Pub Date : 2024-12-20DOI: 10.1016/j.str.2024.11.014
Matthias Günther, Jana Sticht, Christian Freund, Thomas Höfer
Major histocompatibility complex class II (MHC-II) presents antigens to T helper cells. The spectrum of presented peptides is regulated by the exchange catalyst human leukocyte antigen DM (HLA-DM), which dissociates peptide-MHC-II complexes in the endosome. How susceptible a peptide is to HLA-DM is mechanistically not understood. Here, we present a data-driven mathematical model for the conformational landscape of MHC-II that explains the wide range of measured HLA-DM susceptibilities and predicts why some peptides are largely HLA-DM-resistant. We find that the conformational plasticity of MHC-II mediates both allosteric competition and cooperation between peptide and HLA-DM. Competition causes HLA-DM susceptibility to be proportional to the intrinsic peptide off-rate. Remarkably, diverse MHC-II allotypes with conserved HLA-DM interactions show a universal linear susceptibility function. However, HLA-DM-resistant peptides deviate from this susceptibility function; we predict resistance to be caused by fast peptide association with MHC-II. Thus, our study provides quantitative insight into peptide and MHC-II allotype parameters that shape class-II antigen presentation.
{"title":"Antigen presentation by MHC-II is shaped by competitive and cooperative allosteric mechanisms of peptide exchange","authors":"Matthias Günther, Jana Sticht, Christian Freund, Thomas Höfer","doi":"10.1016/j.str.2024.11.014","DOIUrl":"https://doi.org/10.1016/j.str.2024.11.014","url":null,"abstract":"Major histocompatibility complex class II (MHC-II) presents antigens to T helper cells. The spectrum of presented peptides is regulated by the exchange catalyst human leukocyte antigen DM (HLA-DM), which dissociates peptide-MHC-II complexes in the endosome. How susceptible a peptide is to HLA-DM is mechanistically not understood. Here, we present a data-driven mathematical model for the conformational landscape of MHC-II that explains the wide range of measured HLA-DM susceptibilities and predicts why some peptides are largely HLA-DM-resistant. We find that the conformational plasticity of MHC-II mediates both allosteric competition and cooperation between peptide and HLA-DM. Competition causes HLA-DM susceptibility to be proportional to the intrinsic peptide off-rate. Remarkably, diverse MHC-II allotypes with conserved HLA-DM interactions show a universal linear susceptibility function. However, HLA-DM-resistant peptides deviate from this susceptibility function; we predict resistance to be caused by fast peptide association with MHC-II. Thus, our study provides quantitative insight into peptide and MHC-II allotype parameters that shape class-II antigen presentation.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"20 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142857796","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 : 2024-12-20DOI: 10.1016/j.str.2024.11.018
Kavita A. Iyer, Takuya Kobayashi, Takashi Murayama, Montserrat Samsó
Mutations in the skeletal isoform of the ryanodine receptor 1 (RyR1) pose grave risks during anesthesia or treatment with succinylcholine muscle relaxants. These can trigger a potentially lethal malignant hyperthermia (MH) episode via intracellular calcium increase mainly from RyR1 channel leakage. Dantrolene is the only known treatment option to prevent death. The main target of dantrolene is RyR1; however, little is known about the mechanism of inhibition. Cryoelectron microscopy (cryo-EM) structures of dantrolene bound to the severe MH Y522S RyR1 mutant in the closed and open states at 2.5–3.3 Å resolution revealed that the drug binds to the channel’s cytoplasmic assembly, far from the ion gate, interacting with residues W882, W996, and R1000 in the P1 domain. The finding was validated by Ca2+ imaging and [3H]ryanodine binding in wild-type (WT) and alanine mutants. Dantrolene reduced channel opening probability by restricting the central activation module, “cooling down” the primed conformation caused by the mutation.
{"title":"Dantrolene inhibition of ryanodine receptor 1 carrying the severe malignant hyperthermia mutation Y522S visualized by cryo-EM","authors":"Kavita A. Iyer, Takuya Kobayashi, Takashi Murayama, Montserrat Samsó","doi":"10.1016/j.str.2024.11.018","DOIUrl":"https://doi.org/10.1016/j.str.2024.11.018","url":null,"abstract":"Mutations in the skeletal isoform of the ryanodine receptor 1 (RyR1) pose grave risks during anesthesia or treatment with succinylcholine muscle relaxants. These can trigger a potentially lethal malignant hyperthermia (MH) episode via intracellular calcium increase mainly from RyR1 channel leakage. Dantrolene is the only known treatment option to prevent death. The main target of dantrolene is RyR1; however, little is known about the mechanism of inhibition. Cryoelectron microscopy (cryo-EM) structures of dantrolene bound to the severe MH Y522S RyR1 mutant in the closed and open states at 2.5–3.3 Å resolution revealed that the drug binds to the channel’s cytoplasmic assembly, far from the ion gate, interacting with residues W882, W996, and R1000 in the P1 domain. The finding was validated by Ca<sup>2+</sup> imaging and [<sup>3</sup>H]ryanodine binding in wild-type (WT) and alanine mutants. Dantrolene reduced channel opening probability by restricting the central activation module, “cooling down” the primed conformation caused by the mutation.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"112 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142857799","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 : 2024-12-19DOI: 10.1016/j.str.2024.11.017
Sven Larsen-Ledet, Søren Lindemose, Aleksandra Panfilova, Sarah Gersing, Caroline H. Suhr, Aitana Victoria Genzor, Heleen Lanters, Sofie V. Nielsen, Kresten Lindorff-Larsen, Jakob R. Winther, Amelie Stein, Rasmus Hartmann-Petersen
Gene variants resulting in insertions or deletions of amino acid residues (indels) have important consequences for evolution and are often linked to disease, yet, compared to missense variants, the effects of indels are poorly understood and predicted. We developed a sensitive protein folding sensor based on the complementation of uracil auxotrophy in yeast by circular permutated orotate phosphoribosyltransferase (CPOP). The sensor reports on the folding of disease-linked missense variants and de-novo-designed proteins. Applying the folding sensor to a saturated library of single-residue indels in human dihydrofolate reductase (DHFR) revealed that most regions that tolerate indels are confined to internal loops, the termini, and a central α helix. Several indels are temperature sensitive, and folding is rescued upon binding to methotrexate. Rosetta and AlphaFold2 predictions correlate with the observed effects, suggesting that most indels destabilize the native fold and that these computational tools are useful for the classification of indels observed in population sequencing.
{"title":"Systematic characterization of indel variants using a yeast-based protein folding sensor","authors":"Sven Larsen-Ledet, Søren Lindemose, Aleksandra Panfilova, Sarah Gersing, Caroline H. Suhr, Aitana Victoria Genzor, Heleen Lanters, Sofie V. Nielsen, Kresten Lindorff-Larsen, Jakob R. Winther, Amelie Stein, Rasmus Hartmann-Petersen","doi":"10.1016/j.str.2024.11.017","DOIUrl":"https://doi.org/10.1016/j.str.2024.11.017","url":null,"abstract":"Gene variants resulting in insertions or deletions of amino acid residues (indels) have important consequences for evolution and are often linked to disease, yet, compared to missense variants, the effects of indels are poorly understood and predicted. We developed a sensitive protein folding sensor based on the complementation of uracil auxotrophy in yeast by circular permutated orotate phosphoribosyltransferase (CPOP). The sensor reports on the folding of disease-linked missense variants and <em>de</em>-<em>novo</em>-designed proteins. Applying the folding sensor to a saturated library of single-residue indels in human dihydrofolate reductase (DHFR) revealed that most regions that tolerate indels are confined to internal loops, the termini, and a central α helix. Several indels are temperature sensitive, and folding is rescued upon binding to methotrexate. Rosetta and AlphaFold2 predictions correlate with the observed effects, suggesting that most indels destabilize the native fold and that these computational tools are useful for the classification of indels observed in population sequencing.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"87 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142849500","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 : 2024-12-19DOI: 10.1016/j.str.2024.11.016
Reza Kolasangiani, Khashayar Farzanian, Yunfeng Chen, Martin A. Schwartz, Tamara C. Bidone
As major adhesion receptors, integrins transmit biochemical and mechanical signals across the plasma membrane. These functions are regulated by transitions between bent and extended conformations and modulated by force. To understand how force on integrins mediates cellular mechanosensing, we compared two highly homologous integrins, αIIbβ3 and αVβ3. These integrins, expressed in circulating platelets vs. solid tissues, respectively, share the β3 subunit, bind similar ligands and have similar bent and extended conformations. Here, we report that in cells expressing equivalent levels of each integrin, αIIbβ3 mediates spreading on softer substrates than αVβ3. These effects correlate with differences in structural dynamics of the two integrins under force. All-atom simulations show that αIIbβ3 is more flexible than αVβ3 due to correlated residue motions within the α subunit domains. Single molecule measurements confirm that αIIbβ3 extends faster than αVβ3. These results reveal a fundamental relationship between protein function and structural dynamics in cell mechanosensing.
作为主要的粘附受体，整合素通过质膜传递生化和机械信号。这些功能由弯曲构象和伸展构象之间的转换调节，并由力调节。为了了解整合素上的力是如何介导细胞机械传感的，我们比较了两种高度同源的整合素αIIbβ3和αVβ3。这些整合素分别在循环血小板和实体组织中表达，它们共享β3亚基，结合相似的配体，具有相似的弯曲和延伸构象。在这里，我们报告了在表达相同水平的每一种整合素的细胞中，αIIbβ3介导比αVβ3在更软的底物上的扩散。这些效应与两种整合素在作用力作用下的结构动力学差异有关。全原子模拟结果表明，α iib β3比α v β3更灵活，这是由于α亚基域内残基运动的关系。单分子测量证实αIIbβ3的延伸速度比αVβ3快。这些结果揭示了细胞机械传感中蛋白质功能与结构动力学之间的基本关系。
{"title":"Conformational response of αIIbβ3 and αVβ3 integrins to force","authors":"Reza Kolasangiani, Khashayar Farzanian, Yunfeng Chen, Martin A. Schwartz, Tamara C. Bidone","doi":"10.1016/j.str.2024.11.016","DOIUrl":"https://doi.org/10.1016/j.str.2024.11.016","url":null,"abstract":"As major adhesion receptors, integrins transmit biochemical and mechanical signals across the plasma membrane. These functions are regulated by transitions between bent and extended conformations and modulated by force. To understand how force on integrins mediates cellular mechanosensing, we compared two highly homologous integrins, α<sub>IIb</sub>β<sub>3</sub> and α<sub>V</sub>β<sub>3</sub>. These integrins, expressed in circulating platelets vs. solid tissues, respectively, share the β<sub>3</sub> subunit, bind similar ligands and have similar bent and extended conformations. Here, we report that in cells expressing equivalent levels of each integrin, α<sub>IIb</sub>β<sub>3</sub> mediates spreading on softer substrates than α<sub>V</sub>β<sub>3</sub>. These effects correlate with differences in structural dynamics of the two integrins under force. All-atom simulations show that α<sub>IIb</sub>β<sub>3</sub> is more flexible than α<sub>V</sub>β<sub>3</sub> due to correlated residue motions within the α subunit domains. Single molecule measurements confirm that α<sub>IIb</sub>β<sub>3</sub> extends faster than α<sub>V</sub>β<sub>3</sub>. These results reveal a fundamental relationship between protein function and structural dynamics in cell mechanosensing.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"23 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142849503","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 : 2024-12-17DOI: 10.1016/j.str.2024.11.015
Anna D. Burtseva, Timur N. Baymukhametov, Maxim A. Bolshakov, Zoya К. Makhneva, Andrey V. Mardanov, Andrey M. Tsedilin, Huawei Zhang, Vladimir.O. Popov, Aleksandr A. Ashikhmin, Konstantin M. Boyko
Bacteria with the simplest system for solar energy absorption and conversion use various types of light-harvesting complexes for these purposes. Light-harvesting complex 2 (LH2), an important component of the bacterial photosynthetic apparatus, has been structurally well characterized among purple non-sulfur bacteria. In contrast, so far only one high-resolution LH2 structure from sulfur bacteria is known. Here, we report the near-atomic resolution cryoelectron microscopy (cryo-EM) structure of the LH2 complex from the purple sulfur bacterium Ectothiorhodospira haloalkaliphila, which allowed us to determine the predominant polypeptide composition of this complex and the identification of the most probable type of its carotenoid. Comparison of our structure with the only known LH2 complex from a sulfur bacterium revealed severe differences in the overall ring-like organization. Expanding the architectural universe of bacterial light-harvesting complexes, our results demonstrate that, as observed for non-sulfur bacteria, the LH2 complexes of sulfur bacteria may also exhibit various types of spatial organization.
{"title":"Near-atomic cryo-EM structure of the light-harvesting complex LH2 from the sulfur purple bacterium Ectothiorhodospira haloalkaliphila","authors":"Anna D. Burtseva, Timur N. Baymukhametov, Maxim A. Bolshakov, Zoya К. Makhneva, Andrey V. Mardanov, Andrey M. Tsedilin, Huawei Zhang, Vladimir.O. Popov, Aleksandr A. Ashikhmin, Konstantin M. Boyko","doi":"10.1016/j.str.2024.11.015","DOIUrl":"https://doi.org/10.1016/j.str.2024.11.015","url":null,"abstract":"Bacteria with the simplest system for solar energy absorption and conversion use various types of light-harvesting complexes for these purposes. Light-harvesting complex 2 (LH2), an important component of the bacterial photosynthetic apparatus, has been structurally well characterized among purple non-sulfur bacteria. In contrast, so far only one high-resolution LH2 structure from sulfur bacteria is known. Here, we report the near-atomic resolution cryoelectron microscopy (cryo-EM) structure of the LH2 complex from the purple sulfur bacterium <em>Ectothiorhodospira haloalkaliphila</em>, which allowed us to determine the predominant polypeptide composition of this complex and the identification of the most probable type of its carotenoid. Comparison of our structure with the only known LH2 complex from a sulfur bacterium revealed severe differences in the overall ring-like organization. Expanding the architectural universe of bacterial light-harvesting complexes, our results demonstrate that, as observed for non-sulfur bacteria, the LH2 complexes of sulfur bacteria may also exhibit various types of spatial organization.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"48 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142832784","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 : 2024-12-11DOI: 10.1016/j.str.2024.11.013
Alexandra Houser, Isabelle Baconguis
Epithelial sodium channels (ENaCs) play a crucial role in Na+ reabsorption in mammals. To date, four subunits have been identified—α, β, γ, and δ—believed to form different heteromeric complexes. Currently, only the structure of the αβγ complex is known. To investigate the formation of channels with different subunit compositions and to determine how each subunit contributes to distinct channel properties, we co-expressed human δ, β, and γ. Using single-particle cryoelectron microscopy, we observed three distinct ENaC complexes. The structures unveil a pattern in which β and γ positions are conserved among the different complexes while the α position in αβγ trimer is occupied by either δ or another β. The δ subunit induces structural rearrangements in the γ subunit, which may contribute to the differences in channel activity between αβγ and δβγ channels. These structural changes provide molecular insights into how ENaC subunit composition modulates channel function.
{"title":"Structural insights into subunit-dependent functional regulation in epithelial sodium channels","authors":"Alexandra Houser, Isabelle Baconguis","doi":"10.1016/j.str.2024.11.013","DOIUrl":"https://doi.org/10.1016/j.str.2024.11.013","url":null,"abstract":"Epithelial sodium channels (ENaCs) play a crucial role in Na<sup>+</sup> reabsorption in mammals. To date, four subunits have been identified—α, β, γ, and δ—believed to form different heteromeric complexes. Currently, only the structure of the αβγ complex is known. To investigate the formation of channels with different subunit compositions and to determine how each subunit contributes to distinct channel properties, we co-expressed human δ, β, and γ. Using single-particle cryoelectron microscopy, we observed three distinct ENaC complexes. The structures unveil a pattern in which β and γ positions are conserved among the different complexes while the α position in αβγ trimer is occupied by either δ or another β. The δ subunit induces structural rearrangements in the γ subunit, which may contribute to the differences in channel activity between αβγ and δβγ channels. These structural changes provide molecular insights into how ENaC subunit composition modulates channel function.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"9 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142804853","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 : 2024-12-10DOI: 10.1016/j.str.2024.11.011
Alexander M. Barclay, Moses H. Milchberg, Owen A. Warmuth, Marcus D. Tuttle, Christopher J. Dennis, Charles D. Schwieters, Chad M. Rienstra
Amyloid fibrils are protein assemblies that are pathologically linked to neurodegenerative diseases. Fibril structures can aid development of highly specific ligands for diagnostic imaging and therapeutics. Solid-state NMR (SSNMR) is a viable approach to solving fibril structures; however, most SSNMR protocols require manual analysis of extensive spectral data, presenting a major bottleneck to determining structures. Standard automation; routines fall short for symmetric multimeric assemblies like amyloids due to high cross peak degeneracy and the need to account for multiple protein subunits. Here, we employ the probabilistic assignment for structure determination protocol in conjunction with strict; symmetry in Xplor-NIH structure determination software, demonstrating the methodology using data from a previous structure of an α-synuclein (Asyn) fibril implicated in Parkinson disease. The automated protocol generated a structure of comparable, if not superior, quality in a few days of computational time, reducing the manual effort required; to solve amyloid structures by SSNMR.
{"title":"Automated fibril structure calculations in Xplor-NIH","authors":"Alexander M. Barclay, Moses H. Milchberg, Owen A. Warmuth, Marcus D. Tuttle, Christopher J. Dennis, Charles D. Schwieters, Chad M. Rienstra","doi":"10.1016/j.str.2024.11.011","DOIUrl":"https://doi.org/10.1016/j.str.2024.11.011","url":null,"abstract":"Amyloid fibrils are protein assemblies that are pathologically linked to neurodegenerative diseases. Fibril structures can aid development of highly specific ligands for diagnostic imaging and therapeutics. Solid-state NMR (SSNMR) is a viable approach to solving fibril structures; however, most SSNMR protocols require manual analysis of extensive spectral data, presenting a major bottleneck to determining structures. Standard automation; routines fall short for symmetric multimeric assemblies like amyloids due to high cross peak degeneracy and the need to account for multiple protein subunits. Here, we employ the probabilistic assignment for structure determination protocol in conjunction with strict; symmetry in Xplor-NIH structure determination software, demonstrating the methodology using data from a previous structure of an α-synuclein (Asyn) fibril implicated in Parkinson disease. The automated protocol generated a structure of comparable, if not superior, quality in a few days of computational time, reducing the manual effort required; to solve amyloid structures by SSNMR.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"20 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142797529","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 : 2024-12-09DOI: 10.1016/j.str.2024.11.009
Yunlong Qiu, Yiwei Gao, Qinru Bai, Yan Zhao
In cholinergic neurons, choline is the precursor of the excitatory neurotransmitter acetylcholine (ACh), which plays a fundamental role in the brain. The high-affinity choline transporter, CHT1, mediates the efficient recycling of choline to facilitate ACh synthesis in the presynapse. Here, we report high-resolution cryoelectron microscopic (cryo-EM) structures of CHT1 in complex with the inhibitors HC-3 and ML352, the substrate choline, and a substrate-free state. Our structures show distinct binding modes of the inhibitors with different chemical structures, revealing their inhibition mechanisms. Additionally, we observed a chloride ion that directly interacts with the substrate choline, thereby stabilizing its binding with CHT1. Two sodium ions, Na2 and Na3, were clearly identified, which we speculate might be involved in substrate binding and conformational transitions, respectively. Our structures provide molecular insights into the coupling mechanism of ion binding with substrate binding and conformational transitions, promoting our understanding of the ion-coupled substrate transport mechanism.
{"title":"Ion coupling and inhibitory mechanisms of the human presynaptic high-affinity choline transporter CHT1","authors":"Yunlong Qiu, Yiwei Gao, Qinru Bai, Yan Zhao","doi":"10.1016/j.str.2024.11.009","DOIUrl":"https://doi.org/10.1016/j.str.2024.11.009","url":null,"abstract":"In cholinergic neurons, choline is the precursor of the excitatory neurotransmitter acetylcholine (ACh), which plays a fundamental role in the brain. The high-affinity choline transporter, CHT1, mediates the efficient recycling of choline to facilitate ACh synthesis in the presynapse. Here, we report high-resolution cryoelectron microscopic (cryo-EM) structures of CHT1 in complex with the inhibitors HC-3 and ML352, the substrate choline, and a substrate-free state. Our structures show distinct binding modes of the inhibitors with different chemical structures, revealing their inhibition mechanisms. Additionally, we observed a chloride ion that directly interacts with the substrate choline, thereby stabilizing its binding with CHT1. Two sodium ions, Na2 and Na3, were clearly identified, which we speculate might be involved in substrate binding and conformational transitions, respectively. Our structures provide molecular insights into the coupling mechanism of ion binding with substrate binding and conformational transitions, promoting our understanding of the ion-coupled substrate transport mechanism.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"20 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142793553","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 : 2024-12-09DOI: 10.1016/j.str.2024.11.010
Alzbeta Dikunova, Nikola Noskova, Jan H. Overbeck, Martin Polak, David Stelzig, David Zapletal, Karel Kubicek, Jiri Novacek, Remco Sprangers, Richard Stefl
The 5′–3′ exoribonuclease Xrn2, known as Rat1 in yeasts, terminates mRNA transcription by RNA polymerase II (RNAPII). In the torpedo model of termination, the activity of Xrn2/Rat1 is enhanced by Rai1, which is recruited to the termination site by Rtt103, an adaptor protein binding to the RNAPII C-terminal domain (CTD). The overall architecture of the Xrn2/Rat1-Rai1-Rtt103 complex remains unknown. We combined structural biology methods to characterize the torpedo complex from Saccharomyces cerevisiae and Chaetomium thermophilum. Comparison of the structures from these organisms revealed a conserved protein core fold of the subunits, but significant variability in their interaction interfaces. We found that in the mesophile, Rtt103 utilizes an unstructured region to augment a Rai1 β-sheet, while in the thermophile Rtt103 binds to a C-terminal helix of Rai1 via its CTD-interacting domain with an α-helical fold. These different torpedo complex assemblies reflect adaptations to the environment and impact complex recruitment to RNAPII.
5 ‘ -3 ’外核糖核酸酶Xrn2，在酵母中被称为Rat1，通过RNA聚合酶II （RNAPII）终止mRNA转录。在鱼雷终止模型中，Rai1增强了Xrn2/Rat1的活性，Rai1通过Rtt103招募到终止位点，Rtt103是一种结合RNAPII c -末端结构域（CTD）的接头蛋白。Xrn2/Rat1-Rai1-Rtt103复合体的整体结构仍然未知。我们结合结构生物学方法对酿酒酵母和嗜热毛菌的鱼雷复合物进行了表征。这些生物的结构比较揭示了亚基的保守蛋白核心折叠，但它们的相互作用界面有显著的差异。我们发现，在亲介菌中，Rtt103利用一个非结构化区域来增加Rai1 β-片，而在亲热菌中，Rtt103通过其具有α-螺旋折叠的ctd相互作用结构域与Rai1的c端螺旋结合。这些不同的鱼雷复合物装配反映了对环境的适应和影响RNAPII的复合物招募。
{"title":"Assembly of the Xrn2/Rat1–Rai1–Rtt103 termination complexes in mesophilic and thermophilic organisms","authors":"Alzbeta Dikunova, Nikola Noskova, Jan H. Overbeck, Martin Polak, David Stelzig, David Zapletal, Karel Kubicek, Jiri Novacek, Remco Sprangers, Richard Stefl","doi":"10.1016/j.str.2024.11.010","DOIUrl":"https://doi.org/10.1016/j.str.2024.11.010","url":null,"abstract":"The 5′–3′ exoribonuclease Xrn2, known as Rat1 in yeasts, terminates mRNA transcription by RNA polymerase II (RNAPII). In the torpedo model of termination, the activity of Xrn2/Rat1 is enhanced by Rai1, which is recruited to the termination site by Rtt103, an adaptor protein binding to the RNAPII C-terminal domain (CTD). The overall architecture of the Xrn2/Rat1-Rai1-Rtt103 complex remains unknown. We combined structural biology methods to characterize the torpedo complex from <em>Saccharomyces cerevisiae</em> and <em>Chaetomium thermophilum</em>. Comparison of the structures from these organisms revealed a conserved protein core fold of the subunits, but significant variability in their interaction interfaces. We found that in the mesophile, Rtt103 utilizes an unstructured region to augment a Rai1 β-sheet, while in the thermophile Rtt103 binds to a C-terminal helix of Rai1 via its CTD-interacting domain with an α-helical fold. These different torpedo complex assemblies reflect adaptations to the environment and impact complex recruitment to RNAPII.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"34 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142793552","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 : 2024-12-06DOI: 10.1016/j.str.2024.11.008
Yanyan Zhao, Michael F. Schmid, Wah Chiu
Cryogenic electron microscopy single particle analysis (cryoEM-SPA) has evolved into a routine approach for determining macromolecule structures to near-atomic resolution. Cryogenic electron tomography subtomogram averaging (cryoET-STA) toward a similar resolution, in contrast, is still under active development. Here, we use the archeal chaperonin MmCpn as a model macromolecule to quantitatively investigate the resolution limiting factors of cryoET-STA in terms of cumulative electron dose, ice thickness, subtomogram numbers, and tilt angle ranges. By delineating the feasibility and experimental factors of attaining near atomic resolution structure with cryoET-STA, especially the effect of electron damage through the tilt series and inelastic scattering at various ice thickness, we encourage a customized tilt series collection strategy for efficient throughput. This study provides a biophysical basis for the application of cryoET-STA (for highly symmetric molecules like MmCpn) toward high resolution and the rationales in using cryoET-STA to achieve an efficient outcome at the desired resolution.
{"title":"Cost-benefit analysis of cryogenic electron tomography subtomogram averaging of chaperonin MmCpn at near atomic resolution","authors":"Yanyan Zhao, Michael F. Schmid, Wah Chiu","doi":"10.1016/j.str.2024.11.008","DOIUrl":"https://doi.org/10.1016/j.str.2024.11.008","url":null,"abstract":"Cryogenic electron microscopy single particle analysis (cryoEM-SPA) has evolved into a routine approach for determining macromolecule structures to near-atomic resolution. Cryogenic electron tomography subtomogram averaging (cryoET-STA) toward a similar resolution, in contrast, is still under active development. Here, we use the archeal chaperonin MmCpn as a model macromolecule to quantitatively investigate the resolution limiting factors of cryoET-STA in terms of cumulative electron dose, ice thickness, subtomogram numbers, and tilt angle ranges. By delineating the feasibility and experimental factors of attaining near atomic resolution structure with cryoET-STA, especially the effect of electron damage through the tilt series and inelastic scattering at various ice thickness, we encourage a customized tilt series collection strategy for efficient throughput. This study provides a biophysical basis for the application of cryoET-STA (for highly symmetric molecules like MmCpn) toward high resolution and the rationales in using cryoET-STA to achieve an efficient outcome at the desired resolution.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"19 1","pages":""},"PeriodicalIF":5.7,"publicationDate":"2024-12-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142782657","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：