Pub Date : 2025-07-01DOI: 10.1016/j.yjsbx.2025.100133
Xiangrui Shi , Huijuan Yang , Yujie Dai , Hui Zhao , Yuhang Li , Yanxi Li , Xin Zhou , Hailong Yan , Qinghua Zhang , Wei Liu
The global spread of New Delhi metallo-β-lactamases (NDMs) has exacerbated the antimicrobial resistance crisis. This study resolved the crystal structure of NDM-1 hydrolyzing amoxicillin for the first time, revealed that the hydroxyl group in the R1 moiety of amoxicillin anchors a key water molecule (Wat1) via hydrogen bond, inducing a conformational shift in Met67 (average displacement of 3.8 Å compared to its position in complexes with ampicillin, penicillin G, and penicillin V) and impairing the hydrophobic interaction between the loop 3 and the substrate. Molecular dynamics simulations confirmed that the π-π stacking contact time between amoxicillin and the L3 critical residue Phe70 decreased to 4.3 % (ampicillin: 12.3 %), with a binding energy reduction of 10.5 kcal/mol. Steady-state kinetics showed that amoxicillin exhibited a 2.2-fold higher Km and a 5.2-fold higher kcat compared to ampicillin, demonstrating that hydrophilic R1 groups impair enzyme-substrate binding. This work demonstrates the essential role of hydrophobic interactions in L3-mediated substrate binding and provides a novel strategy for designing L3-targeted NDM-1 inhibitors: maximize hydrophobicity and minimize polar surface area in the L3 contact region to block water penetration, thereby stabilizing the inhibitor-L3 interaction.
{"title":"Crystal structure reveals the hydrophilic R1 group impairs NDM-1–ligand binding via water penetration at L3","authors":"Xiangrui Shi , Huijuan Yang , Yujie Dai , Hui Zhao , Yuhang Li , Yanxi Li , Xin Zhou , Hailong Yan , Qinghua Zhang , Wei Liu","doi":"10.1016/j.yjsbx.2025.100133","DOIUrl":"10.1016/j.yjsbx.2025.100133","url":null,"abstract":"<div><div>The global spread of New Delhi metallo-β-lactamases (NDMs) has exacerbated the antimicrobial resistance crisis. This study resolved the crystal structure of NDM-1 hydrolyzing amoxicillin for the first time, revealed that the hydroxyl group in the R1 moiety of amoxicillin anchors a key water molecule (Wat1) via hydrogen bond, inducing a conformational shift in Met67 (average displacement of 3.8 Å compared to its position in complexes with ampicillin, penicillin G, and penicillin V) and impairing the hydrophobic interaction between the loop 3 and the substrate. Molecular dynamics simulations confirmed that the π-π stacking contact time between amoxicillin and the L3 critical residue Phe70 decreased to 4.3 % (ampicillin: 12.3 %), with a binding energy reduction of 10.5 kcal/mol. Steady-state kinetics showed that amoxicillin exhibited a 2.2-fold higher <em>K</em><sub>m</sub> and a 5.2-fold higher <em>k</em><sub>cat</sub> compared to ampicillin, demonstrating that hydrophilic R1 groups impair enzyme-substrate binding. This work demonstrates the essential role of hydrophobic interactions in L3-mediated substrate binding and provides a novel strategy for designing L3-targeted NDM-1 inhibitors: maximize hydrophobicity and minimize polar surface area in the L3 contact region to block water penetration, thereby stabilizing the inhibitor-L3 interaction.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"12 ","pages":"Article 100133"},"PeriodicalIF":3.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144523258","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}
Pub Date : 2025-06-18DOI: 10.1016/j.yjsbx.2025.100132
Nino Tabagari , Franziskus Hauth , Jennifer R. Fleming , Jörg S. Hartig , Olga Mayans
Proteins are heteropolymers composed of twenty standard amino acids, but over 500 non-proteogenic amino acids exist in nature that can be misincorporated into proteins. Canavanine is an antimetabolite of the chemically similar L-arginine. It can be utilized by bacteria such as Pseudomonas canavaninivorans in the legume rhizome as a sole source of carbon and nitrogen. However, canavanine misincorporates in proteins of this bacterium as its arginyl-tRNA synthetase loads tRNAArg with both canavanine and arginine. Canavanyl-tRNAArg deacetylase (CtdA) removes canavanine from misloaded tRNAArg, preventing its protein toxicity, being the first enzyme known to edit tRNA mischarged with a non-proteinogenic amino acid. We have elucidated CtdA’s crystal structure and studied its active site using site-directed mutagenesis. We found that CtdA is a small monomeric enzyme with a central, deep cavity that predictably is the canavanine binding site and a positively charged surface area that likely coordinates the CCA-3′ tRNA attachment sequence. CtdA is distantly related to the B3/B4 cis-editing domains of the multi-subunit enzyme Phenylalanine-tRNA-Synthetase (PheRS). CdtA and B3/B4 domains from bacterial and archaeal/eukaryotic origin are three subclasses of a conserved 3D-fold that differ in type-specific indels, which shape the substrate binding site. We propose a class-unifying nomenclature of secondary structure for this fold. In CtdA, residues Y104, N105, E118 and E191 are relevant for catalysis, of which N105 is conserved in bacterial B3/B4 domains. Residue N105 is in proximity of the canavanyl-ribose junction and might coordinate the nucleophilic water molecule that attacks the substrate, possibly sharing a mechanistic role in CtdA and bacterial B3/B4 editing enzymes.
{"title":"Indel-driven evolution of the canavanine tRNA-editing deacetylase enzyme CtdA","authors":"Nino Tabagari , Franziskus Hauth , Jennifer R. Fleming , Jörg S. Hartig , Olga Mayans","doi":"10.1016/j.yjsbx.2025.100132","DOIUrl":"10.1016/j.yjsbx.2025.100132","url":null,"abstract":"<div><div>Proteins are heteropolymers composed of twenty standard amino acids, but over 500 non-proteogenic amino acids exist in nature that can be misincorporated into proteins. Canavanine is an antimetabolite of the chemically similar L-arginine. It can be utilized by bacteria such as <em>Pseudomonas canavaninivorans</em> in the legume rhizome as a sole source of carbon and nitrogen. However, canavanine misincorporates in proteins of this bacterium as its arginyl-tRNA synthetase loads tRNA<sup>Arg</sup> with both canavanine and arginine. Canavanyl-tRNA<sup>Arg</sup> deacetylase (CtdA) removes canavanine from misloaded tRNA<sup>Arg</sup>, preventing its protein toxicity, being the first enzyme known to edit tRNA mischarged with a non-proteinogenic amino acid. We have elucidated CtdA’s crystal structure and studied its active site using site-directed mutagenesis. We found that CtdA is a small monomeric enzyme with a central, deep cavity that predictably is the canavanine binding site and a positively charged surface area that likely coordinates the CCA-3′ tRNA attachment sequence. CtdA is distantly related to the B3/B4 <em>cis</em>-editing domains of the multi-subunit enzyme Phenylalanine-tRNA-Synthetase (PheRS). CdtA and B3/B4 domains from bacterial and archaeal/eukaryotic origin are three subclasses of a conserved 3D-fold that differ in type-specific indels, which shape the substrate binding site. We propose a class-unifying nomenclature of secondary structure for this fold. In CtdA, residues Y104, N105, E118 and E191 are relevant for catalysis, of which N105 is conserved in bacterial B3/B4 domains. Residue N105 is in proximity of the canavanyl-ribose junction and might coordinate the nucleophilic water molecule that attacks the substrate, possibly sharing a mechanistic role in CtdA and bacterial B3/B4 editing enzymes.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"12 ","pages":"Article 100132"},"PeriodicalIF":3.5,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144490599","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}
Pub Date : 2025-06-01DOI: 10.1016/j.yjsbx.2025.100129
David Melendez-Martinez , Adriana Morales-Martinez , Iliana Vanessa Almanza-Campos , Francisco Sierra-Valdez , Miguel Borja , Alejandro Carbajal-Saucedo , Christopher L. Parkinson , Jorge Benavides
Snake venom defensins are a toxin family found in rattlesnake venoms (Crotalus) which are comprised of crotamine-like peptides and myotoxins. Their tertiary structure resembles the β-defensin family structure. Toxins from this family, such as crotamine (C. durissus terrificus) and myotoxin a (C. viridis viridis), have been described to generate paralysis through Kv 1.3 channel blockade, using three functional basic-hydrophobic dyads (Y-K, R-W, and R-W). However, the structural and functional properties of other snake venom defensins are scarcely described. For that reason, we evaluated the structural–functional characteristics of the rattlesnake venom defensins on the Kv 1.3 channel through in silico analysis. 38 snake venom defensins were found to be peptides from 41 to 48 residues with a highly conserved sequence. The three-dimensional structures had great similitude (RMSD, <1.1 Å). Moreover, molecular dynamics simulations showed that the structures were stable (0.445 ± 0.23 nm). It was found that the snake venom defensins contain two or three basic-hydrophobic dyads, the first one is present in the N-terminal region of the defensin comprised by YK. The dyads two and three are contiguous, forming a motif in the γ-core, of which there are seven phenotypes: RWKW, RWRW, PWRR, PWKR, RWKR, RLGW, and GWRR. These dyads played a key role in the interaction of the defensins with the pore residues of the Kv1.3 channel. These results demonstrated that snake venom defensins have common structural and functional properties, interacting with the Kv 1.3 channel through the basic-hydrophobic dyads.
{"title":"Snake venom defensins: Defining the structural and functional characteristics of the toxin family","authors":"David Melendez-Martinez , Adriana Morales-Martinez , Iliana Vanessa Almanza-Campos , Francisco Sierra-Valdez , Miguel Borja , Alejandro Carbajal-Saucedo , Christopher L. Parkinson , Jorge Benavides","doi":"10.1016/j.yjsbx.2025.100129","DOIUrl":"10.1016/j.yjsbx.2025.100129","url":null,"abstract":"<div><div>Snake venom defensins are a toxin family found in rattlesnake venoms (<em>Crotalus</em>) which are comprised of crotamine-like peptides and myotoxins. Their tertiary structure resembles the β-defensin family structure. Toxins from this family, such as crotamine (<em>C. durissus terrificus</em>) and myotoxin a (<em>C. viridis viridis</em>), have been described to generate paralysis through K<sub>v</sub> 1.3 channel blockade, using three functional basic-hydrophobic dyads (Y-K, R-W, and R-W). However, the structural and functional properties of other snake venom defensins are scarcely described. For that reason, we evaluated the structural–functional characteristics of the rattlesnake venom defensins on the K<sub>v</sub> 1.3 channel through <em>in silico</em> analysis. 38 snake venom defensins were found to be peptides from 41 to 48 residues with a highly conserved sequence. The three-dimensional structures had great similitude (RMSD, <1.1 Å). Moreover, molecular dynamics simulations showed that the structures were stable (0.445 ± 0.23 nm). It was found that the snake venom defensins contain two or three basic-hydrophobic dyads, the first one is present in the N-terminal region of the defensin comprised by YK. The dyads two and three are contiguous, forming a motif in the γ-core, of which there are seven phenotypes: RWKW, RWRW, PWRR, PWKR, RWKR, RLGW, and GWRR. These dyads played a key role in the interaction of the defensins with the pore residues of the K<sub>v</sub>1.3 channel. These results demonstrated that snake venom defensins have common structural and functional properties, interacting with the K<sub>v</sub> 1.3 channel through the basic-hydrophobic dyads.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100129"},"PeriodicalIF":3.5,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144190517","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}
Pub Date : 2025-05-24DOI: 10.1016/j.yjsbx.2025.100128
Abhishek Cukkemane , Nina Becker , Tatsiana Kupreichyk , Henrike Heise , Dieter Willbold , Oliver H. Weiergräber
Disrupted in schizophrenia 1 (DISC1) is a pleiotropic scaffold protein that is postulated to comprise large disordered regions and four distinct structured segments with a high proportion of helical or coiled-coil fold. DISC1 associates with over 300 proteins and is associated with several physiological roles ranging from mitosis to cellular differentiation. Yet, the structural features of the protein are poorly characterized. The C-terminal region (C-region, res. 691–836) forms a tetramer and can also aggregate into amyloid-like fibers, potentially linked to schizophrenia and other chronic mental illnesses. Using a combination of biophysical and structural biology applications, we investigate the structural heterogeneity of three mutants of the C-region, viz., the S713E, S704C and L807-frameshift mutants. We provide evidence for the plasticity of the C region; a thin border separates the conformational flexibility of DISC1 required for interaction with a myriad of partners from disruptive aggregation. Snapshots of aggregates and fibrils growing from a nucleus are presented, along with data supporting the role of the minimal fibrillizing element in the C-region, the β-core. This segment also houses a stretch of residues that is critical for the binding of NDEL1 proteins in the mitotic spindle complex and is absent in the non-binding splice variant DISC1Δ22aa. Physiologically, both the splice variant and the fibers represent loss-of-function states that disrupt cellular division. Our findings highlight the need to decipher the structural elements within the DISC1 C-region to comprehend its physiological role and aggregation-related anomalies, and to establish a rationale for drug development.
{"title":"Tracing the aggregation pathway of the scaffold protein DISC1: Structural implications for chronic mental illnesses","authors":"Abhishek Cukkemane , Nina Becker , Tatsiana Kupreichyk , Henrike Heise , Dieter Willbold , Oliver H. Weiergräber","doi":"10.1016/j.yjsbx.2025.100128","DOIUrl":"10.1016/j.yjsbx.2025.100128","url":null,"abstract":"<div><div>Disrupted in schizophrenia 1 (DISC1) is a pleiotropic scaffold protein that is postulated to comprise large disordered regions and four distinct structured segments with a high proportion of helical or coiled-coil fold. DISC1 associates with over 300 proteins and is associated with several physiological roles ranging from mitosis to cellular differentiation. Yet, the structural features of the protein are poorly characterized. The C-terminal region (C-region, res. 691–836) forms a tetramer and can also aggregate into amyloid-like fibers, potentially linked to schizophrenia and other chronic mental illnesses. Using a combination of biophysical and structural biology applications, we investigate the structural heterogeneity of three mutants of the C-region, viz., the S713E, S704C and L807-frameshift mutants. We provide evidence for the plasticity of the C region; a thin border separates the conformational flexibility of DISC1 required for interaction with a myriad of partners from disruptive aggregation. Snapshots of aggregates and fibrils growing from a nucleus are presented, along with data supporting the role of the minimal fibrillizing element in the C-region, the β-core. This segment also houses a stretch of residues that is critical for the binding of NDEL1 proteins in the mitotic spindle complex and is absent in the non-binding splice variant DISC1Δ22aa. Physiologically, both the splice variant and the fibers represent loss-of-function states that disrupt cellular division. Our findings highlight the need to decipher the structural elements within the DISC1 C-region to comprehend its physiological role and aggregation-related anomalies, and to establish a rationale for drug development.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100128"},"PeriodicalIF":3.5,"publicationDate":"2025-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144137732","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}
Pub Date : 2025-05-23DOI: 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.
{"title":"Crystal structure of the CCA-adding enzyme from Arabidopsis thaliana","authors":"Xiao Wang, Yuan-Yuan Li, Zi-Yan Dou, Jia Wang, 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":"2025-05-23","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}
Pub Date : 2025-05-02DOI: 10.1016/j.yjsbx.2025.100125
Marten L. Chaillet , Sander Roet , Remco C. Veltkamp , Friedrich Förster
Template matching (TM) in cryo-electron tomography (cryo-ET) enables in situ detection and localization of known macromolecules. However, TM faces challenges of weak signal of the macromolecules and interfering features with a high signal-to-noise ratio, which are often addressed by time-consuming, subjective manual curation of results. To improve the detection performance we introduce pytom-match-pick, a GPU-accelerated, open-source command line interface for enhanced TM in cryo-ET. Using pytom-match-pick, we first quantify the effects of point spread function (PSF) weighting and show that a tilt-weighted PSF outperforms a binary wedge with a single defocus estimate. We also assess previously introduced background normalization methods for classification performance. This indicates that phase randomization is more effective than spectrum whitening in reducing false positives. Furthermore, a novel application of the tophat transform on score maps, combined with a dual-constraint thresholding strategy, reduces false positives and improves precision. We benchmarked pytom-match-pick on public datasets, demonstrating improved classification and localization of macromolecules like ribosomal subunits and proteasomes that led to fewer artifacts in subtomogram averages. This tool promises to advance visual proteomics by improving the efficiency and accuracy of macromolecule detection in cellular contexts.
{"title":"pytom-match-pick: A tophat-transform constraint for automated classification in template matching","authors":"Marten L. Chaillet , Sander Roet , Remco C. Veltkamp , Friedrich Förster","doi":"10.1016/j.yjsbx.2025.100125","DOIUrl":"10.1016/j.yjsbx.2025.100125","url":null,"abstract":"<div><div>Template matching (TM) in cryo-electron tomography (cryo-ET) enables <em>in situ</em> detection and localization of known macromolecules. However, TM faces challenges of weak signal of the macromolecules and interfering features with a high signal-to-noise ratio, which are often addressed by time-consuming, subjective manual curation of results. To improve the detection performance we introduce pytom-match-pick, a GPU-accelerated, open-source command line interface for enhanced TM in cryo-ET. Using pytom-match-pick, we first quantify the effects of point spread function (PSF) weighting and show that a tilt-weighted PSF outperforms a binary wedge with a single defocus estimate. We also assess previously introduced background normalization methods for classification performance. This indicates that phase randomization is more effective than spectrum whitening in reducing false positives. Furthermore, a novel application of the tophat transform on score maps, combined with a dual-constraint thresholding strategy, reduces false positives and improves precision. We benchmarked pytom-match-pick on public datasets, demonstrating improved classification and localization of macromolecules like ribosomal subunits and proteasomes that led to fewer artifacts in subtomogram averages. This tool promises to advance visual proteomics by improving the efficiency and accuracy of macromolecule detection in cellular contexts.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100125"},"PeriodicalIF":3.5,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143922897","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}
Pub Date : 2025-04-29DOI: 10.1016/j.yjsbx.2025.100126
Penghui Deng , Xiaoyue Zhang , Jianqing Wen , Mingce Xu , Pengwei Li , Hao Wang , Yunchen Bi
In cartilaginous fish, the immunoglobulin new antigen receptor (IgNAR) is naturally devoid of light chains. The variable regions of IgNAR (VNARs) are solely responsible for antigen recognition, similar to VHHs (variable domain of the heavy chain of heavy-chain antibodies) in camelids. Although VNARs have attracted growing interest, generating VNARs against membrane proteins remains challenging. Furthermore, the structure of a VNAR in complex with a membrane protein has not yet been reported. This study features a membrane protein, Chlorella virus hyaluronan synthase (CvHAS), and provides a comprehensive methodological approach to generate its specific shark VNARs, addressing several major concerns and important optimizations. We showed that shark physiological urea pressure was tolerable for CvHAS, and indirect immobilization was strongly preferred over passive adsorption for membrane proteins. Together with optimizations to improve mononuclear cell (MC) viability and VNAR expression efficiency, we successfully generated S2F6, a CvHAS-specific VNAR with nM-level high affinity. The structure of the CvHAS-S2F6 complex was then determined by cryogenic electron microscopy (cryo-EM), reporting the first membrane protein and VNAR complex structure. It shows that S2F6 binds to the cytoplasmic domain of CvHAS, with a different epitope than the reported CvHAS-specific VHHs. This study provides valuable insights into developing VNARs for membrane proteins and their applications in structural biology.
{"title":"Generation of shark single-domain antibodies as an aid for Cryo-EM structure determination of membrane proteins: Use hyaluronan synthase as an example","authors":"Penghui Deng , Xiaoyue Zhang , Jianqing Wen , Mingce Xu , Pengwei Li , Hao Wang , Yunchen Bi","doi":"10.1016/j.yjsbx.2025.100126","DOIUrl":"10.1016/j.yjsbx.2025.100126","url":null,"abstract":"<div><div>In cartilaginous fish, the immunoglobulin new antigen receptor (IgNAR) is naturally devoid of light chains. The variable regions of IgNAR (VNARs) are solely responsible for antigen recognition, similar to VHHs (variable domain of the heavy chain of heavy-chain antibodies) in camelids. Although VNARs have attracted growing interest, generating VNARs against membrane proteins remains challenging. Furthermore, the structure of a VNAR in complex with a membrane protein has not yet been reported. This study features a membrane protein, Chlorella virus hyaluronan synthase (CvHAS), and provides a comprehensive methodological approach to generate its specific shark VNARs, addressing several major concerns and important optimizations. We showed that shark physiological urea pressure was tolerable for CvHAS, and indirect immobilization was strongly preferred over passive adsorption for membrane proteins. Together with optimizations to improve mononuclear cell (MC) viability and VNAR expression efficiency, we successfully generated S2F6, a CvHAS-specific VNAR with nM-level high affinity. The structure of the CvHAS-S2F6 complex was then determined by cryogenic electron microscopy (cryo-EM), reporting the first membrane protein and VNAR complex structure. It shows that S2F6 binds to the cytoplasmic domain of CvHAS, with a different epitope than the reported CvHAS-specific VHHs. This study provides valuable insights into developing VNARs for membrane proteins and their applications in structural biology.</div></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":"11 ","pages":"Article 100126"},"PeriodicalIF":3.5,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143918313","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}
Pub Date : 2025-03-05DOI: 10.1016/j.yjsbx.2025.100124
Patrick Roth, Dimitrios Fotiadis
The phosphotransferase system glucose-specific transporter IICBGlc serves as a central nutrient uptake system in bacteria. It transports glucose across the plasma membrane via the IICGlc domain and phosphorylates the substrate within the cell to produce the glycolytic intermediate, glucose-6-phosphate, through the IIBGlc domain. Furthermore, IICGlc 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 IICGlc 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 IICBGlc from Escherichia coli. While the IIBGlc protein domain is flexible remaining unresolved, the dimeric IICGlc 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 IICGlc, 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。
{"title":"Cryo-EM structure of a phosphotransferase system glucose transporter stalled in an intermediate conformation","authors":"Patrick Roth, 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<sup>Glc</sup> serves as a central nutrient uptake system in bacteria. It transports glucose across the plasma membrane via the IIC<sup>Glc</sup> domain and phosphorylates the substrate within the cell to produce the glycolytic intermediate, glucose-6-phosphate, through the IIB<sup>Glc</sup> domain. Furthermore, IIC<sup>Glc</sup> 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<sup>Glc</sup> 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<sup>Glc</sup> from <em>Escherichia coli</em>. While the IIB<sup>Glc</sup> protein domain is flexible remaining unresolved, the dimeric IIC<sup>Glc</sup> 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<sup>Glc</sup>, 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":"2025-03-05","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}
Pub Date : 2025-02-11DOI: 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}
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.
{"title":"Molecular biophysics and inhibition mechanism of influenza virus A M2 viroporin by adamantane-based drugs – Challenges in designing antiviral agents","authors":"Kyriakos Georgiou , Dimitrios Kolokouris , 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":"2025-02-04","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}
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