抗真菌药物的可行靶点。

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引用次数: 0

Residues of the ribose binding site are required for human ribokinase activity 核糖结合位点的残基是人类核糖激酶活性所必需的

IF 5.1 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-12-01 Epub Date: 2025-10-28 DOI: 10.1016/j.yjsbx.2025.100137

Juliana C. Ferreira , Lyudmila Nedyalkova , Adrian J. Villanueva , Samar Fadl , Zeynep Nur Cinviz , Kenana Al Adem , Ozge Sensoy , Wael M. Rabeh

Ribokinase (RK) catalyzes the phosphorylation of D-ribose to ribose-5-phosphate, an essential metabolic intermediate for the pentose phosphate pathway, nucleotide biosynthesis, redox balance, and cellular energy metabolism. Despite its central physiological role, the structural and mechanistic bases of human RK activity remain incompletely defined. Here, we present the 2.1 Å resolution crystal structure of human RK bound to ADP and Mg²⁺, revealing a conserved dimeric architecture characteristic of the PfkB family. A conserved β-clasp motif at the dimer interface stabilizes asymmetric conformations between protomers, supporting dynamic active-site gating. Mutagenesis of key ribose-coordinating residues—Asp27, Lys54, Asn57, Glu154, and Ala181—completely abolished catalytic activity, underscoring the essential roles of these residues in substrate binding and positioning. Kinetic analysis showed that the partially active E154A mutant had impaired substrate affinity and catalytic efficiency, indicating that E154 contributes to ribose coordination and active-site geometry. Molecular dynamics simulations further demonstrated that mutations of ribose-coordinating residues disrupt ribose retention and accelerate active-site opening, thereby destabilizing the catalytic pocket. Complementary thermodynamic and kinetic stability measurements revealed that the introduction of E154A reduced both the melting temperature and enzymatic half-life of RK under heat stress, linking structural flexibility to functional robustness. Collectively, these observations define a finely tuned network of ribose-binding interactions that are essential for catalytic activity, conformational regulation, and thermal stability. This work establishes a mechanistic framework for human RK function to support its exploration as a therapeutic target in metabolic and cardiovascular disorders with perturbed ribose metabolism.

核糖激酶（RK）催化d -核糖磷酸化为核糖-5-磷酸，是戊糖磷酸途径、核苷酸生物合成、氧化还原平衡和细胞能量代谢的重要代谢中间体。尽管其核心生理作用，人类RK活性的结构和机制基础仍然不完全确定。在这里，我们展示了人类RK与ADP和Mg2+结合的2.1 Å分辨率晶体结构，揭示了PfkB家族的保守二聚体结构特征。二聚体界面上保守的β-扣基序稳定了原聚体之间的不对称构象，支持动态活性位点门控。关键的核糖协调残基——asp27、Lys54、Asn57、Glu154和ala181的突变完全破坏了催化活性，强调了这些残基在底物结合和定位中的重要作用。动力学分析表明，部分活性的E154A突变体的底物亲和力和催化效率受损，表明E154参与了核糖配位和活性位点的几何结构。分子动力学模拟进一步表明，核糖配位残基的突变破坏了核糖的保留，加速了活性位点的打开，从而破坏了催化袋的稳定。补充的热力学和动力学稳定性测量表明，E154A的引入降低了RK在热应力下的熔化温度和酶促半衰期，将结构灵活性与功能稳健性联系起来。总的来说，这些观察定义了一个精细调整的核糖结合相互作用网络，这对催化活性、构象调节和热稳定性至关重要。这项工作建立了人类RK功能的机制框架，以支持其作为核糖代谢紊乱的代谢和心血管疾病的治疗靶点的探索。

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引用次数: 0

Calcium stabilizes the flexible N-terminal domain of the bacterial ion channel DeCLIC 钙稳定细菌离子通道DeCLIC的柔性n端结构域

IF 5.1 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-12-01 Epub Date: 2025-11-12 DOI: 10.1016/j.yjsbx.2025.100139

Chen Fan , Marie Lycksell , Yuxuan Zhuang , Rebecca J. Howard , Erik Lindahl

Pentameric ligand-gated ion channels (pLGICs) are responsible for the rapid conversion of chemical to electrical signals. In addition to the canonical extracellular and transmembrane domains, some prokaryotic pLGICs contain an N-terminal domain (NTD) of unclear structure and function. In one such case, the calcium-sensitive channel DeCLIC, the NTD appears to accelerate gating; however, its evident flexibility has posed a challenge to model building, and its role in calcium sensitivity is unclear. Here we report cryo-EM structures of DeCLIC in circularized lipid nanodiscs, achieving the highest resolution reported so far, and enabling definition of calcium-binding sites in both the N-terminal and canonical extracellular domains. In addition to the symmetric state, calcium depletion promoted an asymmetric conformation of the NTD, offering a structural rationale for small-angle scattering results. Behavior of these structures in molecular dynamics simulations demonstrated calcium stabilization of the NTD. These features of DeCLIC offer a model system for ion-channel modulation by a flexible accessory domain, potentially conserved in structurally homologous systems across evolution.

五聚体配体门控离子通道（plgic）负责化学信号到电信号的快速转换。除了典型的胞外和跨膜结构域外，一些原核plgic还含有一个结构和功能不明确的n端结构域（NTD）。在一个这样的例子中，钙敏感通道DeCLIC， NTD似乎加速门控；然而，其明显的灵活性对模型构建提出了挑战，其在钙敏感性中的作用尚不清楚。在这里，我们报道了环状脂质纳米圆盘中DeCLIC的低温电镜结构，达到了迄今为止报道的最高分辨率，并能够定义n端和典型细胞外结构域的钙结合位点。除了对称状态外，钙的耗尽还促进了NTD的不对称构象，为小角度散射结果提供了结构原理。这些结构在分子动力学模拟中的行为证明了NTD的钙稳定性。DeCLIC的这些特性为离子通道调制提供了一个灵活的辅助域模型系统，在整个进化过程中可能在结构同源系统中保持保守。

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引用次数: 0

Highly versatile small virus-encoded proteins in cellular membranes: A structural perspective on how proteins’ inherent conformational plasticity couples with host membranes’ properties to control cellular processes 细胞膜中高度通用的小病毒编码蛋白：蛋白质固有构象可塑性如何与宿主膜特性偶联以控制细胞过程的结构视角

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-06-01 Epub Date: 2024-12-11 DOI: 10.1016/j.yjsbx.2024.100117

Arvin Saffarian Delkhosh , Elaheh Hadadianpour , Md Majharul Islam, Elka R. Georgieva

We investigated several small viral proteins that reside and function in cellular membranes. These proteins belong to the viroporin family because they assemble into ion-conducting oligomers. However, despite forming similar oligomeric structures with analogous functions, these proteins have diverse amino acid sequences. In particular, the amino acid compositions of the proposed channel-forming transmembrane (TM) helices are vastly different—some contain residues (e.g., His, Trp, Asp, Ser) that could facilitate cation transport. Still, other viroporins’ TM helices encompass exclusively hydrophobic residues; therefore, it is difficult to explain their channels’ activity, unless other mechanisms (e.g., involving a negative lipid headgroups and/or membrane destabilization) take place. For this study, we selected the M2, Vpu, E, p13II, p7, and 2B proteins from the influenza A, HIV-1, human T-cell leukemia, hepatitis C, and picorna viruses, respectively. We provide a brief overview of the current knowledge about these proteins’ structures as well as remaining questions about more comprehensive understanding of their structures, conformational dynamics, and function. Finally, we outline strategies to utilize a multi-prong structural and computational approach to overcome current deficiencies in the knowledge about these proteins.

我们研究了几种存在于细胞膜上并起作用的小病毒蛋白。这些蛋白质属于毒孔蛋白家族，因为它们组装成离子传导低聚物。然而，尽管形成类似的低聚结构和类似的功能，这些蛋白质具有不同的氨基酸序列。特别是，所提出的形成通道的跨膜（TM）螺旋的氨基酸组成有很大的不同——一些含有可以促进阳离子运输的残基（例如，His, Trp, Asp, Ser）。尽管如此，其他病毒蛋白的TM螺旋只包含疏水性残基；因此，很难解释其通道的活性，除非发生其他机制（例如，涉及负脂质头基团和/或膜不稳定）。在这项研究中，我们分别从甲型流感病毒、HIV-1病毒、人t细胞白血病病毒、丙型肝炎病毒和小核糖核酸病毒中选择了M2、Vpu、E、p13II、p7和2B蛋白。我们简要概述了目前对这些蛋白质结构的认识，以及对其结构，构象动力学和功能的更全面理解的剩余问题。最后，我们概述了利用多管齐下的结构和计算方法来克服目前对这些蛋白质知识的不足的策略。

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引用次数: 0

SidF, a dual substrate N5-acetyl-N5-hydroxy-L-ornithine transacetylase involved in Aspergillus fumigatus siderophore biosynthesis 参与烟曲霉嗜铁菌生物合成的双底物n5 -乙酰- n5 -羟基- l-鸟氨酸转乙酰化酶。

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-06-01 Epub Date: 2024-12-26 DOI: 10.1016/j.yjsbx.2024.100119

Thanalai Poonsiri , Jan Stransky , Nicola Demitri , Hubertus Haas , Michele Cianci , Stefano Benini

Siderophore-mediated iron acquisition is essential for the virulence of Aspergillus fumigatus, a fungus causing life-threatening aspergillosis. Drugs targeting the siderophore biosynthetic pathway could help improve disease management. The transacetylases SidF and SidL generate intermediates for different siderophores in A. fumigatus. A. fumigatus has a yet unidentified transacetylase that complements SidL during iron deficiency in SidL-lacking mutants.

We present the first X-ray structure of SidF, revealing a two-domain architecture with tetrameric assembly. The N-terminal domain contributes to protein solubility and oligomerization, while the C-terminal domain containing the GCN5-related N-acetyltransferase (GNAT) motif is crucial for the enzymatic activity and mediates oligomer formation. Notably, AlphaFold modelling demonstrates structural similarity between SidF and SidL. Enzymatic assays showed that SidF can utilize acetyl-CoA as a donor, previously thought to be a substrate of SidL but not SidF, and selectively uses N5-hydroxy-L-ornithine as an acceptor.

This study elucidates the structure of SidF and reveals its role in siderophore biosynthesis. We propose SidF as the unknown transacetylase complementing SidL activity, highlighting its central role in A. fumigatus siderophore biosynthesis. Investigation of this uncharacterized GNAT protein enhances our understanding of fungal virulence and holds promise for its potential application in developing antifungal therapies.

铁载体介导的铁获取对烟曲霉的毒力至关重要，烟曲霉是一种引起危及生命的曲霉病的真菌。靶向铁载体生物合成途径的药物可以帮助改善疾病管理。烟曲霉转乙酰化酶SidF和SidL产生不同铁载体的中间体。烟曲霉有一种尚未确定的转乙酰化酶，在缺铁的SidL突变体中补充SidL。我们提出了SidF的第一个x射线结构，揭示了具有四聚体组装的双畴结构。n端结构域有助于蛋白质的溶解度和寡聚化，而含有gcn5相关的n -乙酰转移酶（GNAT）基序的c端结构域对于酶活性和介导低聚物的形成至关重要。值得注意的是，AlphaFold模型显示了SidF和SidL之间的结构相似性。酶促实验表明，SidF可以利用乙酰辅酶a作为供体，以前认为乙酰辅酶a是SidL的底物，而不是SidF，并选择性地使用n5 -羟基- l-鸟氨酸作为受体。本研究阐明了SidF的结构，揭示了其在铁载体生物合成中的作用。我们认为SidF是一种未知的转乙酰化酶，与SidL活性互补，突出了其在烟曲霉嗜铁菌生物合成中的核心作用。对这种未表征的GNAT蛋白的研究增强了我们对真菌毒力的理解，并为其在开发抗真菌治疗方面的潜在应用带来了希望。

{"title":"SidF, a dual substrate N5-acetyl-N5-hydroxy-L-ornithine transacetylase involved in Aspergillus fumigatus siderophore biosynthesis","authors":"Thanalai Poonsiri , Jan Stransky , Nicola Demitri , Hubertus Haas , Michele Cianci , Stefano Benini","doi":"10.1016/j.yjsbx.2024.100119","DOIUrl":"10.1016/j.yjsbx.2024.100119","url":null,"abstract":"<div><div>Siderophore-mediated iron acquisition is essential for the virulence of <em>Aspergillus fumigatus</em>, a fungus causing life-threatening aspergillosis. Drugs targeting the siderophore biosynthetic pathway could help improve disease management. The transacetylases SidF and SidL generate intermediates for different siderophores in <em>A. fumigatus</em>. <em>A. fumigatus</em> has a yet unidentified transacetylase that complements SidL during iron deficiency in SidL-lacking mutants.</div><div>We present the first X-ray structure of SidF, revealing a two-domain architecture with tetrameric assembly. The N-terminal domain contributes to protein solubility and oligomerization, while the C-terminal domain containing the GCN5-related N-acetyltransferase (GNAT) motif is crucial for the enzymatic activity and mediates oligomer formation. Notably, AlphaFold modelling demonstrates structural similarity between SidF and SidL. Enzymatic assays showed that SidF can utilize acetyl-CoA as a donor, previously thought to be a substrate of SidL but not SidF, and selectively uses N5-hydroxy-L-ornithine as an acceptor.</div><div>This study elucidates the structure of SidF and reveals its role in siderophore biosynthesis. We propose SidF as the unknown transacetylase complementing SidL activity, highlighting its central role in <em>A. fumigatus</em> siderophore biosynthesis. Investigation of this uncharacterized GNAT protein enhances our understanding of fungal virulence and holds promise for its potential application in developing antifungal therapies.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100119"},"PeriodicalIF":3.5,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11751504/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143023943","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Crystal structure of the CCA-adding enzyme from Arabidopsis thaliana 拟南芥cca添加酶的晶体结构

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-06-01 Epub Date: 2025-05-23 DOI: 10.1016/j.yjsbx.2025.100127

Xiao Wang, Yuan-Yuan Li, Zi-Yan Dou, Jia Wang, Lin Liu

The 3′-terminal CCA-end of tRNA is essential for the attachment of amino acids and correct positioning of the aminoacyl-tRNA in the ribosome. In higher plants, the CCA sequence is synthesized, maintained, and repaired by class-II CCA-adding enzymes encoded by a single nuclear gene but multi-targeted to the nucleus, cytoplasm, plastids, and mitochondria. The structure of plant class-II CCA-adding enzyme remains unsolved. Here we describe the crystal structure of CCA-adding enzyme from Arabidopsis thaliana (AtCCA). The overall structure of AtCCA is similar to other class-II CCA-adding enzymes, but significant differences occur in the body domain. Structural comparison of body and tail domains between AtCCA and other class-II CCA-adding enzymes unravels three specific regions of AtCCA. Based on the modeled AtCCA-tRNA complex, AtCCA may have a different tRNA binding pattern. The three specific regions located in the body domain of AtCCA also provide candidate regions for multi-targeted sorting.

tRNA的3 '端cca端对于氨基酸的附着和氨基酸基tRNA在核糖体中的正确定位至关重要。在高等植物中，CCA序列由一类CCA添加酶合成、维持和修复，这些酶由单个核基因编码，但多靶向细胞核、细胞质、质体和线粒体。植物ⅱ类cca添加酶的结构尚不清楚。本文描述了拟南芥（Arabidopsis thaliana, AtCCA）中cca添加酶的晶体结构。AtCCA的整体结构与其他ii类cca添加酶相似，但在体域上存在显著差异。AtCCA与其他ii类添加cca酶的体和尾结构域的结构比较揭示了AtCCA的三个特定区域。基于建模的AtCCA-tRNA复合物，AtCCA可能具有不同的tRNA结合模式。位于AtCCA体域的三个特定区域也为多目标排序提供了候选区域。

引用次数: 0

Molecular biophysics and inhibition mechanism of influenza virus A M2 viroporin by adamantane-based drugs – Challenges in designing antiviral agents 金刚烷酮类药物抑制流感病毒A - M2病毒孔蛋白的分子生物物理学和机制——设计抗病毒药物的挑战

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-06-01 Epub Date: 2025-02-04 DOI: 10.1016/j.yjsbx.2025.100122

Kyriakos Georgiou , Dimitrios Kolokouris , Antonios Kolocouris

The influenza A matrix 2 (AM2) protein is a prototype viroporin that conducts protons through an array of water molecules and sidechains of ionizable amino acid residues, with His37 being the most important. Amantadine is a prototype AM2 channel blocker and inhibitor of influenza A AM2 wild type (serine-31) replication. Amantadine received approval for prophylaxis against the influenza virus A in 1966. However, the characterization of the mechanism of action of amantadine targeting AM2 came 50 years after its approval as an anti-influenza A drug. We present results from experimental biophysical methods and molecular dynamics simulations for the complexes of the AM2 WT and amantadine-resistant mutant channels (V27A, L26F, S31N) in complex with adamantane-based ligands. Additionally, we describe critical experimental evidence from biochemical/functional and molecular biology experiments. Previous debates on the mechanism of drug binding and inhibition were due to the different membrane mimetic environment, the excess of the drug, and the method used, rather than the accuracy of the experiments. The collective knowledge acquired can inspire research for the development of new antivirals against influenza viruses and provide experience on the application of molecular biophysics to other viroporins.

流感A基质2 （AM2）蛋白是一种原型病毒孔蛋白，它引导质子穿过一系列水分子和可电离氨基酸残基的侧链，其中His37是最重要的。金刚烷胺是一种原型AM2通道阻滞剂和甲型流感AM2野生型（丝氨酸-31）复制抑制剂。金刚烷胺于1966年被批准用于预防甲型流感病毒。然而，金刚烷胺靶向AM2的作用机制是在其被批准为抗甲型流感药物50年后才被确定的。我们展示了AM2 WT和金刚烷胺抗性突变体通道（V27A, L26F, S31N）与金刚烷胺基配体配合物的实验生物物理方法和分子动力学模拟结果。此外，我们还描述了来自生化/功能和分子生物学实验的关键实验证据。以往关于药物结合和抑制机制的争论主要是由于不同的模拟膜环境、药物的过量和使用的方法，而不是实验的准确性。获得的集体知识可以激发针对流感病毒开发新的抗病毒药物的研究，并提供将分子生物物理学应用于其他病毒蛋白的经验。

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引用次数: 0

Cryo-EM structure of a phosphotransferase system glucose transporter stalled in an intermediate conformation 磷酸转移酶系统葡萄糖转运蛋白的低温电镜结构停滞在一个中间构象

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-06-01 Epub Date: 2025-03-05 DOI: 10.1016/j.yjsbx.2025.100124

Patrick Roth, Dimitrios Fotiadis

The phosphotransferase system glucose-specific transporter IICB^Glc serves as a central nutrient uptake system in bacteria. It transports glucose across the plasma membrane via the IIC^Glc domain and phosphorylates the substrate within the cell to produce the glycolytic intermediate, glucose-6-phosphate, through the IIB^Glc domain. Furthermore, IIC^Glc consists of a transport (TD) and a scaffold domain, with the latter being involved in dimer formation. Transport is mediated by an elevator-type mechanism within the IIC^Glc domain, where the substrate binds to the mobile TD. This domain undergoes a large-scale rigid-body movement relative to the static scaffold domain, translocating glucose across the membrane. Structures of elevator-type transporters are typically captured in either inward- or outward-facing conformations. Intermediate states remain elusive, awaiting structural determination and mechanistic interpretation. Here, we present a single-particle cryo-EM structure of purified, n-dodecyl-β-D-maltopyranoside-solubilized IICB^Glc from Escherichia coli. While the IIB^Glc protein domain is flexible remaining unresolved, the dimeric IIC^Glc transporter is found trapped in a hitherto unobserved intermediate conformational state. Specifically, the TD is located halfway between inward- and outward-facing states. Structural analysis revealed a specific n-dodecyl-β-D-maltopyranoside molecule bound to the glucose binding site. The sliding of the TD is potentially impeded halfway due to the bulky nature of the ligand and a shift of the thin gate, thereby stalling the transporter. In conclusion, this study presents a novel conformational state of IIC^Glc, and provides new structural and mechanistic insights into a potential stalling mechanism, paving the way for the rational design of transport inhibitors targeting this critical bacterial metabolic process.

磷酸转移酶系统葡萄糖特异性转运体IICBGlc是细菌的中心营养摄取系统。它通过IIBGlc结构域在质膜上运输葡萄糖，并通过IIBGlc结构域使细胞内的底物磷酸化，产生糖酵解中间体葡萄糖-6-磷酸。此外，IICGlc由转运（TD）和支架结构域组成，后者参与二聚体的形成。转运由IICGlc结构域内的升降机式机制介导，其中底物与可移动的TD结合。相对于静态支架结构域，该结构域经历了大规模的刚体运动，使葡萄糖跨膜转运。电梯式转运体的结构通常是向内或向外的构象。中间状态仍然难以捉摸，等待结构确定和机制解释。在这里，我们展示了从大肠杆菌中纯化的n-十二烷基-β- d -麦芽吡喃苷溶解的IICBGlc的单颗粒低温电镜结构。虽然IIBGlc蛋白结构域仍然是柔性的，但二聚体IICGlc转运体被发现处于迄今为止未观察到的中间构象状态。具体来说，TD位于朝内和朝外状态之间。结构分析显示一个特定的n-十二烷基-β- d -麦芽吡喃苷分子与葡萄糖结合位点结合。由于配体的体积和薄栅的移位，TD的滑动可能在中途受阻，从而使转运体停滞。总之，本研究提出了IICGlc的一种新的构象状态，并为潜在的阻滞机制提供了新的结构和机制见解，为合理设计针对这一关键细菌代谢过程的转运抑制剂铺平了道路。

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引用次数: 0

Non-uniform Fourier transform based image classification in single-particle Cryo-EM 基于非均匀傅里叶变换的单粒子冷冻电镜图像分类

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-06-01 Epub Date: 2025-02-03 DOI: 10.1016/j.yjsbx.2025.100121

ZiJian Bai, Jian Huang

In the single-particle Cryo-EM projection image classification, it is a common practice to apply the Fourier transform to the images and extract rotation-invariant features in the frequency domain. However, this process involves interpolation, which can reduce the accuracy of the results. In contrast, the non-uniform Fourier transform provides more direct and accurate computation of rotation-invariant features without the need for interpolation in the computation process. Leveraging the capabilities of the non-uniform discrete Fourier transform (NUDFT), we have developed an algorithm for the rotation-invariant classification. To highlight its potential and applicability in the field of single-particle Cryo-EM, we conducted a direct comparison with the traditional Fourier transform and other methods, demonstrating the superior performance of the NUDFT.

在单粒子冷冻电镜投影图像分类中，常用的方法是对图像进行傅里叶变换，在频域提取旋转不变性特征。然而，这个过程涉及到插值，这可能会降低结果的准确性。相比之下，非均匀傅里叶变换提供了更直接和准确的旋转不变特征计算，而不需要在计算过程中进行插值。利用非均匀离散傅里叶变换（NUDFT）的能力，我们开发了一种旋转不变分类算法。为了突出其在单粒子Cryo-EM领域的潜力和适用性，我们与传统的傅里叶变换等方法进行了直接比较，证明了NUDFT的优越性能。

引用次数: 0

Production and cryo-electron microscopy structure of an internally tagged SARS-CoV-2 spike ecto-domain construct 内部标记的SARS-CoV-2刺状结构域结构体的制备和低温电镜结构

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-06-01 Epub Date: 2025-02-11 DOI: 10.1016/j.yjsbx.2025.100123

Suruchi Singh , Yi Liu , Meghan Burke , Vamseedhar Rayaprolu , Stephen E. Stein , S. Saif Hasan

The SARS-CoV-2 spike protein is synthesized in the endoplasmic reticulum of host cells, from where it undergoes export to the Golgi and the plasma membrane or retrieval from the Golgi to the endoplasmic reticulum. Elucidating the fundamental principles of this bidirectional secretion are pivotal to understanding virus assembly and designing the next generation of spike genetic vaccine with enhanced export properties. However, the widely used strategy of C-terminal affinity tagging of the spike cytosolic tail interferes with proper bidirectional trafficking. Hence, the structural and biophysical investigations of spike protein trafficking have been hindered by a lack of appropriate spike constructs. Here we describe a strategy for the internal tagging of the spike protein. Using sequence analyses and AlphaFold modeling, we identified a site down-stream of the signal sequence for the insertion of a twin-strep-tag, which facilitates purification of an ecto-domain construct from the extra-cellular medium of mammalian Expi293F cells. Mass spectrometry analyses show that the internal tag has minimal impact on N-glycan modifications, which are pivotal for spike-host interactions. Single particle cryo-electron microscopy reconstructions of the spike ecto-domain reveal conformational states compatible for ACE2 receptor interactions, further solidifying the feasibility of the internal tagging strategy. Collectively, these results present a substantial advance towards reagent development for the investigations of spike protein trafficking during coronavirus infection and genetic vaccination.

SARS-CoV-2刺突蛋白在宿主细胞的内质网合成，从那里输出到高尔基体和质膜，或从高尔基体返回到内质网。阐明这种双向分泌的基本原理对于理解病毒组装和设计具有增强出口特性的下一代刺突基因疫苗至关重要。然而，广泛使用的穗胞质尾部c端亲和标记策略干扰了正常的双向运输。因此，由于缺乏合适的刺突结构，刺突蛋白运输的结构和生物物理研究受到阻碍。在这里，我们描述了刺突蛋白的内部标记策略。利用序列分析和AlphaFold模型，我们确定了信号序列下游的一个位置，用于插入双链标签，这有助于从哺乳动物Expi293F细胞的细胞外培养基中纯化外结构域结构体。质谱分析表明，内部标签对n -聚糖修饰的影响最小，而n -聚糖修饰是刺-宿主相互作用的关键。单粒子低温电镜重建的尖峰外位结构域揭示了与ACE2受体相互作用相容的构象状态，进一步巩固了内部标记策略的可行性。总的来说，这些结果为研究冠状病毒感染和遗传疫苗接种期间刺突蛋白运输的试剂开发提供了实质性的进展。

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引用次数: 0