Journal of Structural Biology: X最新文献

英文中文

Cryo-EM structure of a phosphotransferase system glucose transporter stalled in an intermediate conformation

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub 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.

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引用次数: 0

Production and cryo-electron microscopy structure of an internally tagged SARS-CoV-2 spike ecto-domain construct

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub 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.

{"title":"Production and cryo-electron microscopy structure of an internally tagged SARS-CoV-2 spike ecto-domain construct","authors":"Suruchi Singh , Yi Liu , Meghan Burke , Vamseedhar Rayaprolu , Stephen E. Stein , S. Saif Hasan","doi":"10.1016/j.yjsbx.2025.100123","DOIUrl":"10.1016/j.yjsbx.2025.100123","url":null,"abstract":"<div><div>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 <em>N</em>-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.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100123"},"PeriodicalIF":3.5,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143437729","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

Molecular biophysics and inhibition mechanism of influenza virus A M2 viroporin by adamantane-based drugs – Challenges in designing antiviral agents

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub 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.

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引用次数: 0

Non-uniform Fourier transform based image classification in single-particle Cryo-EM

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub 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.

引用次数: 0

Protein identification using Cryo-EM and artificial intelligence guides improved sample purification

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-01-21 DOI: 10.1016/j.yjsbx.2025.100120

Kenneth D. Carr , Dane Evan D. Zambrano , Connor Weidle , Alex Goodson , Helen E. Eisenach , Harley Pyles , Alexis Courbet , Neil P. King , Andrew J. Borst

Protein purification is essential in protein biochemistry, structural biology, and protein design, enabling the determination of protein structures, the study of biological mechanisms, and the characterization of both natural and de novo designed proteins. However, standard purification strategies often encounter challenges, such as unintended co-purification of contaminants alongside the target protein. This issue is particularly problematic for self-assembling protein nanomaterials, where unexpected geometries may reflect novel assembly states, cross-contamination, or native proteins originating from the expression host. Here, we used an automated structure-to-sequence pipeline to first identify an unknown co-purifying protein found in several purified designed protein samples. By integrating cryo-electron microscopy (Cryo-EM), ModelAngelo’s sequence-agnostic model-building, and Protein BLAST, we identified the contaminant as dihydrolipoamide succinyltransferase (DLST). This identification was validated through comparisons with DLST structures in the Protein Data Bank, AlphaFold 3 predictions based on the DLST sequence from our E. coli expression vector, and traditional biochemical methods. The identification informed subsequent modifications to our purification protocol, which successfully excluded DLST from future preparations. To explore the potential broader utility of this approach, we benchmarked four computational methods for DLST identification across varying resolution ranges. This study demonstrates the successful application of a structure-to-sequence protein identification workflow, integrating Cryo-EM, ModelAngelo, Protein BLAST, and AlphaFold 3 predictions, to identify and ultimately help guide the removal of DLST from sample purification efforts. It highlights the potential of combining Cryo-EM with AI-driven tools for accurate protein identification and addressing purification challenges across diverse contexts in protein science.

{"title":"Protein identification using Cryo-EM and artificial intelligence guides improved sample purification","authors":"Kenneth D. Carr , Dane Evan D. Zambrano , Connor Weidle , Alex Goodson , Helen E. Eisenach , Harley Pyles , Alexis Courbet , Neil P. King , Andrew J. Borst","doi":"10.1016/j.yjsbx.2025.100120","DOIUrl":"10.1016/j.yjsbx.2025.100120","url":null,"abstract":"<div><div>Protein purification is essential in protein biochemistry, structural biology, and protein design, enabling the determination of protein structures, the study of biological mechanisms, and the characterization of both natural and de novo designed proteins. However, standard purification strategies often encounter challenges, such as unintended co-purification of contaminants alongside the target protein. This issue is particularly problematic for self-assembling protein nanomaterials, where unexpected geometries may reflect novel assembly states, cross-contamination, or native proteins originating from the expression host. Here, we used an automated structure-to-sequence pipeline to first identify an unknown co-purifying protein found in several purified designed protein samples. By integrating cryo-electron microscopy (Cryo-EM), ModelAngelo’s sequence-agnostic model-building, and Protein BLAST, we identified the contaminant as dihydrolipoamide succinyltransferase (DLST). This identification was validated through comparisons with DLST structures in the Protein Data Bank, AlphaFold 3 predictions based on the DLST sequence from our E. coli expression vector, and traditional biochemical methods. The identification informed subsequent modifications to our purification protocol, which successfully excluded DLST from future preparations. To explore the potential broader utility of this approach, we benchmarked four computational methods for DLST identification across varying resolution ranges. This study demonstrates the successful application of a structure-to-sequence protein identification workflow, integrating Cryo-EM, ModelAngelo, Protein BLAST, and AlphaFold 3 predictions, to identify and ultimately help guide theremoval of DLST from sample purification efforts. It highlights the potential of combining Cryo-EM with AI-driven tools for accurate protein identification and addressing purification challenges across diverse contexts in protein science.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100120"},"PeriodicalIF":3.5,"publicationDate":"2025-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143130613","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

SidF, a dual substrate N5-acetyl-N5-hydroxy-L-ornithine transacetylase involved in Aspergillus fumigatus siderophore biosynthesis

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub 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.

{"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":"2024-12-26","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

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 : 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

Corrigendum to “Minimizing ice contamination during specimen preparation for cryo-soft X-ray tomography and cryo-electron tomography” [J. Struct. Biol.: X 10(2024) 100113] 对 "尽量减少低温软 X 射线断层成像和低温电子断层成像标本制备过程中的冰污染 "的更正[J. Struct.

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2024-10-30 DOI: 10.1016/j.yjsbx.2024.100115

Chia-Chun Hsieh, Zi-Jing Lin, Lee-Jene Lai

引用次数: 0

Editorial by Natalie Reznikov [for Buss et al., “Hierarchical organization of bone in three dimensions: A twist of twists” (2022)] Natalie Reznikov [为 Buss 等人撰写的社论《骨骼的三维分层组织：扭曲的扭曲"（2022 年］

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2024-10-30 DOI: 10.1016/j.yjsbx.2024.100116

引用次数: 0

Structural analysis of the stable form of fibroblast growth factor 2 – FGF2-STAB 成纤维细胞生长因子 2（FGF2-STAB）稳定形式的结构分析

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2024-10-24 DOI: 10.1016/j.yjsbx.2024.100112

Gabin de La Bourdonnaye , Martin Marek , Tereza Ghazalova , Jiri Damborsky , Petr Pachl , Jiri Brynda , Veronika Stepankova , Radka Chaloupkova

Fibroblast growth factor 2 (FGF2) is a signaling protein that plays a significant role in tissue development and repair. FGF2 binds to fibroblast growth factor receptors (FGFRs) alongside its co-factor heparin, which protects FGF2 from degradation. The binding between FGF2 and FGFRs induces intracellular signaling pathways such as RAS-MAPK, PI3K-AKT, and STAT. FGF2 has strong potential for application in cell culturing, wound healing, and cosmetics but the potential is severely limited by its low protein stability. The thermostable variant FGF2-STAB was constructed by computer-assisted protein engineering to overcome the natural limitation of FGF2. Previously reported characterization of FGF2-STAB revealed an enhanced ability to induce MAP/ERK signaling while having a lower dependence on heparin when compared with FGF2-wt. Here we report the crystal structure of FGF2-STAB solved at 1.3 Å resolution. Protein stabilization is achieved by newly formed hydrophobic interactions, polar contacts, and one additional hydrogen bond. The overall structure of FGF2-STAB is similar to FGF2-wt and does not reveal information on the experimentally observed lower dependence on heparin. A noticeable difference in flexibility in the receptor binding region can explain the differences in signaling between FGF2-STAB and its wild-type counterpart. Our structural analysis provided molecular insights into the stabilization and unique biological properties of FGF2-STAB.

成纤维细胞生长因子 2（FGF2）是一种信号蛋白，在组织发育和修复中发挥着重要作用。FGF2 与成纤维细胞生长因子受体（FGFRs）结合，其辅助因子肝素可保护 FGF2 免受降解。FGF2 与 FGFRs 之间的结合可诱导细胞内信号通路，如 RAS-MAPK、PI3K-AKT 和 STAT。FGF2 在细胞培养、伤口愈合和美容方面有很大的应用潜力，但由于其蛋白质稳定性较低，应用潜力受到严重限制。为了克服 FGF2 的天然限制，我们通过计算机辅助蛋白质工程构建了恒温变体 FGF2-STAB。与 FGF2-wt 相比，FGF2-STAB 诱导 MAP/ERK 信号转导的能力更强，同时对肝素的依赖性更低。蛋白质的稳定是通过新形成的疏水相互作用、极性接触和一个额外的氢键实现的。FGF2-STAB 的整体结构与 FGF2-wt 相似，并没有揭示实验观察到的肝素依赖性较低的信息。受体结合区灵活性的明显差异可以解释 FGF2-STAB 与野生型受体之间信号传导的差异。我们的结构分析提供了有关 FGF2-STAB 稳定性和独特生物特性的分子见解。

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