Pub Date : 2025-09-29DOI: 10.1016/j.str.2025.09.003
Chenyao Li, Rouslan G. Efremov
Ryanodine receptors (RyRs) are intracellular tetrameric ion channels responsible for Ca2+ release from the sarcoplasmic and endoplasmic reticulum. Ryanodine receptor 1 (RyR1) isoform, critical for muscle contraction, has been studied most extensively. While cryoelectron microscopy (cryo-EM) has been instrumental in revealing near-atomic details of RyR gating mechanisms, the open probability of RyR1 under cryo-EM conditions is notably lower than that observed in electrophysiological studies. Here, we present a cryo-EM study examining the open probability of RyR1 solubilized in CHAPS with varying lipid concentrations. We found that increasing lipid concentration from 0.001% to 0.05% raised the RyR1 open probability from 16% to 84%, whereas RyR1 reconstituted into lipid nanodiscs remained closed. We modeled 72 lipid molecules in the map reconstructed at the highest lipid concentration. These findings demonstrate the important role of lipids in modulating the open fraction of solubilized RyR1 channels under cryo-EM conditions and suggest optimal lipid mimetics for structural studies of RyR1 gating.
{"title":"Lipids modulate the open probability of RyR1 under cryo-EM conditions","authors":"Chenyao Li, Rouslan G. Efremov","doi":"10.1016/j.str.2025.09.003","DOIUrl":"https://doi.org/10.1016/j.str.2025.09.003","url":null,"abstract":"Ryanodine receptors (RyRs) are intracellular tetrameric ion channels responsible for Ca<sup>2+</sup> release from the sarcoplasmic and endoplasmic reticulum. Ryanodine receptor 1 (RyR1) isoform, critical for muscle contraction, has been studied most extensively. While cryoelectron microscopy (cryo-EM) has been instrumental in revealing near-atomic details of RyR gating mechanisms, the open probability of RyR1 under cryo-EM conditions is notably lower than that observed in electrophysiological studies. Here, we present a cryo-EM study examining the open probability of RyR1 solubilized in CHAPS with varying lipid concentrations. We found that increasing lipid concentration from 0.001% to 0.05% raised the RyR1 open probability from 16% to 84%, whereas RyR1 reconstituted into lipid nanodiscs remained closed. We modeled 72 lipid molecules in the map reconstructed at the highest lipid concentration. These findings demonstrate the important role of lipids in modulating the open fraction of solubilized RyR1 channels under cryo-EM conditions and suggest optimal lipid mimetics for structural studies of RyR1 gating.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"67 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145183026","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Interleukin-31 (IL-31) signals through the IL-31 receptor alpha (IL-31RA) and oncostatin M receptor beta (OSMRβ) heterodimer, mediating pruritus, dermatitis, inflammatory responses, neuroimmune interactions, and certain cancers. Here, we present the crystal structure of canine IL-31 (cIL-31) in complex with a neutralizing caninized monoclonal antibody (2D10-2). This antibody competitively inhibited cIL-31 binding to canine OSMRβ (cOSMRβ) but not to canine IL-31RA (cIL-31RA). Moreover, it effectively blocked cIL-31-induced STAT5 phosphorylation in vitro and alleviated cIL-31-induced pruritus in beagle dogs. Structural analysis identified key antibody-binding residues in α-helical A, α-helical D, and the AB loop of cIL-31. Systematic mutagenesis based on the complex structure further defined the conformational epitopes of cIL-31 recognized by cOSMRβ. In summary, this study reports the IL-31 structure, revealing a four-α-helical bundle cytokine, and elucidates 2D10-2’s neutralizing mechanism by targeting the cIL-31-cOSMRβ interaction. These findings advance our understanding of IL-31 and offer insights for developing IL-31-targeted therapeutics.
{"title":"Structural insights into IL-31 signaling inhibition by a neutralizing antibody","authors":"Tianling Guo, Yuxin Zheng, Zheng Fan, Ping Liu, Yan Chai, Xiaoping Liao, Caili Zhang, Xuefei Pang, Delin Li, Feng Gao, Haixia Xiao","doi":"10.1016/j.str.2025.09.002","DOIUrl":"https://doi.org/10.1016/j.str.2025.09.002","url":null,"abstract":"Interleukin-31 (IL-31) signals through the IL-31 receptor alpha (IL-31RA) and oncostatin M receptor beta (OSMRβ) heterodimer, mediating pruritus, dermatitis, inflammatory responses, neuroimmune interactions, and certain cancers. Here, we present the crystal structure of canine IL-31 (cIL-31) in complex with a neutralizing caninized monoclonal antibody (2D10-2). This antibody competitively inhibited cIL-31 binding to canine OSMRβ (cOSMRβ) but not to canine IL-31RA (cIL-31RA). Moreover, it effectively blocked cIL-31-induced STAT5 phosphorylation <em>in vitro</em> and alleviated cIL-31-induced pruritus in beagle dogs. Structural analysis identified key antibody-binding residues in α-helical A, α-helical D, and the AB loop of cIL-31. Systematic mutagenesis based on the complex structure further defined the conformational epitopes of cIL-31 recognized by cOSMRβ. In summary, this study reports the IL-31 structure, revealing a four-α-helical bundle cytokine, and elucidates 2D10-2’s neutralizing mechanism by targeting the cIL-31-cOSMRβ interaction. These findings advance our understanding of IL-31 and offer insights for developing IL-31-targeted therapeutics.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"17 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145140951","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Release of AlphaFold 2 and subsequent development of AlphaFold 3 had a profound impact on protein structure prediction, providing near-experimental accuracy. However, the utility of AF2’s confidence index (pLDDT) as indicators of protein flexibility remains underexplored and debated. In this large-scale study, we evaluate AF2’s pLDDT as a predictor of protein flexibility by comparing it with flexibility metrics derived from molecular dynamics (MD) simulations from the ATLAS dataset, NMR ensembles, and experimental B-factors. We also assess the efficiency of ESMFold pLDDT and AlphaFold 3 in this context. Our findings reveal that AF2 pLDDT reasonably correlates with MD and NMR-derived flexibility metrics, but fails to capture flexibility in the presence of interacting partners, and therefore need to be cautiously interpreted. Furthermore, AF2 pLDDT appears more relevant than B-factor values for evaluation of protein flexibility. While AF3 shows slight improvements in capturing protein dynamics, MD simulations remain superior for comprehensive flexibility assessment.
{"title":"Flexibility or uncertainty? A critical assessment of AlphaFold 2 pLDDT","authors":"Yann Vander Meersche, Julien Diharce, Jean-Christophe Gelly, Tatiana Galochkina","doi":"10.1016/j.str.2025.09.001","DOIUrl":"https://doi.org/10.1016/j.str.2025.09.001","url":null,"abstract":"Release of AlphaFold 2 and subsequent development of AlphaFold 3 had a profound impact on protein structure prediction, providing near-experimental accuracy. However, the utility of AF2’s confidence index (pLDDT) as indicators of protein flexibility remains underexplored and debated. In this large-scale study, we evaluate AF2’s pLDDT as a predictor of protein flexibility by comparing it with flexibility metrics derived from molecular dynamics (MD) simulations from the ATLAS dataset, NMR ensembles, and experimental B-factors. We also assess the efficiency of ESMFold pLDDT and AlphaFold 3 in this context. Our findings reveal that AF2 pLDDT reasonably correlates with MD and NMR-derived flexibility metrics, but fails to capture flexibility in the presence of interacting partners, and therefore need to be cautiously interpreted. Furthermore, AF2 pLDDT appears more relevant than B-factor values for evaluation of protein flexibility. While AF3 shows slight improvements in capturing protein dynamics, MD simulations remain superior for comprehensive flexibility assessment.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"85 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145134461","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-17DOI: 10.1016/j.str.2025.08.019
Yilun Han, Mohamed Ghadie, Yu Xia
Protein-protein interactions (PPIs) and genetic interactions are central to cellular function. We investigate their relationship in the structurally resolved yeast PPI network, specifically the relationship between PPI structural divergence and functional divergence. For pairs of proteins (“interactor pairs”) binding to the same target protein, we measure PPI structural divergence using interfacial overlap (the number of interfacial residues in the target protein shared between the interactor pair), and functional divergence using genetic interaction profile similarity. We find a significant and robust negative correlation between interfacial overlap and genetic interaction profile similarity, where interactor pairs with large shared interface on the target protein tend to perform divergent phenotypic-level functions. This relationship is the strongest when functional similarity is measured by genetic interaction profile similarity, rather than by gene ontology-based functional similarity. Our findings suggest that competitive binding drives functional divergence of proteins at the phenotypic level.
{"title":"The relationship between interfacial overlap and functional divergence in the yeast protein-protein interaction network","authors":"Yilun Han, Mohamed Ghadie, Yu Xia","doi":"10.1016/j.str.2025.08.019","DOIUrl":"https://doi.org/10.1016/j.str.2025.08.019","url":null,"abstract":"Protein-protein interactions (PPIs) and genetic interactions are central to cellular function. We investigate their relationship in the structurally resolved yeast PPI network, specifically the relationship between PPI structural divergence and functional divergence. For pairs of proteins (“interactor pairs”) binding to the same target protein, we measure PPI structural divergence using interfacial overlap (the number of interfacial residues in the target protein shared between the interactor pair), and functional divergence using genetic interaction profile similarity. We find a significant and robust negative correlation between interfacial overlap and genetic interaction profile similarity, where interactor pairs with large shared interface on the target protein tend to perform divergent phenotypic-level functions. This relationship is the strongest when functional similarity is measured by genetic interaction profile similarity, rather than by gene ontology-based functional similarity. Our findings suggest that competitive binding drives functional divergence of proteins at the phenotypic level.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"64 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145072048","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-17DOI: 10.1016/j.str.2025.08.018
Jonathan Webb, Jeremy J.M. Liew, Andrew D. Gnann, Khandan Ilkhani, MacKenzie Patterson, Sayantanee Paul, Marta Forés, Gerardo Jiménez, Alexey Veraksa, Daniel P. Dowling
The HMG-box protein capicua (CIC) is a conserved transcriptional repressor with key functions in development and disease. CIC binding of DNA requires both its HMG-box and a separate domain called C1. How these domains cooperate to recognize specific DNA sequences is not known. Here, we report the crystal structure of the human CIC HMG-box and C1 domains complexed with a DNA oligomer containing a consensus octameric binding site. We find that both domains adopt tri-helical structures that pack against opposite sides of the DNA helix. The C1 domain folds into a helix-turn-helix (HTH) structure, inserting into the DNA major groove to enhance affinity. We investigate the system using molecular dynamics simulations and binding assays that interrogate the observed HMG-box and C1 domain interface and prominent cancer variants. Our results reveal a unique bipartite DNA-binding module and provide insights into the effects of cancer and domain interface mutations.
{"title":"Molecular basis of DNA recognition by the HMG-box-C1 module of capicua","authors":"Jonathan Webb, Jeremy J.M. Liew, Andrew D. Gnann, Khandan Ilkhani, MacKenzie Patterson, Sayantanee Paul, Marta Forés, Gerardo Jiménez, Alexey Veraksa, Daniel P. Dowling","doi":"10.1016/j.str.2025.08.018","DOIUrl":"https://doi.org/10.1016/j.str.2025.08.018","url":null,"abstract":"The HMG-box protein capicua (CIC) is a conserved transcriptional repressor with key functions in development and disease. CIC binding of DNA requires both its HMG-box and a separate domain called C1. How these domains cooperate to recognize specific DNA sequences is not known. Here, we report the crystal structure of the human CIC HMG-box and C1 domains complexed with a DNA oligomer containing a consensus octameric binding site. We find that both domains adopt tri-helical structures that pack against opposite sides of the DNA helix. The C1 domain folds into a helix-turn-helix (HTH) structure, inserting into the DNA major groove to enhance affinity. We investigate the system using molecular dynamics simulations and binding assays that interrogate the observed HMG-box and C1 domain interface and prominent cancer variants. Our results reveal a unique bipartite DNA-binding module and provide insights into the effects of cancer and domain interface mutations.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"92 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145072046","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-12DOI: 10.1016/j.str.2025.08.017
Pooja Asthana, Sonya Lee, Christian M. MacDonald, Ian B. Seiple, James S. Fraser
Streptogramins are potent antibiotics targeting bacterial ribosome. The synergistic binding of group A and B streptogramins to 50S-ribosome yields bactericidal effects. However, their efficacy is compromised by resistance mechanisms, including enzymatic acetylation of group A streptogramins by virginiamycin acetyltransferase (Vat) enzymes, which reduces their affinity for ribosomes. Using fragment-based drug discovery we identified starting points for development of VatD inhibitors. X-ray crystallography screening revealed three primary fragment-binding sites on VatD. In the acetyl-binding subsite, fragments stabilized distinct conformational states in critical residues, His82 and Trp121. In the antibiotic-binding site, two fragments formed interactions that could be leveraged for competitive inhibition. Elaborations of these fragments showed weak inhibition of VatD activity, indicating potential for further optimization. These findings establish initial hits that could restore streptogramin efficacy by targeting VatD directly, providing a structural foundation for inhibitor development against resistant bacterial strains.
{"title":"Initial leads to combat streptogramin resistance generated from X-ray fragment screening against VatD","authors":"Pooja Asthana, Sonya Lee, Christian M. MacDonald, Ian B. Seiple, James S. Fraser","doi":"10.1016/j.str.2025.08.017","DOIUrl":"https://doi.org/10.1016/j.str.2025.08.017","url":null,"abstract":"Streptogramins are potent antibiotics targeting bacterial ribosome. The synergistic binding of group A and B streptogramins to 50S-ribosome yields bactericidal effects. However, their efficacy is compromised by resistance mechanisms, including enzymatic acetylation of group A streptogramins by virginiamycin acetyltransferase (Vat) enzymes, which reduces their affinity for ribosomes. Using fragment-based drug discovery we identified starting points for development of VatD inhibitors. X-ray crystallography screening revealed three primary fragment-binding sites on VatD. In the acetyl-binding subsite, fragments stabilized distinct conformational states in critical residues, His82 and Trp121. In the antibiotic-binding site, two fragments formed interactions that could be leveraged for competitive inhibition. Elaborations of these fragments showed weak inhibition of VatD activity, indicating potential for further optimization. These findings establish initial hits that could restore streptogramin efficacy by targeting VatD directly, providing a structural foundation for inhibitor development against resistant bacterial strains.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"67 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145043308","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
ASC-1 homology (ASCH) domain family proteins are believed to play essential roles in RNA metabolism, but detailed structural and functional information is limited. Research has shown that the E. coli enzyme YqfB, which contains an ASCH domain, has amidohydrolase activity, converting N4-acetylcytidine (ac4C) RNA nucleoside into cytidine. Here, we present the crystal structures of EcYqfB both in its unbound state and bound to a substrate. Our analysis reveals how the substrate interacts with the enzyme, offering insights into its catalytic mechanism. In vivo experiments further show that deleting EcYqfB does not change overall ac4C levels across various RNA types, indicating that EcYqfB specifically functions in ac4C nucleoside metabolism. We also determined the structures of two homologous proteins: mouse EOLA1 and the human TRIP4-ASCH domain, highlighting differences in their substrate preferences. These findings offer important insights for future research into the structure and function of the ASCH domain protein family.
{"title":"Structural analysis of ASCH domain-containing proteins and their implications for nucleotide processing","authors":"Chunyan Meng, Xiaoyan Shi, Wenting Guo, Xing Jian, Jie Zhao, Yan Wen, Ruiqi Wang, Yu Li, Sha Xu, Haitao Chen, Jiayu Zhang, Mingjia Chen, Hao Chen, Baixing Wu","doi":"10.1016/j.str.2025.08.015","DOIUrl":"https://doi.org/10.1016/j.str.2025.08.015","url":null,"abstract":"ASC-1 homology (ASCH) domain family proteins are believed to play essential roles in RNA metabolism, but detailed structural and functional information is limited. Research has shown that the <em>E. coli</em> enzyme YqfB, which contains an ASCH domain, has amidohydrolase activity, converting <em>N</em><sup>4</sup>-acetylcytidine (ac<sup>4</sup>C) RNA nucleoside into cytidine. Here, we present the crystal structures of <em>Ec</em>YqfB both in its unbound state and bound to a substrate. Our analysis reveals how the substrate interacts with the enzyme, offering insights into its catalytic mechanism. <em>In vivo</em> experiments further show that deleting <em>Ec</em>YqfB does not change overall ac<sup>4</sup>C levels across various RNA types, indicating that <em>Ec</em>YqfB specifically functions in ac<sup>4</sup>C nucleoside metabolism. We also determined the structures of two homologous proteins: mouse EOLA1 and the human TRIP4-ASCH domain, highlighting differences in their substrate preferences. These findings offer important insights for future research into the structure and function of the ASCH domain protein family.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"24 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145043309","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-08DOI: 10.1016/j.str.2025.08.013
Chang Wang, Amin Khosrozadeh, Ioan Iacovache, Benoît Zuber
Cryo-electron tomography (cryoET) provides 3D datasets of organelles and proteins at nanometer and sub-nanometer resolution. However, locating target proteins in live cells remains a significant challenge. Conventional labeling methods, such as fluorescent protein tagging and immunogold labeling, are unsuitable for small structures in vitrified samples at molecular resolution. Directly linking large, visually identifiable proteins to target proteins may alter their structure, localization, and function. To overcome this, we employed a rapamycin-induced oligomer formation system involving two tags, FK506 binding protein (FKBP) and FKBP-rapamycin binding (FRB), which bind in the presence of rapamycin. FKBP is linked to the target protein, while FRB is linked to ferritin, a large (10–12 nm) iron-binding complex that creates strong contrast in cryoET. Upon adding rapamycin to the cell medium, the iron-loaded ferritin accurately marks the target protein location. As in situ cryoET with subtomogram averaging advances, our method addresses the persistent challenge of locating target proteins in live cells.
{"title":"Genetically encoded FerriTag as a specific label for cryo-electron tomography","authors":"Chang Wang, Amin Khosrozadeh, Ioan Iacovache, Benoît Zuber","doi":"10.1016/j.str.2025.08.013","DOIUrl":"https://doi.org/10.1016/j.str.2025.08.013","url":null,"abstract":"Cryo-electron tomography (cryoET) provides 3D datasets of organelles and proteins at nanometer and sub-nanometer resolution. However, locating target proteins in live cells remains a significant challenge. Conventional labeling methods, such as fluorescent protein tagging and immunogold labeling, are unsuitable for small structures in vitrified samples at molecular resolution. Directly linking large, visually identifiable proteins to target proteins may alter their structure, localization, and function. To overcome this, we employed a rapamycin-induced oligomer formation system involving two tags, FK506 binding protein (FKBP) and FKBP-rapamycin binding (FRB), which bind in the presence of rapamycin. FKBP is linked to the target protein, while FRB is linked to ferritin, a large (10–12 nm) iron-binding complex that creates strong contrast in cryoET. Upon adding rapamycin to the cell medium, the iron-loaded ferritin accurately marks the target protein location. As <em>in situ</em> cryoET with subtomogram averaging advances, our method addresses the persistent challenge of locating target proteins in live cells.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"69 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145009471","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-05DOI: 10.1016/j.str.2025.08.012
Hao Xu, Yimin Zhang, Qinru Bai, Linli He, Qihao Chen, Yunlong Qiu, Renjie Li, Jie Yu, Jun Zhao, Yan Zhao
GABA (g-aminobutyric acid) transporter 3 (GAT3) is primarily found in glial cells and is essential for regulating GABA homeostasis in the central nervous system by mediating GABA uptake. Consequently, GAT3 has emerged as a significant therapeutic target for the treatment of epilepsy. In this study, we present the cryoelectron microscopy (cryo-EM) structures of GAT3 bound to its substrate GABA, the selective inhibitor SNAP-5114, and in the substrate-free state. GAT3 binds to GABA in an inward-facing conformation, while SNAP-5114 occupies the GABA-binding pocket and is stabilized by extensive interactions with surrounding residues. Functional studies reveal that E66 plays a pivotal role in determining the substrate-binding mode and specificity of SNAP-5114 binding. Taken together, our study clarifies the GABA binding mechanism of GAT3 and reveals the molecular basis for the specific inhibition of SNAP-5114, offering valuable insights for developing GAT3 subtypes selective inhibitors, which hold potential as a treatment for epilepsy.
{"title":"Substrate and inhibitor binding of human GABA transporter 3","authors":"Hao Xu, Yimin Zhang, Qinru Bai, Linli He, Qihao Chen, Yunlong Qiu, Renjie Li, Jie Yu, Jun Zhao, Yan Zhao","doi":"10.1016/j.str.2025.08.012","DOIUrl":"https://doi.org/10.1016/j.str.2025.08.012","url":null,"abstract":"GABA (g-aminobutyric acid) transporter 3 (GAT3) is primarily found in glial cells and is essential for regulating GABA homeostasis in the central nervous system by mediating GABA uptake. Consequently, GAT3 has emerged as a significant therapeutic target for the treatment of epilepsy. In this study, we present the cryoelectron microscopy (cryo-EM) structures of GAT3 bound to its substrate GABA, the selective inhibitor SNAP-5114, and in the substrate-free state. GAT3 binds to GABA in an inward-facing conformation, while SNAP-5114 occupies the GABA-binding pocket and is stabilized by extensive interactions with surrounding residues. Functional studies reveal that E66 plays a pivotal role in determining the substrate-binding mode and specificity of SNAP-5114 binding. Taken together, our study clarifies the GABA binding mechanism of GAT3 and reveals the molecular basis for the specific inhibition of SNAP-5114, offering valuable insights for developing GAT3 subtypes selective inhibitors, which hold potential as a treatment for epilepsy.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"39 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144996090","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-04DOI: 10.1016/j.str.2025.08.005
Zhenmei Xu, Yuanzheng He
Rhodopsins typically harness light energy through the covalently bound retinal cofactor. However, some rhodopsins have lost this ability during evolution. In this issue of Structure, Kovalev et al.1 present the cryo-electron microscopy (cryo-EM) structure of a retinal-free flotillin-associated rhodopsin (FArhodopsin), providing new insights into their architecture and potential non-photochemical functions.
{"title":"Structural insights into retinal-free microbial rhodopsins","authors":"Zhenmei Xu, Yuanzheng He","doi":"10.1016/j.str.2025.08.005","DOIUrl":"https://doi.org/10.1016/j.str.2025.08.005","url":null,"abstract":"Rhodopsins typically harness light energy through the covalently bound retinal cofactor. However, some rhodopsins have lost this ability during evolution. In this issue of <em>Structure</em>, Kovalev et al.<span><span><sup>1</sup></span></span> present the cryo-electron microscopy (cryo-EM) structure of a retinal-free flotillin-associated rhodopsin (FArhodopsin), providing new insights into their architecture and potential non-photochemical functions.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"29 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144987575","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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