Journal of Structural Biology: X最新文献

{"title":"Structural characterization and inhibition of carbonic anhydrase from Candida parapsilosis","authors":"Jiří Dostál ,&nbsp;Zdeňka Uhrová ,&nbsp;Magdalena Škrlová ,&nbsp;Stanislav Macháček ,&nbsp;Kamila Clarová ,&nbsp;Martin Lepšík ,&nbsp;Ondřej Bulvas ,&nbsp;Milan Vrábel ,&nbsp;Olga Heidingsfeld ,&nbsp;Iva Pichová","doi":"10.1016/j.yjsbx.2025.100140","DOIUrl":"10.1016/j.yjsbx.2025.100140","url":null,"abstract":"<div><div>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 <em>Candida parapsilosis</em> 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 <em>C. albicans</em>. 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.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"12 ","pages":"Article 100140"},"PeriodicalIF":5.1,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145568484","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Residues of the ribose binding site are required for human ribokinase activity","authors":"Juliana C. Ferreira ,&nbsp;Lyudmila Nedyalkova ,&nbsp;Adrian J. Villanueva ,&nbsp;Samar Fadl ,&nbsp;Zeynep Nur Cinviz ,&nbsp;Kenana Al Adem ,&nbsp;Ozge Sensoy ,&nbsp;Wael M. Rabeh","doi":"10.1016/j.yjsbx.2025.100137","DOIUrl":"10.1016/j.yjsbx.2025.100137","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"12 ","pages":"Article 100137"},"PeriodicalIF":5.1,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145415076","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Calcium stabilizes the flexible N-terminal domain of the bacterial ion channel DeCLIC","authors":"Chen Fan ,&nbsp;Marie Lycksell ,&nbsp;Yuxuan Zhuang ,&nbsp;Rebecca J. Howard ,&nbsp;Erik Lindahl","doi":"10.1016/j.yjsbx.2025.100139","DOIUrl":"10.1016/j.yjsbx.2025.100139","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"12 ","pages":"Article 100139"},"PeriodicalIF":5.1,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145568486","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"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","authors":"Arvin Saffarian Delkhosh ,&nbsp;Elaheh Hadadianpour ,&nbsp;Md Majharul Islam,&nbsp;Elka R. Georgieva","doi":"10.1016/j.yjsbx.2024.100117","DOIUrl":"10.1016/j.yjsbx.2024.100117","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100117"},"PeriodicalIF":3.5,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11714672/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142971424","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}

{"title":"SidF, a dual substrate N5-acetyl-N5-hydroxy-L-ornithine transacetylase involved in Aspergillus fumigatus siderophore biosynthesis","authors":"Thanalai Poonsiri ,&nbsp;Jan Stransky ,&nbsp;Nicola Demitri ,&nbsp;Hubertus Haas ,&nbsp;Michele Cianci ,&nbsp;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}

{"title":"Crystal structure of the CCA-adding enzyme from Arabidopsis thaliana","authors":"Xiao Wang,&nbsp;Yuan-Yuan Li,&nbsp;Zi-Yan Dou,&nbsp;Jia Wang,&nbsp;Lin Liu","doi":"10.1016/j.yjsbx.2025.100127","DOIUrl":"10.1016/j.yjsbx.2025.100127","url":null,"abstract":"<div><div>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 <em>Arabidopsis thaliana</em> (<em>At</em>CCA)<em>.</em> The overall structure of <em>At</em>CCA is similar to other class-II CCA-adding enzymes<em>,</em> but significant differences occur in the body domain. Structural comparison of body and tail domains between <em>At</em>CCA and other class-II CCA-adding enzymes unravels three specific regions of <em>At</em>CCA. Based on the modeled <em>At</em>CCA-tRNA complex, <em>At</em>CCA may have a different tRNA binding pattern. The three specific regions located in the body domain of <em>At</em>CCA also provide candidate regions for multi-targeted sorting.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100127"},"PeriodicalIF":3.5,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144168679","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular biophysics and inhibition mechanism of influenza virus A M2 viroporin by adamantane-based drugs – Challenges in designing antiviral agents","authors":"Kyriakos Georgiou ,&nbsp;Dimitrios Kolokouris ,&nbsp;Antonios Kolocouris","doi":"10.1016/j.yjsbx.2025.100122","DOIUrl":"10.1016/j.yjsbx.2025.100122","url":null,"abstract":"<div><div>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<strong>,</strong> 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.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100122"},"PeriodicalIF":3.5,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143445911","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}

{"title":"Cryo-EM structure of a phosphotransferase system glucose transporter stalled in an intermediate conformation","authors":"Patrick Roth,&nbsp;Dimitrios Fotiadis","doi":"10.1016/j.yjsbx.2025.100124","DOIUrl":"10.1016/j.yjsbx.2025.100124","url":null,"abstract":"<div><div>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, <em>n</em>-dodecyl-β-D-maltopyranoside-solubilized IICB^Glc from <em>Escherichia coli</em>. 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 <em>n</em>-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.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100124"},"PeriodicalIF":3.5,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143580418","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}

{"title":"Non-uniform Fourier transform based image classification in single-particle Cryo-EM","authors":"ZiJian Bai,&nbsp;Jian Huang","doi":"10.1016/j.yjsbx.2025.100121","DOIUrl":"10.1016/j.yjsbx.2025.100121","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100121"},"PeriodicalIF":3.5,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143378637","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}

{"title":"Production and cryo-electron microscopy structure of an internally tagged SARS-CoV-2 spike ecto-domain construct","authors":"Suruchi Singh ,&nbsp;Yi Liu ,&nbsp;Meghan Burke ,&nbsp;Vamseedhar Rayaprolu ,&nbsp;Stephen E. Stein ,&nbsp;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":"2025-06-01","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}