{"title":"Structural basis of hydride and proton transfer reactions revealed by the detection of hydrogen atoms in mammalian NADH-cytochrome b5 reductase","authors":"Yu Hirano, Kazuo Kurihara, Katsuhiro Kusaka, Andreas Ostermann, Masahide Hikita, Shigenobu Kimura, Kunio Miki, Taro Tamada","doi":"10.1016/j.str.2025.10.006","DOIUrl":"https://doi.org/10.1016/j.str.2025.10.006","url":null,"abstract":"","PeriodicalId":22168,"journal":{"name":"Structure","volume":"151 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145396753","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 : 2025-10-28DOI: 10.1016/j.str.2025.10.004
Peixuan Yu, Bradon S. Krah, Melanie A. Orlando, Sundharraman Subramanian, Benjamin J. Orlando
Bacteria utilize a variety of mechanisms to remodel the cell wall in response to environmental and antimicrobial stress. In the model organism Bacillus subtilis, the ytr operon encoding putative ATP-binding cassette (ABC) transporter(s) is highly upregulated in response to cell wall-targeting antibiotics. Here we show that the ytr operon encodes two distinct ABC transporters: YtrBCD and YtrEF. Using cryo-electron microscopy(cryo-EM), we determined the structures of YtrEF in nucleotide-free and ADP-vanadate bound states. The structures demonstrate that YtrEF adopts a type VII ABC transporter fold. Nucleotide binding induced conformational changes that propagate from the cytosolic region through the transmembrane helices to ultimately reorient the extracellular domains. Extended bacterial growth assays and suppressor mutation identification indicated that YtrEF contributes to alteration of colony morphology. These findings establish YtrEF as a type VII ABC transporter that is induced by cell wall-targeting antibiotics and a new avenue to phenotypically assess the ytr operon.
{"title":"Structural analysis of a Gram-positive type VII ABC transporter induced by cell wall-targeting antibiotics","authors":"Peixuan Yu, Bradon S. Krah, Melanie A. Orlando, Sundharraman Subramanian, Benjamin J. Orlando","doi":"10.1016/j.str.2025.10.004","DOIUrl":"https://doi.org/10.1016/j.str.2025.10.004","url":null,"abstract":"Bacteria utilize a variety of mechanisms to remodel the cell wall in response to environmental and antimicrobial stress. In the model organism <em>Bacillus subtilis</em>, the <em>ytr</em> operon encoding putative ATP-binding cassette (ABC) transporter(s) is highly upregulated in response to cell wall-targeting antibiotics. Here we show that the <em>ytr</em> operon encodes two distinct ABC transporters: YtrBCD and YtrEF. Using cryo-electron microscopy(cryo-EM), we determined the structures of YtrEF in nucleotide-free and ADP-vanadate bound states. The structures demonstrate that YtrEF adopts a type VII ABC transporter fold. Nucleotide binding induced conformational changes that propagate from the cytosolic region through the transmembrane helices to ultimately reorient the extracellular domains. Extended bacterial growth assays and suppressor mutation identification indicated that YtrEF contributes to alteration of colony morphology. These findings establish YtrEF as a type VII ABC transporter that is induced by cell wall-targeting antibiotics and a new avenue to phenotypically assess the <em>ytr</em> operon.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"177 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145382148","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 : 2025-10-27DOI: 10.1016/j.str.2025.10.002
Yu Xu,Kaushik Thakkar,Li Guan,Yu Miao,Manal Mehibel,Robert B Lee,David Marciano,Vignesh Viswanathan,Ziwei Wang,Jinglong Wang,Lu Ji,Hongbin Cao,Camille Fisher Petrakian,Jocelyn Valenzuela,Edward LaGory,Xianglian Jia,Eui Jung Moon,Rodolph Martinez,Fang Wu,Richard L Frock,Everett J Moding,Quynh-Thu Le,Erinn B Rankin,Cheng Zhang,Possu Huang,Monica M Olcina,Amato J Giaccia,Edward E Graves
High-throughput mutagenesis approaches are widely employed to systematically characterize protein functions and play a critical role in therapeutic developments. As the largest class of membrane receptors, G protein-coupled receptors (GPCRs) are a primary focus of these studies. However, while significant progress has been made in understanding GPCRs themselves, mutagenesis studies on their ligands have lagged behind, because of the difficulties in solubilizing the target receptor. In this study, we present a novel approach that employs lipid vesicles to embed and stabilize target membrane receptors, allowing direct ligand screening. We applied this platform to investigate the anaphylatoxin complement 5a (C5a) and examined how mutations affect binding to its two native GPCRs: complement 5a receptor 1 (C5aR1) and complement 5a receptor 2 (C5aR2). The screening revealed new insights into the molecular basis of the interaction and led to the discovery of novel ligands that selectively activate C5aR2, but not C5aR1.
{"title":"High throughput mutational characterization of the GPCR ligand C5a using yeast display and deep sequencing.","authors":"Yu Xu,Kaushik Thakkar,Li Guan,Yu Miao,Manal Mehibel,Robert B Lee,David Marciano,Vignesh Viswanathan,Ziwei Wang,Jinglong Wang,Lu Ji,Hongbin Cao,Camille Fisher Petrakian,Jocelyn Valenzuela,Edward LaGory,Xianglian Jia,Eui Jung Moon,Rodolph Martinez,Fang Wu,Richard L Frock,Everett J Moding,Quynh-Thu Le,Erinn B Rankin,Cheng Zhang,Possu Huang,Monica M Olcina,Amato J Giaccia,Edward E Graves","doi":"10.1016/j.str.2025.10.002","DOIUrl":"https://doi.org/10.1016/j.str.2025.10.002","url":null,"abstract":"High-throughput mutagenesis approaches are widely employed to systematically characterize protein functions and play a critical role in therapeutic developments. As the largest class of membrane receptors, G protein-coupled receptors (GPCRs) are a primary focus of these studies. However, while significant progress has been made in understanding GPCRs themselves, mutagenesis studies on their ligands have lagged behind, because of the difficulties in solubilizing the target receptor. In this study, we present a novel approach that employs lipid vesicles to embed and stabilize target membrane receptors, allowing direct ligand screening. We applied this platform to investigate the anaphylatoxin complement 5a (C5a) and examined how mutations affect binding to its two native GPCRs: complement 5a receptor 1 (C5aR1) and complement 5a receptor 2 (C5aR2). The screening revealed new insights into the molecular basis of the interaction and led to the discovery of novel ligands that selectively activate C5aR2, but not C5aR1.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"7 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-10-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145380919","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 : 2025-10-24DOI: 10.1016/j.str.2025.09.011
Andrew D Huber,Efren Garcia-Maldonado,Wenwei Lin,Shyaron Poudel,Jing Wu,Darcie J Miller,Taosheng Chen
Nuclear receptor antagonists are used to treat various diseases, but the precise antagonist mechanisms differ among receptors and compounds. Understanding the interplay between ligand-receptor interactions and transcriptional outcomes is critical. The nuclear receptor pregnane X receptor (PXR) is activated by many medicinal compounds and upregulates drug metabolism genes in response, decreasing efficacy and/or increasing toxicity of drugs. Co-administered PXR antagonists could reduce these effects, but such compounds have only recently been identified, and molecular elements governing their actions remain largely unknown. Here, we show chemically similar PXR ligands with three distinct activities (agonist, antagonist, and inverse agonist) that are altered by PXR mutations. These diverging activities are linked to ligand-induced changes at the intersection of ligand, receptor ligand-binding pocket, and receptor surface where transcriptional coregulators are recruited. We also find that antagonists can act by multiple mechanisms regarding coregulator recruitment, highlighting the complexity of ligand-receptor interactions that influence transcriptional activity.
{"title":"Subtle changes in ligand-receptor interactions dramatically alter transcriptional outcomes of pregnane X receptor modulators.","authors":"Andrew D Huber,Efren Garcia-Maldonado,Wenwei Lin,Shyaron Poudel,Jing Wu,Darcie J Miller,Taosheng Chen","doi":"10.1016/j.str.2025.09.011","DOIUrl":"https://doi.org/10.1016/j.str.2025.09.011","url":null,"abstract":"Nuclear receptor antagonists are used to treat various diseases, but the precise antagonist mechanisms differ among receptors and compounds. Understanding the interplay between ligand-receptor interactions and transcriptional outcomes is critical. The nuclear receptor pregnane X receptor (PXR) is activated by many medicinal compounds and upregulates drug metabolism genes in response, decreasing efficacy and/or increasing toxicity of drugs. Co-administered PXR antagonists could reduce these effects, but such compounds have only recently been identified, and molecular elements governing their actions remain largely unknown. Here, we show chemically similar PXR ligands with three distinct activities (agonist, antagonist, and inverse agonist) that are altered by PXR mutations. These diverging activities are linked to ligand-induced changes at the intersection of ligand, receptor ligand-binding pocket, and receptor surface where transcriptional coregulators are recruited. We also find that antagonists can act by multiple mechanisms regarding coregulator recruitment, highlighting the complexity of ligand-receptor interactions that influence transcriptional activity.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"109 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145369499","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 : 2025-10-24DOI: 10.1016/j.str.2025.09.012
Daniel Yoon,Kamil Nosol,Ali Rasouli,Rose Bang-Sørensen,Rossitza N Irobalieva,Hongtao Liu,Emad Tajkhorshid,Kaspar P Locher
Sodium-taurocholate co-transporting polypeptide (NTCP) is a sodium-dependent transporter mediating the hepatic uptake of bile salts and serving as the receptor of hepatitis B and D viruses. While previous studies identified binding sites for sodium ions and substrates, the mechanism remains controversial. We here report a high-resolution structure of NTCP in a closed-tunnel conformation that does not feature substrate binding sites but reveals evidence of two bound sodium ions. To evaluate the functional relevance of this state and gain insight into the transport mechanism, we performed μs-scale molecular dynamics simulations of NTCP starting from distinct conformations and substrate and ion configurations. We observed that both the closed-tunnel and open-tunnel conformations are highly stable, but that the sodium ions and bile salt molecules can shift positions without substantial conformational changes. Our results suggest that the closed-tunnel conformation might represents an inactive state rather than an essential component of a productive transport cycle.
{"title":"Structure of nanobody-inhibited state of human bile salt transporter NTCP.","authors":"Daniel Yoon,Kamil Nosol,Ali Rasouli,Rose Bang-Sørensen,Rossitza N Irobalieva,Hongtao Liu,Emad Tajkhorshid,Kaspar P Locher","doi":"10.1016/j.str.2025.09.012","DOIUrl":"https://doi.org/10.1016/j.str.2025.09.012","url":null,"abstract":"Sodium-taurocholate co-transporting polypeptide (NTCP) is a sodium-dependent transporter mediating the hepatic uptake of bile salts and serving as the receptor of hepatitis B and D viruses. While previous studies identified binding sites for sodium ions and substrates, the mechanism remains controversial. We here report a high-resolution structure of NTCP in a closed-tunnel conformation that does not feature substrate binding sites but reveals evidence of two bound sodium ions. To evaluate the functional relevance of this state and gain insight into the transport mechanism, we performed μs-scale molecular dynamics simulations of NTCP starting from distinct conformations and substrate and ion configurations. We observed that both the closed-tunnel and open-tunnel conformations are highly stable, but that the sodium ions and bile salt molecules can shift positions without substantial conformational changes. Our results suggest that the closed-tunnel conformation might represents an inactive state rather than an essential component of a productive transport cycle.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"52 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145369490","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}
The CRISPR-Cas system is crucial for the adaptive immune response of prokaryotes and has been widely applied for genetic engineering. Cas13d, a type VI-D CRISPR-Cas effector, functions as RNA-guided ribonuclease and has been engineered for programmable RNA editing, which is a commonly used, active, and well-characterized small type VI editor. Here, we determined cryoelectron microscopy (cryo-EM) structures of Ruminococcus flavefaciens Cas13d in a RfxCas13d-crRNA-off-target-RNA ternary complex and RfxCas13d-crRNA binary complex at 3.10 and 3.13 Å resolution. The ternary complex consists of RfxCas13d, crRNA, and a captured short off-target ssRNA at a complex state of binding proximal mismatched RNA. RfxCas13d undergoes conformational changes with or without the off-target RNA, but the catalytic sites remain unchanged. Mg2+ aids in stabilizing the crRNA repeat region structure, which may be crucial for RNA binding. This discovery provides the foundation for developing RfxCas13d as a mature tool and offers a framework for advancing transcriptome engineering.
{"title":"Cryo-EM structure of the RfxCas13d-crRNA-off-target-RNA complex.","authors":"Qianxi Yang,Yifang Sun,Lei Sun,Tian Chi,Zhenguo Chen","doi":"10.1016/j.str.2025.09.010","DOIUrl":"https://doi.org/10.1016/j.str.2025.09.010","url":null,"abstract":"The CRISPR-Cas system is crucial for the adaptive immune response of prokaryotes and has been widely applied for genetic engineering. Cas13d, a type VI-D CRISPR-Cas effector, functions as RNA-guided ribonuclease and has been engineered for programmable RNA editing, which is a commonly used, active, and well-characterized small type VI editor. Here, we determined cryoelectron microscopy (cryo-EM) structures of Ruminococcus flavefaciens Cas13d in a RfxCas13d-crRNA-off-target-RNA ternary complex and RfxCas13d-crRNA binary complex at 3.10 and 3.13 Å resolution. The ternary complex consists of RfxCas13d, crRNA, and a captured short off-target ssRNA at a complex state of binding proximal mismatched RNA. RfxCas13d undergoes conformational changes with or without the off-target RNA, but the catalytic sites remain unchanged. Mg2+ aids in stabilizing the crRNA repeat region structure, which may be crucial for RNA binding. This discovery provides the foundation for developing RfxCas13d as a mature tool and offers a framework for advancing transcriptome engineering.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"1 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145357625","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}
The subcortical maternal complex (SCMC) is essential for mammalian preimplantation development, yet how SCMCcore (MATER/NLRP5, TLE6, FLOPED/OOEP) engages regulatory partners remains unclear. We determined cryo-EM structures of mouse SCMC bound to ZBED3 and human SCMC bound to NLRP2. Our structure reveals that ZBED3 interacts with all three SCMCcore subunits via its zinc finger domain, with conserved residue Phe73 mediating specific contacts. In contrast, human NLRP2 only binds to the WD40 domain of TLE6 through its leucine-rich repeat (LRR) domain. Similar interactions were also confirmed for NLRP7 with TLE6. These findings were cross-validated by in vivo proximity ligation and in vitro pull-down assays. Our work proposes a paradigmatic "Lego-like" assembly model, where the SCMCcore sequentially recruits different partners through diverse molecular interfaces. These findings provide critical structural insights into the SCMC's architecture and its multifaceted regulatory roles in early mammalian embryogenesis.
{"title":"Structural assembly of the subcortical maternal complex SCMC.","authors":"Guojin Ou,Qingting Liu,Haizhan Jiao,Zhuo Han,Jinhong Li,Ling Min,Pengliang Chi,Sibei Liu,Jialu Li,Qianqian Qi,Zihan Zhang,Li Guo,Xiang Wang,Lei Li,Jing Chen,Hongli Hu,Dong Deng","doi":"10.1016/j.str.2025.09.009","DOIUrl":"https://doi.org/10.1016/j.str.2025.09.009","url":null,"abstract":"The subcortical maternal complex (SCMC) is essential for mammalian preimplantation development, yet how SCMCcore (MATER/NLRP5, TLE6, FLOPED/OOEP) engages regulatory partners remains unclear. We determined cryo-EM structures of mouse SCMC bound to ZBED3 and human SCMC bound to NLRP2. Our structure reveals that ZBED3 interacts with all three SCMCcore subunits via its zinc finger domain, with conserved residue Phe73 mediating specific contacts. In contrast, human NLRP2 only binds to the WD40 domain of TLE6 through its leucine-rich repeat (LRR) domain. Similar interactions were also confirmed for NLRP7 with TLE6. These findings were cross-validated by in vivo proximity ligation and in vitro pull-down assays. Our work proposes a paradigmatic \"Lego-like\" assembly model, where the SCMCcore sequentially recruits different partners through diverse molecular interfaces. These findings provide critical structural insights into the SCMC's architecture and its multifaceted regulatory roles in early mammalian embryogenesis.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"108 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145338595","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 : 2025-10-16DOI: 10.1016/j.str.2025.09.008
Maria del Carmen Fernandez-Ramirez, Binh A. Nguyen, Shumaila Afrin, Virender Singh, Bret Evers, John M. Shelton, Christian Lopez Escobar, Parker Bassett, Lanie Wang, Maja Pękała, Yasmin Ahmed, Luis O. Cabrera Hernandez, Rose Pedretti, Preeti Singh, Jacob Canepa, Aleksandra Wosztyl, Yang Li, David R. Boyer, Qin Cao, Lorena Saelices
Transthyretin amyloidosis is a systemic protein misfolding disorder with diverse clinical phenotypes, including cardiomyopathy, polyneuropathy, or a combination of both. While structural polymorphism of amyloid fibrils has been linked to disease heterogeneity in neurodegenerative disorders, its role in transthyretin amyloidosis remains unclear. Here, we used cryo-electron microscopy to analyze ex vivo fibrils extracted from the hearts of three patients carrying the T60A mutation, a variant associated with mixed cardiac and neuropathic symptoms. In one patient, we additionally examined fibrils from the thyroid, kidney, and liver. All fibrils across patients and tissues adopted a single morphology previously associated with cardiomyopathy. Complementary molecular analyses revealed high compositional homogeneity. Notably, we extracted fibrils from the liver, an organ considered fibril-free, with seeding capacity in vitro. These findings suggest structural homogeneity as a hallmark of cardiac and mixed phenotypes, and provide a mechanistic rationale for the transmission of amyloidosis following domino liver transplantation.
{"title":"Structural and molecular homogeneity of ATTRv-T60A amyloid fibrils across patients and organs","authors":"Maria del Carmen Fernandez-Ramirez, Binh A. Nguyen, Shumaila Afrin, Virender Singh, Bret Evers, John M. Shelton, Christian Lopez Escobar, Parker Bassett, Lanie Wang, Maja Pękała, Yasmin Ahmed, Luis O. Cabrera Hernandez, Rose Pedretti, Preeti Singh, Jacob Canepa, Aleksandra Wosztyl, Yang Li, David R. Boyer, Qin Cao, Lorena Saelices","doi":"10.1016/j.str.2025.09.008","DOIUrl":"https://doi.org/10.1016/j.str.2025.09.008","url":null,"abstract":"Transthyretin amyloidosis is a systemic protein misfolding disorder with diverse clinical phenotypes, including cardiomyopathy, polyneuropathy, or a combination of both. While structural polymorphism of amyloid fibrils has been linked to disease heterogeneity in neurodegenerative disorders, its role in transthyretin amyloidosis remains unclear. Here, we used cryo-electron microscopy to analyze <em>ex vivo</em> fibrils extracted from the hearts of three patients carrying the T60A mutation, a variant associated with mixed cardiac and neuropathic symptoms. In one patient, we additionally examined fibrils from the thyroid, kidney, and liver. All fibrils across patients and tissues adopted a single morphology previously associated with cardiomyopathy. Complementary molecular analyses revealed high compositional homogeneity. Notably, we extracted fibrils from the liver, an organ considered fibril-free, with seeding capacity <em>in vitro</em>. These findings suggest structural homogeneity as a hallmark of cardiac and mixed phenotypes, and provide a mechanistic rationale for the transmission of amyloidosis following domino liver transplantation.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"9 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145295468","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 : 2025-10-09DOI: 10.1016/j.str.2025.09.007
William S. Henriques, Jarrett Bowman, Laina N. Hall, Colin C. Gauvin, Hui Wei, Huihui Kuang, Christina M. Zimanyi, Edward T. Eng, Andrew Santiago-Frangos, Blake Wiedenheft
Cas1 and Cas2 are the hallmark proteins of prokaryotic adaptive immunity. However, these two proteins are often fused to other proteins and the functional association of these fusions often remain poorly understood. Here we purify and determine structures of Cas1 and the Cas2/3 fusion proteins from Pseudomonas aeruginosa at distinct stages of CRISPR adaptation. Collectively, these structures reveal a prominent, positively charged channel on one face of the integration complex that captures short fragments of foreign DNA. Foreign DNA binding triggers conformational changes in Cas2/3 that expose new DNA binding surfaces necessary for homing the DNA-bound integrase to specific CRISPR loci. The length of the foreign DNA substrate determines if Cas1-2/3 docks completely onto the CRISPR repeat to successfully catalyze two sequential transesterification reactions required for integration. Together, these structures clarify how the Cas1-2/3 proteins orchestrate foreign DNA capture, site-specific delivery, and integration of new DNA into the bacterial genome.
{"title":"Structures reveal how the Cas1-2/3 integrase captures, delivers, and integrates foreign DNA into CRISPR loci","authors":"William S. Henriques, Jarrett Bowman, Laina N. Hall, Colin C. Gauvin, Hui Wei, Huihui Kuang, Christina M. Zimanyi, Edward T. Eng, Andrew Santiago-Frangos, Blake Wiedenheft","doi":"10.1016/j.str.2025.09.007","DOIUrl":"https://doi.org/10.1016/j.str.2025.09.007","url":null,"abstract":"Cas1 and Cas2 are the hallmark proteins of prokaryotic adaptive immunity. However, these two proteins are often fused to other proteins and the functional association of these fusions often remain poorly understood. Here we purify and determine structures of Cas1 and the Cas2/3 fusion proteins from <em>Pseudomonas aeruginosa</em> at distinct stages of CRISPR adaptation. Collectively, these structures reveal a prominent, positively charged channel on one face of the integration complex that captures short fragments of foreign DNA. Foreign DNA binding triggers conformational changes in Cas2/3 that expose new DNA binding surfaces necessary for homing the DNA-bound integrase to specific CRISPR loci. The length of the foreign DNA substrate determines if Cas1-2/3 docks completely onto the CRISPR repeat to successfully catalyze two sequential transesterification reactions required for integration. Together, these structures clarify how the Cas1-2/3 proteins orchestrate foreign DNA capture, site-specific delivery, and integration of new DNA into the bacterial genome.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"16 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145247581","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 : 2025-10-03DOI: 10.1016/j.str.2025.09.006
Evan J. McMahon, Alexander G. Cioffi, Patrick R. Visperas, Yueqing Lin, Michael Shaghafi, Courtney M. Daczkowski, Johannes C. Hermann, Robert A. Everley, Richard M. Neve, Daniel A. Erlanson, Kevin R. Webster, Vikram Narayan, Weiru Wang
Targeted protein degradation (TPD) leverages the ubiquitin-proteasome system to eliminate disease-causing proteins via E3 ligases. To date, the field is limited to utilizing a few of the over 600 human E3 ligases. To expand this repertoire, we conducted structural and functional validation of DDB1 (Damage-specific DNA binding protein 1) and Cullin-associated factor (DCAF)2 (DTL/CDT2), a Cullin4-RING ligase substrate adaptor implicated in DNA damage response and cancer, as a novel E3 for TPD. Cryoelectron microscopy (cryo-EM) structures of the DCAF2:DDB1:DDA1 complex (3.3 Å), a ligand bound complex (3.1 Å), and a ternary complex with a covalent proteolysis-targeting chimera (PROTAC) and BRD4 (3.4 Å) reveal PROTAC-mediated substrate recruitment. Using covalent bifunctional tool compounds engaging residue C141 in the WD40 domain, we demonstrate robust ubiquitination in biochemical assays and cellular TPD using the COFFEE (covalent functionalization followed by E3 electroporation) method. These findings position DCAF2 as a promising E3 adaptor for PROTAC strategies and identify C141 as a relevant site for future PROTAC discovery.
{"title":"Structural basis for DCAF2 as a novel E3 ligase for PROTAC-mediated targeted protein degradation","authors":"Evan J. McMahon, Alexander G. Cioffi, Patrick R. Visperas, Yueqing Lin, Michael Shaghafi, Courtney M. Daczkowski, Johannes C. Hermann, Robert A. Everley, Richard M. Neve, Daniel A. Erlanson, Kevin R. Webster, Vikram Narayan, Weiru Wang","doi":"10.1016/j.str.2025.09.006","DOIUrl":"https://doi.org/10.1016/j.str.2025.09.006","url":null,"abstract":"Targeted protein degradation (TPD) leverages the ubiquitin-proteasome system to eliminate disease-causing proteins via E3 ligases. To date, the field is limited to utilizing a few of the over 600 human E3 ligases. To expand this repertoire, we conducted structural and functional validation of DDB1 (Damage-specific DNA binding protein 1) and Cullin-associated factor (DCAF)2 (DTL/CDT2), a Cullin4-RING ligase substrate adaptor implicated in DNA damage response and cancer, as a novel E3 for TPD. Cryoelectron microscopy (cryo-EM) structures of the DCAF2:DDB1:DDA1 complex (3.3 Å), a ligand bound complex (3.1 Å), and a ternary complex with a covalent proteolysis-targeting chimera (PROTAC) and BRD4 (3.4 Å) reveal PROTAC-mediated substrate recruitment. Using covalent bifunctional tool compounds engaging residue C141 in the WD40 domain, we demonstrate robust ubiquitination in biochemical assays and cellular TPD using the COFFEE (covalent functionalization followed by E3 electroporation) method. These findings position DCAF2 as a promising E3 adaptor for PROTAC strategies and identify C141 as a relevant site for future PROTAC discovery.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"52 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145209661","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}
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：