Pub Date : 2025-09-25DOI: 10.1038/s41594-025-01681-8
Anindita Nayak, Digant Nayak, Lijia Jia, Eliza A. Ruben, Suryavathi Viswanadhapalli, Priscila dos Santos Bury, Khaled Mohamed Nassar, Corey H. Yu, Anna A. Tumanova, Caleb M. Stratton, Pirouz Ebadi, Dmitri N. Ivanov, Patrick Sung, Ratna K. Vadlamudi, Elizabeth V. Wasmuth, Shaun K. Olsen
Post-translational modification of proteins by SUMO (small ubiquitin-like modifier) regulates fundamental cellular processes and occurs through the sequential interactions and activities of three enzymes: E1, E2 and E3. SUMO E1 activates SUMO in a two-step process involving adenylation and thioester bond formation, followed by transfer of SUMO to its dedicated E2 enzyme, UBC9. This process is termed E1–E2 thioester transfer (or transthioesterification). Despite its fundamental importance, the molecular basis for SUMO E1–UBC9 thioester transfer and the molecular rules governing SUMO E1–UBC9 specificity are poorly understood. Here we present cryo-EM reconstructions of human SUMO E1 in complex with UBC9, SUMO1 adenylate and SUMO1 thioester intermediate. Our structures reveal drastic conformational changes that accompany thioester transfer, providing insights into the molecular recognition of UBC9 by SUMO E1 and delineating the rules that govern SUMO E1–UBC9 specificity. Collectively, our structural, biochemical and cell-based studies elucidate the molecular mechanisms by which SUMOylation exerts its essential biological functions. This study reveals how human small ubiquitin-like modifier (SUMO) E1 recruits its E2 partner UBC9 and transfers SUMO1 through large structural changes, uncovering key mechanisms that ensure specificity and fidelity in SUMOylation, an essential protein modification pathway.
{"title":"Cryo-EM structures reveal the molecular mechanism of SUMO E1–E2 thioester transfer","authors":"Anindita Nayak, Digant Nayak, Lijia Jia, Eliza A. Ruben, Suryavathi Viswanadhapalli, Priscila dos Santos Bury, Khaled Mohamed Nassar, Corey H. Yu, Anna A. Tumanova, Caleb M. Stratton, Pirouz Ebadi, Dmitri N. Ivanov, Patrick Sung, Ratna K. Vadlamudi, Elizabeth V. Wasmuth, Shaun K. Olsen","doi":"10.1038/s41594-025-01681-8","DOIUrl":"10.1038/s41594-025-01681-8","url":null,"abstract":"Post-translational modification of proteins by SUMO (small ubiquitin-like modifier) regulates fundamental cellular processes and occurs through the sequential interactions and activities of three enzymes: E1, E2 and E3. SUMO E1 activates SUMO in a two-step process involving adenylation and thioester bond formation, followed by transfer of SUMO to its dedicated E2 enzyme, UBC9. This process is termed E1–E2 thioester transfer (or transthioesterification). Despite its fundamental importance, the molecular basis for SUMO E1–UBC9 thioester transfer and the molecular rules governing SUMO E1–UBC9 specificity are poorly understood. Here we present cryo-EM reconstructions of human SUMO E1 in complex with UBC9, SUMO1 adenylate and SUMO1 thioester intermediate. Our structures reveal drastic conformational changes that accompany thioester transfer, providing insights into the molecular recognition of UBC9 by SUMO E1 and delineating the rules that govern SUMO E1–UBC9 specificity. Collectively, our structural, biochemical and cell-based studies elucidate the molecular mechanisms by which SUMOylation exerts its essential biological functions. This study reveals how human small ubiquitin-like modifier (SUMO) E1 recruits its E2 partner UBC9 and transfers SUMO1 through large structural changes, uncovering key mechanisms that ensure specificity and fidelity in SUMOylation, an essential protein modification pathway.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"32 12","pages":"2441-2453"},"PeriodicalIF":10.1,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.comhttps://www.nature.com/articles/s41594-025-01681-8.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145140445","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-22DOI: 10.1038/s41594-025-01659-6
Taylor L. Mighell, Ben Lehner
Reduced protein abundance is the most frequent mechanism by which rare missense variants cause disease. A promising therapeutic avenue for treating reduced abundance variants is pharmacological chaperones (PCs, also known as correctors or stabilizers), small molecules that bind to and stabilize target proteins. PCs have been approved as clinical treatments for specific variants, but protein energetics suggest their effects might be much more general. To comprehensively assess PC efficacy for variation in a given protein, it is necessary to first assign the molecular mechanism explaining all pathogenic variants, then measure the response to the PC. Here we establish such a framework for the vasopressin 2 receptor (V2R), a G-protein-coupled receptor in which loss-of-function variants cause nephrogenic diabetes insipidus (NDI). Our data show that more than half of NDI variants are poorly expressed, highlighting loss of stability as the major pathogenic mechanism. Treatment with a PC rescues the expression of 87% of destabilized variants. The non-rescued variants identify the drug’s predicted binding site. Our results provide proof-of-principle that small molecule binding can rescue destabilizing variants throughout a protein’s structure. The application of this principle to other proteins should allow the development of effective therapies for many different rare diseases. Many mutations cause disease because they destabilize proteins. Here, Mighell and Lehner show that a single small molecule can correct the destabilization caused by nearly all pathogenic mutations in a human GPCR.
{"title":"A small molecule stabilizer rescues the surface expression of nearly all missense variants in a GPCR","authors":"Taylor L. Mighell, Ben Lehner","doi":"10.1038/s41594-025-01659-6","DOIUrl":"10.1038/s41594-025-01659-6","url":null,"abstract":"Reduced protein abundance is the most frequent mechanism by which rare missense variants cause disease. A promising therapeutic avenue for treating reduced abundance variants is pharmacological chaperones (PCs, also known as correctors or stabilizers), small molecules that bind to and stabilize target proteins. PCs have been approved as clinical treatments for specific variants, but protein energetics suggest their effects might be much more general. To comprehensively assess PC efficacy for variation in a given protein, it is necessary to first assign the molecular mechanism explaining all pathogenic variants, then measure the response to the PC. Here we establish such a framework for the vasopressin 2 receptor (V2R), a G-protein-coupled receptor in which loss-of-function variants cause nephrogenic diabetes insipidus (NDI). Our data show that more than half of NDI variants are poorly expressed, highlighting loss of stability as the major pathogenic mechanism. Treatment with a PC rescues the expression of 87% of destabilized variants. The non-rescued variants identify the drug’s predicted binding site. Our results provide proof-of-principle that small molecule binding can rescue destabilizing variants throughout a protein’s structure. The application of this principle to other proteins should allow the development of effective therapies for many different rare diseases. Many mutations cause disease because they destabilize proteins. Here, Mighell and Lehner show that a single small molecule can correct the destabilization caused by nearly all pathogenic mutations in a human GPCR.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"32 12","pages":"2429-2440"},"PeriodicalIF":10.1,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.comhttps://www.nature.com/articles/s41594-025-01659-6.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145117045","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-19DOI: 10.1038/s41594-025-01676-5
Grant A. Pellowe, Tomas B. Voisin, Laura Karpauskaite, Sarah L. Maslen, Alžběta Roeselová, J. Mark Skehel, Chloe Roustan, Roger George, Andrea Nans, Svend Kjær, Ian A. Taylor, David Balchin
Proteins with multiple domains are intrinsically prone to misfold, yet fold efficiently during their synthesis on the ribosome. This is especially important in eukaryotes, where multidomain proteins predominate. Here we sought to understand how multidomain protein folding is modulated by the eukaryotic ribosome. We used hydrogen–deuterium exchange mass spectrometry and cryo-electron microscopy to characterize the structure and dynamics of partially synthesized intermediates of a model multidomain protein. We find that nascent subdomains fold progressively during synthesis on the human ribosome, templated by interactions across domain interfaces. The conformational ensemble of the nascent chain is tuned by its unstructured C-terminal segments, which keep interfaces between folded domains in dynamic equilibrium until translation termination. This contrasts with the bacterial ribosome, on which domain interfaces form early and remain stable during synthesis. Delayed domain docking may avoid interdomain misfolding to promote the maturation of multidomain proteins in eukaryotes. By studying dynamic folding intermediates on the human ribosome, Pellowe et al. show that newly made domains help each other to fold but do not stably interact until synthesis is complete, avoiding interdomain misfolding.
{"title":"The human ribosome modulates multidomain protein biogenesis by delaying cotranslational domain docking","authors":"Grant A. Pellowe, Tomas B. Voisin, Laura Karpauskaite, Sarah L. Maslen, Alžběta Roeselová, J. Mark Skehel, Chloe Roustan, Roger George, Andrea Nans, Svend Kjær, Ian A. Taylor, David Balchin","doi":"10.1038/s41594-025-01676-5","DOIUrl":"10.1038/s41594-025-01676-5","url":null,"abstract":"Proteins with multiple domains are intrinsically prone to misfold, yet fold efficiently during their synthesis on the ribosome. This is especially important in eukaryotes, where multidomain proteins predominate. Here we sought to understand how multidomain protein folding is modulated by the eukaryotic ribosome. We used hydrogen–deuterium exchange mass spectrometry and cryo-electron microscopy to characterize the structure and dynamics of partially synthesized intermediates of a model multidomain protein. We find that nascent subdomains fold progressively during synthesis on the human ribosome, templated by interactions across domain interfaces. The conformational ensemble of the nascent chain is tuned by its unstructured C-terminal segments, which keep interfaces between folded domains in dynamic equilibrium until translation termination. This contrasts with the bacterial ribosome, on which domain interfaces form early and remain stable during synthesis. Delayed domain docking may avoid interdomain misfolding to promote the maturation of multidomain proteins in eukaryotes. By studying dynamic folding intermediates on the human ribosome, Pellowe et al. show that newly made domains help each other to fold but do not stably interact until synthesis is complete, avoiding interdomain misfolding.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"32 11","pages":"2296-2307"},"PeriodicalIF":10.1,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.comhttps://www.nature.com/articles/s41594-025-01676-5.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145092139","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-18DOI: 10.1038/s41594-025-01646-x
Eli Arama, Katia Cosentino, Peter E. Czabotar, Boyi Gan, Elizabeth Hartland, Xuejun Jiang, Jonathan C. Kagan, Shigekazu Nagata, Kate Schroder, Liming Sun, Daichao Xu, Junying Yuan
Cell death contributes to tissue homeostasis and plays critical roles in inflammation and host defense. Our increasing understanding of the physiological importance of cell death underlines the need to more fully elucidate its underlying mechanisms in health and disease. Molecular and structural insight into the cell death apparatus could provide strategies to target the loss of cells in pathophysiological contexts. We asked experts studying a range of cell death types to share with us what they are most excited to tackle and what the field needs for progress.
{"title":"Towards a molecular and structural definition of cell death","authors":"Eli Arama, Katia Cosentino, Peter E. Czabotar, Boyi Gan, Elizabeth Hartland, Xuejun Jiang, Jonathan C. Kagan, Shigekazu Nagata, Kate Schroder, Liming Sun, Daichao Xu, Junying Yuan","doi":"10.1038/s41594-025-01646-x","DOIUrl":"10.1038/s41594-025-01646-x","url":null,"abstract":"Cell death contributes to tissue homeostasis and plays critical roles in inflammation and host defense. Our increasing understanding of the physiological importance of cell death underlines the need to more fully elucidate its underlying mechanisms in health and disease. Molecular and structural insight into the cell death apparatus could provide strategies to target the loss of cells in pathophysiological contexts. We asked experts studying a range of cell death types to share with us what they are most excited to tackle and what the field needs for progress.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"32 10","pages":"1854-1858"},"PeriodicalIF":10.1,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145083356","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Upon starvation, the autophagy-initiating Atg1 complex undergoes phase separation to organize the preautophagosomal structure (PAS) in Saccharomyces cerevisiae, from which autophagosome formation is considered to proceed. However, the physiological roles of the PAS droplet remain unclear. Here we show that core Atg proteins are recruited into early PAS droplets that are formed by phase separation of the Atg1 complex with different efficiencies in vitro. The Atg12–Atg5–Atg16 E3 ligase complex for Atg8 lipidation is the most efficiently condensed in the droplets through specific Atg12–Atg17 interaction, which is also important for the PAS targeting of the E3 complex in vivo. In vitro reconstitution demonstrates that E3-enriched early PAS droplets promote Atg8 lipidation and that Atg8 coating of the vesicle membrane is both necessary and sufficient for their condensation into the droplets. These data suggest that the PAS functions as an efficient production site for lipidated Atg8 and pools membrane seeds to drive autophagosome formation. Fujioka et al. show that, under starvation, yeast Atg1 forms droplets that concentrate autophagy factors, boosting Atg8 lipidation and clustering vesicles. These droplets likely serve as hubs that generate membrane seeds for autophagosome formation.
{"title":"Phase separation promotes Atg8 lipidation and vesicle condensation for autophagy progression","authors":"Yuko Fujioka, Takuma Tsuji, Tetsuya Kotani, Hiroyuki Kumeta, Chika Kakuta, Junko Shimasaki, Toyoshi Fujimoto, Hitoshi Nakatogawa, Nobuo N. Noda","doi":"10.1038/s41594-025-01678-3","DOIUrl":"10.1038/s41594-025-01678-3","url":null,"abstract":"Upon starvation, the autophagy-initiating Atg1 complex undergoes phase separation to organize the preautophagosomal structure (PAS) in Saccharomyces cerevisiae, from which autophagosome formation is considered to proceed. However, the physiological roles of the PAS droplet remain unclear. Here we show that core Atg proteins are recruited into early PAS droplets that are formed by phase separation of the Atg1 complex with different efficiencies in vitro. The Atg12–Atg5–Atg16 E3 ligase complex for Atg8 lipidation is the most efficiently condensed in the droplets through specific Atg12–Atg17 interaction, which is also important for the PAS targeting of the E3 complex in vivo. In vitro reconstitution demonstrates that E3-enriched early PAS droplets promote Atg8 lipidation and that Atg8 coating of the vesicle membrane is both necessary and sufficient for their condensation into the droplets. These data suggest that the PAS functions as an efficient production site for lipidated Atg8 and pools membrane seeds to drive autophagosome formation. Fujioka et al. show that, under starvation, yeast Atg1 forms droplets that concentrate autophagy factors, boosting Atg8 lipidation and clustering vesicles. These droplets likely serve as hubs that generate membrane seeds for autophagosome formation.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"32 11","pages":"2285-2295"},"PeriodicalIF":10.1,"publicationDate":"2025-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145067759","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-16DOI: 10.1038/s41594-025-01672-9
We highlight primary research and commissioned content that delve into the biology of ubiquitylation and degradation mechanisms.
我们强调主要的研究和委托的内容，深入到泛素化和降解机制的生物学。
{"title":"Focus on ubiquitylation and protein degradation","authors":"","doi":"10.1038/s41594-025-01672-9","DOIUrl":"10.1038/s41594-025-01672-9","url":null,"abstract":"We highlight primary research and commissioned content that delve into the biology of ubiquitylation and degradation mechanisms.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"32 9","pages":"1581-1581"},"PeriodicalIF":10.1,"publicationDate":"2025-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.comhttps://www.nature.com/articles/s41594-025-01672-9.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145067758","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-15DOI: 10.1038/s41594-025-01665-8
Tianyang Liu, Luyan Cao, Miroslav Mladenov, Guillaume Romet-Lemonne, Michael Way, Carolyn A. Moores
Branched actin networks nucleated by the Arp2/3 complex have critical roles in various cellular processes, from cell migration to intracellular transport. However, when activated by WISH/DIP/SPIN90-family proteins, Arp2/3 nucleates linear actin filaments. Here we found that human SPIN90 is a dimer that can nucleate bidirectional actin filaments. To understand the basis for this, we determined a 3-Å-resolution structure of human SPIN90–Arp2/3 complex nucleating actin filaments. Our structure shows that SPIN90 dimerizes through a three-helix bundle and interacts with two Arp2/3 complexes. Each SPIN90 molecule binds both Arp2/3 complexes to promote their activation. Our analysis demonstrates that single-filament nucleation by Arp2/3 is mechanistically more like branch formation than previously appreciated. The dimerization domain in SPIN90 orthologs is conserved in metazoans, suggesting that this mode of bidirectional nucleation is a common strategy to generate antiparallel actin filaments. Liu et al. show that SPIN90 dimerizes and binds two Arp2/3 complexes to nucleate two bidirectional actin filaments and the dimerization domain is conserved in multicellular animals, suggesting that the mechanism of bidirectional actin filament nucleation is conserved.
{"title":"Arp2/3-mediated bidirectional actin assembly by SPIN90 dimers","authors":"Tianyang Liu, Luyan Cao, Miroslav Mladenov, Guillaume Romet-Lemonne, Michael Way, Carolyn A. Moores","doi":"10.1038/s41594-025-01665-8","DOIUrl":"10.1038/s41594-025-01665-8","url":null,"abstract":"Branched actin networks nucleated by the Arp2/3 complex have critical roles in various cellular processes, from cell migration to intracellular transport. However, when activated by WISH/DIP/SPIN90-family proteins, Arp2/3 nucleates linear actin filaments. Here we found that human SPIN90 is a dimer that can nucleate bidirectional actin filaments. To understand the basis for this, we determined a 3-Å-resolution structure of human SPIN90–Arp2/3 complex nucleating actin filaments. Our structure shows that SPIN90 dimerizes through a three-helix bundle and interacts with two Arp2/3 complexes. Each SPIN90 molecule binds both Arp2/3 complexes to promote their activation. Our analysis demonstrates that single-filament nucleation by Arp2/3 is mechanistically more like branch formation than previously appreciated. The dimerization domain in SPIN90 orthologs is conserved in metazoans, suggesting that this mode of bidirectional nucleation is a common strategy to generate antiparallel actin filaments. Liu et al. show that SPIN90 dimerizes and binds two Arp2/3 complexes to nucleate two bidirectional actin filaments and the dimerization domain is conserved in multicellular animals, suggesting that the mechanism of bidirectional actin filament nucleation is conserved.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"32 11","pages":"2262-2271"},"PeriodicalIF":10.1,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.comhttps://www.nature.com/articles/s41594-025-01665-8.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145059291","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Arp2/3 complex is a key nucleator of actin filaments. It requires activation by nucleation-promoting factors (NPFs). WISH/DIP1/SPIN90 (WDS) proteins represent a unique class of NPFs that activate the Arp2/3 complex independently of preexisting filaments, promoting linear actin-filament nucleation. In fission yeast, Dip1 binds to the clamp subunits in Arp2/3 complex to induce the short-pitch conformation, where Arp2 moves closer to Arp3 to mimic a filamentous actin dimer. However, how WDS proteins stimulate subunit flattening in Arp subunits, a ‘scissor-like’ conformational change akin to what is observed in an actin monomer during filament formation, remained unclear. Here we present cryo-electron microscopy structures of human SPIN90 bound to activated bovine Arp2/3 complex on an actin filament pointed end. The structures show that SPIN90 dimerizes through a metazoan-specific domain in the middle segment, engaging both the clamp and the Arp3/ARPC3 interface, to drive the activating conformational changes in Arp2/3 complex. Remarkably, a single SPIN90 dimer can also bridge two Arp2/3 complexes, enabling bidirectional actin nucleation and suggesting a mechanism for rapidly assembling complex actin network architectures. Francis et al. used cryo-electron microscopy to show how a SPIN90 dimer activates the metazoan Arp2/3 complex to nucleate linear actin filaments for unidirectional and bidirectional growth, forming potential scaffolds for rapid assembly of dynamic actin networks.
{"title":"Activation of Arp2/3 complex by a SPIN90 dimer in linear actin-filament nucleation","authors":"Justus Francis, Achyutha Krishna Pathri, Kankipati Teja Shyam, Sridhar Sripada, Rishav Mitra, Heidy Y. Narvaez-Ortiz, Kiran Vyshnav Eliyan, Brad J. Nolen, Saikat Chowdhury","doi":"10.1038/s41594-025-01673-8","DOIUrl":"10.1038/s41594-025-01673-8","url":null,"abstract":"Arp2/3 complex is a key nucleator of actin filaments. It requires activation by nucleation-promoting factors (NPFs). WISH/DIP1/SPIN90 (WDS) proteins represent a unique class of NPFs that activate the Arp2/3 complex independently of preexisting filaments, promoting linear actin-filament nucleation. In fission yeast, Dip1 binds to the clamp subunits in Arp2/3 complex to induce the short-pitch conformation, where Arp2 moves closer to Arp3 to mimic a filamentous actin dimer. However, how WDS proteins stimulate subunit flattening in Arp subunits, a ‘scissor-like’ conformational change akin to what is observed in an actin monomer during filament formation, remained unclear. Here we present cryo-electron microscopy structures of human SPIN90 bound to activated bovine Arp2/3 complex on an actin filament pointed end. The structures show that SPIN90 dimerizes through a metazoan-specific domain in the middle segment, engaging both the clamp and the Arp3/ARPC3 interface, to drive the activating conformational changes in Arp2/3 complex. Remarkably, a single SPIN90 dimer can also bridge two Arp2/3 complexes, enabling bidirectional actin nucleation and suggesting a mechanism for rapidly assembling complex actin network architectures. Francis et al. used cryo-electron microscopy to show how a SPIN90 dimer activates the metazoan Arp2/3 complex to nucleate linear actin filaments for unidirectional and bidirectional growth, forming potential scaffolds for rapid assembly of dynamic actin networks.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"32 11","pages":"2272-2284"},"PeriodicalIF":10.1,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145059293","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-15DOI: 10.1038/s41594-025-01666-7
Carlos Vega-Gutiérrez, Javier Picañol-Párraga, Irene Sánchez-Valls, Victoria del Pilar Ribón-Fuster, David Soto, Beatriz Herguedas
AMPA-type glutamate receptors, fundamental ion channels for fast excitatory neurotransmission and synaptic plasticity, contain a GluA tetrameric core surrounded by auxiliary proteins such as transmembrane AMPA receptor regulatory proteins (TARPs) or Cornichons. Their exact composition and stoichiometry govern functional properties, including kinetics, calcium permeability and trafficking. The GluA1–GluA3 subunits predominate in the adult forebrain and are well characterized. However, we lack structural information on full-length GluA4-containing AMPARs, a subtype that has specific roles in brain development and specific cell types in mammals. Here we present the cryo-electron microscopy structures of rat GluA4:TARP-γ2 trapped in active, resting and desensitized states, covering a full gating cycle. Additionally, we describe the structure of GluA4 alone, which displays a classical Y-shaped conformation. In resting conditions, GluA4:TARP-γ2 adopts two conformations, one resembling the desensitized states of other GluA subunits. Moreover, we identify a regulatory site for TARP-γ2 in the ligand-binding domain that modulates gating kinetics. Our findings uncover distinct features of GluA4, highlighting how subunit composition and auxiliary proteins shape receptor structure and dynamics, expanding glutamatergic signaling diversity. Vega-Gutiérrez et al. present cryo-electron microscopy structures of GluA4-containing AMPA receptors, which are key for brain signaling. They show GluA4-specific conformations and explain how subunit composition shapes receptor architecture, dynamics and function.
{"title":"GluA4 AMPA receptor gating mechanisms and modulation by auxiliary proteins","authors":"Carlos Vega-Gutiérrez, Javier Picañol-Párraga, Irene Sánchez-Valls, Victoria del Pilar Ribón-Fuster, David Soto, Beatriz Herguedas","doi":"10.1038/s41594-025-01666-7","DOIUrl":"10.1038/s41594-025-01666-7","url":null,"abstract":"AMPA-type glutamate receptors, fundamental ion channels for fast excitatory neurotransmission and synaptic plasticity, contain a GluA tetrameric core surrounded by auxiliary proteins such as transmembrane AMPA receptor regulatory proteins (TARPs) or Cornichons. Their exact composition and stoichiometry govern functional properties, including kinetics, calcium permeability and trafficking. The GluA1–GluA3 subunits predominate in the adult forebrain and are well characterized. However, we lack structural information on full-length GluA4-containing AMPARs, a subtype that has specific roles in brain development and specific cell types in mammals. Here we present the cryo-electron microscopy structures of rat GluA4:TARP-γ2 trapped in active, resting and desensitized states, covering a full gating cycle. Additionally, we describe the structure of GluA4 alone, which displays a classical Y-shaped conformation. In resting conditions, GluA4:TARP-γ2 adopts two conformations, one resembling the desensitized states of other GluA subunits. Moreover, we identify a regulatory site for TARP-γ2 in the ligand-binding domain that modulates gating kinetics. Our findings uncover distinct features of GluA4, highlighting how subunit composition and auxiliary proteins shape receptor structure and dynamics, expanding glutamatergic signaling diversity. Vega-Gutiérrez et al. present cryo-electron microscopy structures of GluA4-containing AMPA receptors, which are key for brain signaling. They show GluA4-specific conformations and explain how subunit composition shapes receptor architecture, dynamics and function.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"32 12","pages":"2416-2428"},"PeriodicalIF":10.1,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145059292","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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: 10.1038/s41594-025-01669-4
Cameron J. Glasscock, Robert J. Pecoraro, Ryan McHugh, Lindsey A. Doyle, Wei Chen, Olivier Boivin, Beau Lonnquist, Emily Na, Yuliya Politanska, Hugh K. Haddox, David Cox, Christoffer Norn, Brian Coventry, Inna Goreshnik, Dionne Vafeados, Gyu Rie Lee, Raluca Gordân, Barry L. Stoddard, Frank DiMaio, David Baker
Sequence-specific DNA-binding proteins (DBPs) have critical roles in biology and biotechnology and there has been considerable interest in the engineering of DBPs with new or altered specificities for genome editing and other applications. While there has been some success in reprogramming naturally occurring DBPs using selection methods, the computational design of new DBPs that recognize arbitrary target sites remains an outstanding challenge. We describe a computational method for the design of small DBPs that recognize short specific target sequences through interactions with bases in the major groove and use this method to generate binders for five distinct DNA targets with mid-nanomolar to high-nanomolar affinities. The individual binding modules have specificity closely matching the computational models at as many as six base-pair positions and higher-order specificity can be achieved by rigidly positioning the binders along the DNA double helix using RFdiffusion. The crystal structure of a designed DBP–target site complex is in close agreement with the design model and the designed DBPs function in both Escherichia coli and mammalian cells to repress and activate transcription of neighboring genes. Our method provides a route to small and, hence, readily deliverable sequence-specific DBPs for gene regulation and editing. The authors develop a computational method to design small DNA-binding proteins (DBPs) that target specific sequences. Designed DBPs show structural accuracy and function in both bacterial and mammalian cells for transcriptional regulation.
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