Pub Date : 2024-07-19DOI: 10.1038/s41594-024-01360-0
Li Li, Mariia Yu Rybak, Jinzhong Lin, Matthieu G Gagnon
Translation termination involves release factors RF1, RF2 and the GTPase RF3 that recycles RF1 and RF2 from the ribosome. RF3 dissociates from the ribosome in the GDP-bound form and must then exchange GDP for GTP. The 70S ribosome termination complex (70S-TC) accelerates GDP exchange in RF3, suggesting that the 70S-TC can function as the guanine nucleotide exchange factor for RF3. Here, we use cryogenic-electron microscopy to elucidate the mechanism of GDP dissociation from RF3 catalyzed by the Escherichia coli 70S-TC. The non-rotated ribosome bound to RF1 remodels RF3 and induces a peptide flip in the phosphate-binding loop, efficiently ejecting GDP. Binding of GTP allows RF3 to dock at the GTPase center, promoting the dissociation of RF1 from the ribosome. The structures recapitulate the functional cycle of RF3 on the ribosome and uncover the mechanism by which the 70S-TC allosterically dismantles the phosphate-binding groove in RF3, a previously overlooked function of the ribosome.
{"title":"The ribosome termination complex remodels release factor RF3 and ejects GDP.","authors":"Li Li, Mariia Yu Rybak, Jinzhong Lin, Matthieu G Gagnon","doi":"10.1038/s41594-024-01360-0","DOIUrl":"10.1038/s41594-024-01360-0","url":null,"abstract":"<p><p>Translation termination involves release factors RF1, RF2 and the GTPase RF3 that recycles RF1 and RF2 from the ribosome. RF3 dissociates from the ribosome in the GDP-bound form and must then exchange GDP for GTP. The 70S ribosome termination complex (70S-TC) accelerates GDP exchange in RF3, suggesting that the 70S-TC can function as the guanine nucleotide exchange factor for RF3. Here, we use cryogenic-electron microscopy to elucidate the mechanism of GDP dissociation from RF3 catalyzed by the Escherichia coli 70S-TC. The non-rotated ribosome bound to RF1 remodels RF3 and induces a peptide flip in the phosphate-binding loop, efficiently ejecting GDP. Binding of GTP allows RF3 to dock at the GTPase center, promoting the dissociation of RF1 from the ribosome. The structures recapitulate the functional cycle of RF3 on the ribosome and uncover the mechanism by which the 70S-TC allosterically dismantles the phosphate-binding groove in RF3, a previously overlooked function of the ribosome.</p>","PeriodicalId":18836,"journal":{"name":"Nature Structural &Molecular Biology","volume":" ","pages":""},"PeriodicalIF":16.8,"publicationDate":"2024-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141727534","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 : 2022-01-22DOI: 10.1101/2022.01.21.477284
D. Snead, M. Matyszewski, Andrea M. Dickey, Yu Lin, A. Leschziner, Samara L. Reck-Peterson
Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most commonly mutated genes in familial Parkinson’s Disease (PD). Under some circumstances, LRRK2 co-localizes with microtubules in cells, an association enhanced by PD mutations. We report a cryo-electron microscopy structure of the catalytic half of LRRK2, containing its kinase, which is in a closed conformation, and GTPase domains, bound to microtubules. We also report a structure of the catalytic half of LRRK1, which is closely related to LRRK2, but is not linked to PD. LRRK1’s structure is similar to LRRK2, but LRRK1 does not interact with microtubules. Guided by these structures, we identify amino acids in LRRK2’s GTPase domain that mediate microtubule binding; mutating them disrupts microtubule binding in vitro and in cells, without affecting LRRK2’s kinase activity. Our results have implications for the design of therapeutic LRRK2 kinase inhibitors.
{"title":"Structural basis for Parkinson’s disease-linked LRRK2’s binding to microtubules","authors":"D. Snead, M. Matyszewski, Andrea M. Dickey, Yu Lin, A. Leschziner, Samara L. Reck-Peterson","doi":"10.1101/2022.01.21.477284","DOIUrl":"https://doi.org/10.1101/2022.01.21.477284","url":null,"abstract":"Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most commonly mutated genes in familial Parkinson’s Disease (PD). Under some circumstances, LRRK2 co-localizes with microtubules in cells, an association enhanced by PD mutations. We report a cryo-electron microscopy structure of the catalytic half of LRRK2, containing its kinase, which is in a closed conformation, and GTPase domains, bound to microtubules. We also report a structure of the catalytic half of LRRK1, which is closely related to LRRK2, but is not linked to PD. LRRK1’s structure is similar to LRRK2, but LRRK1 does not interact with microtubules. Guided by these structures, we identify amino acids in LRRK2’s GTPase domain that mediate microtubule binding; mutating them disrupts microtubule binding in vitro and in cells, without affecting LRRK2’s kinase activity. Our results have implications for the design of therapeutic LRRK2 kinase inhibitors.","PeriodicalId":18836,"journal":{"name":"Nature Structural &Molecular Biology","volume":"29 1","pages":"1196 - 1207"},"PeriodicalIF":16.8,"publicationDate":"2022-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41446644","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 : 2021-08-10DOI: 10.1101/2021.08.10.455846
K. Tsai, Vanja Stojković, D. J. Lee, Iris D. Young, Teresa Szal, N. Vázquez-Laslop, A. Mankin, James S. Fraser, D. Fujimori
The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. In this study, we determined that the second-generation oxazolidinone radezolid also induces stalling with alanine at the penultimate position. However, the molecular basis for context-specificity of these inhibitors has not been elucidated. In this study, we determined high-resolution cryo-EM structures of both linezolid and radezolid-stalled ribosome complexes. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling by forcing the antibiotic to adopt a conformation that narrows the hydrophobic alanine pocket. Together, the structural and biochemical findings presented in this work provide molecular understanding of context-specific inhibition of translation by clinically important oxazolidinone antibiotics.
{"title":"Structural basis for context-specific inhibition of translation by oxazolidinone antibiotics","authors":"K. Tsai, Vanja Stojković, D. J. Lee, Iris D. Young, Teresa Szal, N. Vázquez-Laslop, A. Mankin, James S. Fraser, D. Fujimori","doi":"10.1101/2021.08.10.455846","DOIUrl":"https://doi.org/10.1101/2021.08.10.455846","url":null,"abstract":"The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. In this study, we determined that the second-generation oxazolidinone radezolid also induces stalling with alanine at the penultimate position. However, the molecular basis for context-specificity of these inhibitors has not been elucidated. In this study, we determined high-resolution cryo-EM structures of both linezolid and radezolid-stalled ribosome complexes. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling by forcing the antibiotic to adopt a conformation that narrows the hydrophobic alanine pocket. Together, the structural and biochemical findings presented in this work provide molecular understanding of context-specific inhibition of translation by clinically important oxazolidinone antibiotics.","PeriodicalId":18836,"journal":{"name":"Nature Structural &Molecular Biology","volume":"1 1","pages":""},"PeriodicalIF":16.8,"publicationDate":"2021-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"62332424","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 : 2021-06-18DOI: 10.1101/2021.06.18.449003
Yahui Yan, H. Harding, D. Ron
Many regulatory PPP1R subunits join few catalytic PP1c subunits to mediate phosphoserine and phosphothreonine dephosphorylation in metazoans. Regulatory subunits engage the surface of PP1c, locally affecting flexible access of the phosphopeptide to the active site. However, catalytic efficiency of holophosphatases towards their phosphoprotein substrates remains unexplained. Here we present a cryo-EM structure of the tripartite PP1c–PPP1R15A–G-actin holophosphatase that terminates signaling in the mammalian integrated stress response (ISR) in the pre-dephosphorylation complex with its substrate, translation initiation factor 2α (eIF2α). G-actin, whose essential role in eIF2α dephosphorylation is supported crystallographically, biochemically and genetically, aligns the catalytic and regulatory subunits, creating a composite surface that engages the N-terminal domain of eIF2α to position the distant phosphoserine-51 at the active site. Substrate residues that mediate affinity for the holophosphatase also make critical contacts with eIF2α kinases. Thus, a convergent process of higher-order substrate recognition specifies functionally antagonistic phosphorylation and dephosphorylation in the ISR. Structures of the dephosphorylation complex for phosphorylated eIF2α reveal how contacts with the regulatory PPP1R15A subunit mediate substrate selectivity, providing a paradigm for dephosphorylation reactions by diverse combinatorially assembled holophosphatases.
{"title":"Higher-order phosphatase–substrate contacts terminate the integrated stress response","authors":"Yahui Yan, H. Harding, D. Ron","doi":"10.1101/2021.06.18.449003","DOIUrl":"https://doi.org/10.1101/2021.06.18.449003","url":null,"abstract":"Many regulatory PPP1R subunits join few catalytic PP1c subunits to mediate phosphoserine and phosphothreonine dephosphorylation in metazoans. Regulatory subunits engage the surface of PP1c, locally affecting flexible access of the phosphopeptide to the active site. However, catalytic efficiency of holophosphatases towards their phosphoprotein substrates remains unexplained. Here we present a cryo-EM structure of the tripartite PP1c–PPP1R15A–G-actin holophosphatase that terminates signaling in the mammalian integrated stress response (ISR) in the pre-dephosphorylation complex with its substrate, translation initiation factor 2α (eIF2α). G-actin, whose essential role in eIF2α dephosphorylation is supported crystallographically, biochemically and genetically, aligns the catalytic and regulatory subunits, creating a composite surface that engages the N-terminal domain of eIF2α to position the distant phosphoserine-51 at the active site. Substrate residues that mediate affinity for the holophosphatase also make critical contacts with eIF2α kinases. Thus, a convergent process of higher-order substrate recognition specifies functionally antagonistic phosphorylation and dephosphorylation in the ISR. Structures of the dephosphorylation complex for phosphorylated eIF2α reveal how contacts with the regulatory PPP1R15A subunit mediate substrate selectivity, providing a paradigm for dephosphorylation reactions by diverse combinatorially assembled holophosphatases.","PeriodicalId":18836,"journal":{"name":"Nature Structural &Molecular Biology","volume":"28 1","pages":"835 - 846"},"PeriodicalIF":16.8,"publicationDate":"2021-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49110087","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 : 2020-11-30DOI: 10.1101/2020.11.30.403857
L. Farnung, M. Ochmann, M. Engeholm, P. Cramer
Efficient transcription of RNA polymerase II (Pol II) through nucleosomes requires the help of various factors. Here we show biochemically that Pol II transcription through a nucleosome is facilitated by the chromatin remodeler Chd1 and the histone chaperone FACT when the elongation factors Spt4/5 and TFIIS are present. We report cryo-EM structures of transcribing Saccharomyces cerevisiae Pol II−Spt4/5−nucleosome complexes with bound Chd1 or FACT. In the first structure, Pol II transcription exposes the proximal histone H2A−H2B dimer that is bound by Spt5. Pol II has also released the inhibitory DNA-binding region of Chd1 that is poised to pump DNA toward Pol II. In the second structure, Pol II has generated a partially unraveled nucleosome that binds FACT, which excludes Chd1 and Spt5. These results suggest that Pol II progression through a nucleosome activates Chd1, enables FACT binding and eventually triggers transfer of FACT together with histones to upstream DNA. Structural and functional analyses of RNA polymerase II−nucleosome complexes reveal how the chromatin remodeler Chd1 and the histone chaperone FACT mediate Pol II transcription through a nucleosome.
{"title":"Structural basis of nucleosome transcription mediated by Chd1 and FACT","authors":"L. Farnung, M. Ochmann, M. Engeholm, P. Cramer","doi":"10.1101/2020.11.30.403857","DOIUrl":"https://doi.org/10.1101/2020.11.30.403857","url":null,"abstract":"Efficient transcription of RNA polymerase II (Pol II) through nucleosomes requires the help of various factors. Here we show biochemically that Pol II transcription through a nucleosome is facilitated by the chromatin remodeler Chd1 and the histone chaperone FACT when the elongation factors Spt4/5 and TFIIS are present. We report cryo-EM structures of transcribing Saccharomyces cerevisiae Pol II−Spt4/5−nucleosome complexes with bound Chd1 or FACT. In the first structure, Pol II transcription exposes the proximal histone H2A−H2B dimer that is bound by Spt5. Pol II has also released the inhibitory DNA-binding region of Chd1 that is poised to pump DNA toward Pol II. In the second structure, Pol II has generated a partially unraveled nucleosome that binds FACT, which excludes Chd1 and Spt5. These results suggest that Pol II progression through a nucleosome activates Chd1, enables FACT binding and eventually triggers transfer of FACT together with histones to upstream DNA. Structural and functional analyses of RNA polymerase II−nucleosome complexes reveal how the chromatin remodeler Chd1 and the histone chaperone FACT mediate Pol II transcription through a nucleosome.","PeriodicalId":18836,"journal":{"name":"Nature Structural &Molecular Biology","volume":"28 1","pages":"382 - 387"},"PeriodicalIF":16.8,"publicationDate":"2020-11-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42190486","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 : 2020-06-29DOI: 10.1101/2020.06.29.177642
M. Girbig, A. Misiaszek, Matthias K. Vorländer, Aleix Lafita, H. Grötsch, F. Baudin, A. Bateman, C. Müller
RNA polymerase III (Pol III) synthesizes transfer RNAs and other short, essential RNAs. Human Pol III misregulation is linked to tumor transformation, neurodegenerative and developmental disorders, and increased sensitivity to viral infections. Here, we present cryo-electron microscopy structures at 2.8 to 3.3 Å resolution of transcribing and unbound human Pol III. We observe insertion of the TFIIS-like subunit RPC10 into the polymerase funnel, providing insights into how RPC10 triggers transcription termination. Our structures resolve elements absent from Saccharomyces cerevisiae Pol III such as the winged-helix domains of RPC5 and an iron–sulfur cluster, which tethers the heterotrimer subcomplex to the core. The cancer-associated RPC7α isoform binds the polymerase clamp, potentially interfering with Pol III inhibition by tumor suppressor MAF1, which may explain why overexpressed RPC7α enhances tumor transformation. Finally, the human Pol III structure allows mapping of disease-related mutations and may contribute to the development of inhibitors that selectively target Pol III for therapeutic interventions. Cryo-EM structures of human Pol III in both apo- and elongating states reveal metazoan-specific differences in the regulation of transcription termination and identify mutations relevant to human disease.
RNA聚合酶III (RNA polymerase III, Pol III)合成转移RNA和其他短而必需的RNA。人类Pol III的失调与肿瘤转化、神经退行性和发育障碍以及对病毒感染的敏感性增加有关。在这里，我们展示了2.8至3.3 Å分辨率的转录和未结合的人Pol III的低温电镜结构。我们观察到tfiis样亚基RPC10插入到聚合酶漏斗中，为RPC10如何触发转录终止提供了见解。我们的结构解决了酿酒酵母Pol III中缺乏的元素，如RPC5的翼状螺旋结构域和铁硫簇，它将异源三聚体亚络合物连接到核心。癌症相关的RPC7α亚型结合聚合酶钳，可能干扰肿瘤抑制因子MAF1对Pol III的抑制，这可能解释了为什么过表达的RPC7α促进肿瘤转化。最后，人类Pol III结构允许绘制疾病相关突变，并可能有助于开发选择性靶向Pol III进行治疗干预的抑制剂。人类Pol III在载子状态和延长状态下的低温电镜结构揭示了转录终止调控的后生动物特异性差异，并鉴定了与人类疾病相关的突变。
{"title":"Cryo-EM structures of human RNA polymerase III in its unbound and transcribing states","authors":"M. Girbig, A. Misiaszek, Matthias K. Vorländer, Aleix Lafita, H. Grötsch, F. Baudin, A. Bateman, C. Müller","doi":"10.1101/2020.06.29.177642","DOIUrl":"https://doi.org/10.1101/2020.06.29.177642","url":null,"abstract":"RNA polymerase III (Pol III) synthesizes transfer RNAs and other short, essential RNAs. Human Pol III misregulation is linked to tumor transformation, neurodegenerative and developmental disorders, and increased sensitivity to viral infections. Here, we present cryo-electron microscopy structures at 2.8 to 3.3 Å resolution of transcribing and unbound human Pol III. We observe insertion of the TFIIS-like subunit RPC10 into the polymerase funnel, providing insights into how RPC10 triggers transcription termination. Our structures resolve elements absent from Saccharomyces cerevisiae Pol III such as the winged-helix domains of RPC5 and an iron–sulfur cluster, which tethers the heterotrimer subcomplex to the core. The cancer-associated RPC7α isoform binds the polymerase clamp, potentially interfering with Pol III inhibition by tumor suppressor MAF1, which may explain why overexpressed RPC7α enhances tumor transformation. Finally, the human Pol III structure allows mapping of disease-related mutations and may contribute to the development of inhibitors that selectively target Pol III for therapeutic interventions. Cryo-EM structures of human Pol III in both apo- and elongating states reveal metazoan-specific differences in the regulation of transcription termination and identify mutations relevant to human disease.","PeriodicalId":18836,"journal":{"name":"Nature Structural &Molecular Biology","volume":"28 1","pages":"210 - 219"},"PeriodicalIF":16.8,"publicationDate":"2020-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42310095","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 : 2020-02-24DOI: 10.1101/2020.02.21.960211
Corentin Claeys Bouuaert, Sam E. Tischfield, Stephen Pu, Eleni P. Mimitou, E. Arias-Palomo, J. Berger, S. Keeney
Spo11, which makes DNA double-strand breaks (DSBs) that are essential for meiotic recombination, has long been recalcitrant to biochemical study. We provide molecular analysis of Saccharomyces cerevisiae Spo11 purified with partners Rec102, Rec104 and Ski8. Rec102 and Rec104 jointly resemble the B subunit of archaeal topoisomerase VI, with Rec104 occupying a position similar to the Top6B GHKL-type ATPase domain. Unexpectedly, the Spo11 complex is monomeric (1:1:1:1 stoichiometry), consistent with dimerization controlling DSB formation. Reconstitution of DNA binding reveals topoisomerase-like preferences for duplex–duplex junctions and bent DNA. Spo11 also binds noncovalently but with high affinity to DNA ends mimicking cleavage products, suggesting a mechanism to cap DSB ends. Mutations that reduce DNA binding in vitro attenuate DSB formation, alter DSB processing and reshape the DSB landscape in vivo. Our data reveal structural and functional similarities between the Spo11 core complex and Topo VI, but also highlight differences reflecting their distinct biological roles. Biochemical and structural characterization of the meiotic DSB core complex of budding yeast reveals molecular architecture and DNA-binding properties similar to those of ancestral Topo VI.
{"title":"Structural and functional characterization of the Spo11 core complex","authors":"Corentin Claeys Bouuaert, Sam E. Tischfield, Stephen Pu, Eleni P. Mimitou, E. Arias-Palomo, J. Berger, S. Keeney","doi":"10.1101/2020.02.21.960211","DOIUrl":"https://doi.org/10.1101/2020.02.21.960211","url":null,"abstract":"Spo11, which makes DNA double-strand breaks (DSBs) that are essential for meiotic recombination, has long been recalcitrant to biochemical study. We provide molecular analysis of Saccharomyces cerevisiae Spo11 purified with partners Rec102, Rec104 and Ski8. Rec102 and Rec104 jointly resemble the B subunit of archaeal topoisomerase VI, with Rec104 occupying a position similar to the Top6B GHKL-type ATPase domain. Unexpectedly, the Spo11 complex is monomeric (1:1:1:1 stoichiometry), consistent with dimerization controlling DSB formation. Reconstitution of DNA binding reveals topoisomerase-like preferences for duplex–duplex junctions and bent DNA. Spo11 also binds noncovalently but with high affinity to DNA ends mimicking cleavage products, suggesting a mechanism to cap DSB ends. Mutations that reduce DNA binding in vitro attenuate DSB formation, alter DSB processing and reshape the DSB landscape in vivo. Our data reveal structural and functional similarities between the Spo11 core complex and Topo VI, but also highlight differences reflecting their distinct biological roles. Biochemical and structural characterization of the meiotic DSB core complex of budding yeast reveals molecular architecture and DNA-binding properties similar to those of ancestral Topo VI.","PeriodicalId":18836,"journal":{"name":"Nature Structural &Molecular Biology","volume":"28 1","pages":"92 - 102"},"PeriodicalIF":16.8,"publicationDate":"2020-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49195616","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}
Introduction In Chapter 2 we discussed the fundamentals of visioning and building a strong collaborative partnership—especially among key local agencies and organizations. But members of the public at large also need and increasingly want to reconnect with their community through food and agriculture—whether as voters, tourists or farm neighbors. In this section we describe some strategies for engaing the public in local food and agriculture system development.
{"title":"Engaging the public","authors":"Emily Troshynski","doi":"10.1201/9781315384467-5","DOIUrl":"https://doi.org/10.1201/9781315384467-5","url":null,"abstract":"Introduction In Chapter 2 we discussed the fundamentals of visioning and building a strong collaborative partnership—especially among key local agencies and organizations. But members of the public at large also need and increasingly want to reconnect with their community through food and agriculture—whether as voters, tourists or farm neighbors. In this section we describe some strategies for engaing the public in local food and agriculture system development.","PeriodicalId":18836,"journal":{"name":"Nature Structural &Molecular Biology","volume":"39 1","pages":"1287-1287"},"PeriodicalIF":16.8,"publicationDate":"2020-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74000721","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}
Xuepeng Wei, Kollin Schultz, Gleb A. Bazilevsky, Austin D. Vogt, R. Marmorstein
ATP-citrate lyase (ACLY) synthesizes cytosolic acetyl coenzyme A (acetyl-CoA), a fundamental cellular building block. Accordingly, aberrant ACLY activity is observed in many diseases. Here we report cryo-EM structures of human ACLY, alone or bound to substrates or products. ACLY forms a homotetramer with a rigid citrate synthase homology (CSH) module, flanked by four flexible acetyl-CoA synthetase homology (ASH) domains; CoA is bound at the CSH-ASH interface in mutually exclusive productive or unproductive conformations. The structure of a catalytic mutant of ACLY in the presence of ATP, citrate and CoA substrates reveals a phospho-citryl-CoA intermediate in the ASH domain. ACLY with acetyl-CoA and oxaloacetate products shows the products bound in the ASH domain, with an additional oxaloacetate in the CSH domain, which could function in ACLY autoinhibition. These structures, which are supported by biochemical and biophysical data, challenge previous proposals of the ACLY catalytic mechanism and suggest additional therapeutic possibilities for ACLY-associated metabolic disorders.
atp -柠檬酸解酶(ACLY)合成胞浆乙酰辅酶A (acetyl- coa)，这是细胞的基本组成部分。因此，在许多疾病中观察到异常的ACLY活性。在这里，我们报告了人类ACLY的低温电镜结构，单独或结合底物或产物。ACLY形成一个具有刚性柠檬酸合成酶同源性(CSH)模块的同聚体，两侧是四个柔性乙酰辅酶a合成酶同源性(ASH)结构域;CoA以互斥的生产性或非生产性构象结合在CSH-ASH界面上。在ATP、柠檬酸盐和CoA底物存在下，ACLY的催化突变体的结构揭示了ASH域中的磷酸柠檬酸辅酶a中间体。含有乙酰辅酶a和草酰乙酸产物的ACLY表明产物结合在ASH结构域，在CSH结构域有一个额外的草酰乙酸，这可能在ACLY中起自抑制作用。这些结构得到了生化和生物物理数据的支持，挑战了之前关于ACLY催化机制的建议，并为ACLY相关代谢疾病的治疗提供了额外的可能性。
{"title":"Structure of ACLY in complex with CoA","authors":"Xuepeng Wei, Kollin Schultz, Gleb A. Bazilevsky, Austin D. Vogt, R. Marmorstein","doi":"10.2210/pdb6poe/pdb","DOIUrl":"https://doi.org/10.2210/pdb6poe/pdb","url":null,"abstract":"ATP-citrate lyase (ACLY) synthesizes cytosolic acetyl coenzyme A (acetyl-CoA), a fundamental cellular building block. Accordingly, aberrant ACLY activity is observed in many diseases. Here we report cryo-EM structures of human ACLY, alone or bound to substrates or products. ACLY forms a homotetramer with a rigid citrate synthase homology (CSH) module, flanked by four flexible acetyl-CoA synthetase homology (ASH) domains; CoA is bound at the CSH-ASH interface in mutually exclusive productive or unproductive conformations. The structure of a catalytic mutant of ACLY in the presence of ATP, citrate and CoA substrates reveals a phospho-citryl-CoA intermediate in the ASH domain. ACLY with acetyl-CoA and oxaloacetate products shows the products bound in the ASH domain, with an additional oxaloacetate in the CSH domain, which could function in ACLY autoinhibition. These structures, which are supported by biochemical and biophysical data, challenge previous proposals of the ACLY catalytic mechanism and suggest additional therapeutic possibilities for ACLY-associated metabolic disorders.","PeriodicalId":18836,"journal":{"name":"Nature Structural &Molecular Biology","volume":"27 1","pages":"33-41"},"PeriodicalIF":16.8,"publicationDate":"2019-12-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44608200","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}
Y. Park, A. Walls, Z. Wang, M. Sauer, W. Li, M. Tortorici, B. Bosch, F. DiMaio, D. Veesler
{"title":"MERS-CoV S structure in complex with 2,3-sialyl-N-acetyl-lactosamine","authors":"Y. Park, A. Walls, Z. Wang, M. Sauer, W. Li, M. Tortorici, B. Bosch, F. DiMaio, D. Veesler","doi":"10.2210/pdb6q06/pdb","DOIUrl":"https://doi.org/10.2210/pdb6q06/pdb","url":null,"abstract":"","PeriodicalId":18836,"journal":{"name":"Nature Structural &Molecular Biology","volume":"1 1","pages":""},"PeriodicalIF":16.8,"publicationDate":"2019-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"68201933","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}
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