[{"ddc":["570","572"],"title":"Structural and mechanistic study of bacterial complex I and its cyanobacterial ortholog","status":"public","user_id":"8b945eb4-e2f2-11eb-945a-df72226e66a9","_id":"12781","oa_version":"Published Version","file":[{"relation":"main_file","file_id":"12852","embargo":"2024-04-20","date_updated":"2023-04-19T14:33:41Z","date_created":"2023-04-19T14:33:41Z","checksum":"5ebb6345cb4119f93460c81310265a6d","embargo_to":"local","file_name":"VladyslavKravchuk_PhD_Thesis_PostSub_Final_1.pdf","access_level":"closed","file_size":6071553,"content_type":"application/pdf","creator":"vkravchu"},{"date_updated":"2023-04-20T07:02:59Z","date_created":"2023-04-19T14:33:52Z","checksum":"c12055c48411d030d2afa51de2166221","relation":"source_file","file_id":"12853","embargo":"2024-04-20","file_size":19468766,"content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","creator":"vkravchu","embargo_to":"local","file_name":"VladyslavKravchuk_PhD_Thesis_PostSub_Final.docx","access_level":"closed"}],"alternative_title":["ISTA Thesis"],"type":"dissertation","abstract":[{"lang":"eng","text":"Most energy in humans is produced in form of ATP by the mitochondrial respiratory chain consisting of several protein assemblies embedded into lipid membrane (complexes I-V). Complex I is the first and the largest enzyme of the respiratory chain which is essential for energy production. It couples the transfer of two electrons from NADH to ubiquinone with proton translocation across bacterial or inner mitochondrial membrane. The coupling mechanism between electron transfer and proton translocation is one of the biggest enigma in bioenergetics and structural biology. Even though the enzyme has been studied for decades, only recent technological advances in cryo-EM allowed its extensive structural investigation. \r\n\r\nComplex I from E.coli appears to be of special importance because it is a perfect model system with a rich mutant library, however the structure of the entire complex was unknown. In this thesis I have resolved structures of the minimal complex I version from E. coli in different states including reduced, inhibited, under reaction turnover and several others. Extensive structural analyses of these structures and comparison to structures from other species allowed to derive general features of conformational dynamics and propose a universal coupling mechanism. The mechanism is straightforward, robust and consistent with decades of experimental data available for complex I from different species. \r\n\r\nCyanobacterial NDH (cyanobacterial complex I) is a part of broad complex I superfamily and was studied as well in this thesis. It plays an important role in cyclic electron transfer (CET), during which electrons are cycled within PSI through ferredoxin and plastoquinone to generate proton gradient without NADPH production. Here, I solved structure of NDH and revealed additional state, which was not observed before. The novel “resting” state allowed to propose the mechanism of CET regulation. Moreover, conformational dynamics of NDH resembles one in complex I which suggest more broad universality of the proposed coupling mechanism.\r\n\r\nIn summary, results presented here helped to interpret decades of experimental data for complex I and contributed to fundamental mechanistic understanding of protein function.\r\n"}],"page":"127","citation":{"ama":"Kravchuk V. Structural and mechanistic study of bacterial complex I and its cyanobacterial ortholog. 2023. doi:10.15479/at:ista:12781","apa":"Kravchuk, V. (2023). Structural and mechanistic study of bacterial complex I and its cyanobacterial ortholog. Institute of Science and Technology Austria. https://doi.org/10.15479/at:ista:12781","ieee":"V. Kravchuk, “Structural and mechanistic study of bacterial complex I and its cyanobacterial ortholog,” Institute of Science and Technology Austria, 2023.","ista":"Kravchuk V. 2023. Structural and mechanistic study of bacterial complex I and its cyanobacterial ortholog. Institute of Science and Technology Austria.","short":"V. Kravchuk, Structural and Mechanistic Study of Bacterial Complex I and Its Cyanobacterial Ortholog, Institute of Science and Technology Austria, 2023.","mla":"Kravchuk, Vladyslav. Structural and Mechanistic Study of Bacterial Complex I and Its Cyanobacterial Ortholog. Institute of Science and Technology Austria, 2023, doi:10.15479/at:ista:12781.","chicago":"Kravchuk, Vladyslav. “Structural and Mechanistic Study of Bacterial Complex I and Its Cyanobacterial Ortholog.” Institute of Science and Technology Austria, 2023. https://doi.org/10.15479/at:ista:12781."},"date_published":"2023-03-23T00:00:00Z","day":"23","has_accepted_license":"1","article_processing_charge":"No","publication_status":"published","publisher":"Institute of Science and Technology Austria","department":[{"_id":"GradSch"},{"_id":"LeSa"}],"year":"2023","date_created":"2023-03-31T12:24:42Z","date_updated":"2023-08-04T08:54:51Z","author":[{"id":"4D62F2A6-F248-11E8-B48F-1D18A9856A87","last_name":"Kravchuk","first_name":"Vladyslav","full_name":"Kravchuk, Vladyslav"}],"related_material":{"record":[{"id":"12138","relation":"part_of_dissertation","status":"public"}]},"file_date_updated":"2023-04-20T07:02:59Z","ec_funded":1,"project":[{"grant_number":"25541","_id":"238A0A5A-32DE-11EA-91FC-C7463DDC885E","name":"Structural characterization of E. coli complex I: an important mechanistic model"},{"name":"Structure and mechanism of respiratory chain molecular machines","call_identifier":"H2020","grant_number":"101020697","_id":"627abdeb-2b32-11ec-9570-ec31a97243d3"}],"acknowledged_ssus":[{"_id":"EM-Fac"}],"supervisor":[{"full_name":"Sazanov, Leonid A","first_name":"Leonid A","last_name":"Sazanov","id":"338D39FE-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-0977-7989"}],"degree_awarded":"PhD","language":[{"iso":"eng"}],"doi":"10.15479/at:ista:12781","month":"03","publication_identifier":{"isbn":["978-3-99078-029-9"],"issn":["2663-337X"]}},{"file_date_updated":"2023-05-30T17:07:05Z","ec_funded":1,"publication_status":"published","publisher":"Springer Nature","department":[{"_id":"LeSa"}],"acknowledgement":"This research was supported by the Scientific Service Units (SSU) of IST Austria through resources provided by the Electron Microscopy Facility (EMF), the Life Science Facility (LSF) and the IST high-performance computing cluster. We thank V.-V. Hodirnau from IST Austria EMF, M. Babiak from CEITEC for assistance with collecting cryo-EM data and A. Charnagalov for the assistance with protein purification. V.K. was a recipient of a DOC Fellowship of the Austrian Academy of Sciences at the Institute of Science and Technology, Austria. V.K. and O.P. are funded by the ERC Advanced Grant 101020697 RESPICHAIN to L.S. This work was also supported by the Medical Research Council (UK).","year":"2022","pmid":1,"date_updated":"2023-08-04T08:54:52Z","date_created":"2023-01-12T12:04:33Z","volume":609,"author":[{"full_name":"Kravchuk, Vladyslav","id":"4D62F2A6-F248-11E8-B48F-1D18A9856A87","last_name":"Kravchuk","first_name":"Vladyslav"},{"id":"5D8C9660-5D49-11EA-8188-567B3DDC885E","first_name":"Olga","last_name":"Petrova","full_name":"Petrova, Olga"},{"full_name":"Kampjut, Domen","id":"37233050-F248-11E8-B48F-1D18A9856A87","last_name":"Kampjut","first_name":"Domen"},{"first_name":"Anna","last_name":"Wojciechowska-Bason","full_name":"Wojciechowska-Bason, Anna"},{"full_name":"Breese, Zara","last_name":"Breese","first_name":"Zara"},{"id":"338D39FE-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-0977-7989","first_name":"Leonid A","last_name":"Sazanov","full_name":"Sazanov, Leonid A"}],"related_material":{"record":[{"id":"12781","relation":"dissertation_contains","status":"public"}],"link":[{"url":"https://doi.org/10.1038/s41586-022-05457-8","relation":"erratum"},{"url":"https://ista.ac.at/en/news/proton-dominos-kick-off-life/","description":"News on ISTA website","relation":"press_release"}]},"month":"09","publication_identifier":{"eissn":["1476-4687"],"issn":["0028-0836"]},"quality_controlled":"1","isi":1,"project":[{"grant_number":"25541","_id":"238A0A5A-32DE-11EA-91FC-C7463DDC885E","name":"Structural characterization of E. coli complex I: an important mechanistic model"},{"grant_number":"101020697","_id":"627abdeb-2b32-11ec-9570-ec31a97243d3","call_identifier":"H2020","name":"Structure and mechanism of respiratory chain molecular machines"}],"external_id":{"pmid":["36104567"],"isi":["000854788200001"]},"oa":1,"acknowledged_ssus":[{"_id":"EM-Fac"},{"_id":"LifeSc"},{"_id":"ScienComp"}],"language":[{"iso":"eng"}],"doi":"10.1038/s41586-022-05199-7","type":"journal_article","abstract":[{"lang":"eng","text":"Complex I is the first enzyme in the respiratory chain, which is responsible for energy production in mitochondria and bacteria1. Complex I couples the transfer of two electrons from NADH to quinone and the translocation of four protons across the membrane2, but the coupling mechanism remains contentious. Here we present cryo-electron microscopy structures of Escherichia coli complex I (EcCI) in different redox states, including catalytic turnover. EcCI exists mostly in the open state, in which the quinone cavity is exposed to the cytosol, allowing access for water molecules, which enable quinone movements. Unlike the mammalian paralogues3, EcCI can convert to the closed state only during turnover, showing that closed and open states are genuine turnover intermediates. The open-to-closed transition results in the tightly engulfed quinone cavity being connected to the central axis of the membrane arm, a source of substrate protons. Consistently, the proportion of the closed state increases with increasing pH. We propose a detailed but straightforward and robust mechanism comprising a ‘domino effect’ series of proton transfers and electrostatic interactions: the forward wave (‘dominoes stacking’) primes the pump, and the reverse wave (‘dominoes falling’) results in the ejection of all pumped protons from the distal subunit NuoL. This mechanism explains why protons exit exclusively from the NuoL subunit and is supported by our mutagenesis data. We contend that this is a universal coupling mechanism of complex I and related enzymes."}],"issue":"7928","status":"public","ddc":["572"],"title":"A universal coupling mechanism of respiratory complex I","intvolume":" 609","_id":"12138","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","oa_version":"Submitted Version","file":[{"creator":"lsazanov","file_size":1425655,"content_type":"application/pdf","access_level":"open_access","file_name":"EcCxI_manuscript_rev3_noSI_updated_withFigs_opt.pdf","success":1,"checksum":"d42a93e24f59e883ef0b5429832391d0","date_created":"2023-05-30T17:05:31Z","date_updated":"2023-05-30T17:05:31Z","file_id":"13104","relation":"main_file"},{"checksum":"5422bc0a73b3daadafa262c7ea6deae3","success":1,"date_updated":"2023-05-30T17:07:05Z","date_created":"2023-05-30T17:07:05Z","relation":"main_file","file_id":"13105","file_size":9842513,"content_type":"application/pdf","creator":"lsazanov","access_level":"open_access","file_name":"EcCxI_manuscript_rev3_SI_All_opt_upd.pdf"}],"keyword":["Multidisciplinary"],"scopus_import":"1","day":"22","has_accepted_license":"1","article_processing_charge":"No","article_type":"original","page":"808-814","publication":"Nature","citation":{"ama":"Kravchuk V, Petrova O, Kampjut D, Wojciechowska-Bason A, Breese Z, Sazanov LA. A universal coupling mechanism of respiratory complex I. Nature. 2022;609(7928):808-814. doi:10.1038/s41586-022-05199-7","apa":"Kravchuk, V., Petrova, O., Kampjut, D., Wojciechowska-Bason, A., Breese, Z., & Sazanov, L. A. (2022). A universal coupling mechanism of respiratory complex I. Nature. Springer Nature. https://doi.org/10.1038/s41586-022-05199-7","ieee":"V. Kravchuk, O. Petrova, D. Kampjut, A. Wojciechowska-Bason, Z. Breese, and L. A. Sazanov, “A universal coupling mechanism of respiratory complex I,” Nature, vol. 609, no. 7928. Springer Nature, pp. 808–814, 2022.","ista":"Kravchuk V, Petrova O, Kampjut D, Wojciechowska-Bason A, Breese Z, Sazanov LA. 2022. A universal coupling mechanism of respiratory complex I. Nature. 609(7928), 808–814.","short":"V. Kravchuk, O. Petrova, D. Kampjut, A. Wojciechowska-Bason, Z. Breese, L.A. Sazanov, Nature 609 (2022) 808–814.","mla":"Kravchuk, Vladyslav, et al. “A Universal Coupling Mechanism of Respiratory Complex I.” Nature, vol. 609, no. 7928, Springer Nature, 2022, pp. 808–14, doi:10.1038/s41586-022-05199-7.","chicago":"Kravchuk, Vladyslav, Olga Petrova, Domen Kampjut, Anna Wojciechowska-Bason, Zara Breese, and Leonid A Sazanov. “A Universal Coupling Mechanism of Respiratory Complex I.” Nature. Springer Nature, 2022. https://doi.org/10.1038/s41586-022-05199-7."},"date_published":"2022-09-22T00:00:00Z"},{"_id":"7464","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","title":"Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly","ddc":["570"],"status":"public","intvolume":" 16","oa_version":"Published Version","file":[{"access_level":"open_access","file_name":"2020_PLOSPatho_Dick.pdf","creator":"dernst","file_size":4551246,"content_type":"application/pdf","file_id":"7484","relation":"main_file","checksum":"a297f54d1fef0efe4789ca00f37f241e","date_updated":"2020-07-14T12:47:59Z","date_created":"2020-02-11T10:07:28Z"}],"type":"journal_article","abstract":[{"text":"Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CANTD). For HIV-1 the cellular molecule inositol hexakisphosphate (IP6) interacts with and stabilizes the immature hexamer, and is required for production of infectious virus particles. We have used in vitro assembly, cryo-electron tomography and subtomogram averaging, atomistic molecular dynamics simulations and mutational analyses to study the HIV-related lentivirus equine infectious anemia virus (EIAV). In particular, we sought to understand the structural conservation of the immature lentivirus lattice and the role of IP6 in EIAV assembly. Similar to HIV-1, IP6 strongly promoted in vitro assembly of EIAV Gag proteins into virus-like particles (VLPs), which took three morphologically highly distinct forms: narrow tubes, wide tubes, and spheres. Structural characterization of these VLPs to sub-4Å resolution unexpectedly showed that all three morphologies are based on an immature lattice with preserved key structural components, highlighting the structural versatility of CA to form immature assemblies. A direct comparison between EIAV and HIV revealed that both lentiviruses maintain similar immature interfaces, which are established by both conserved and non-conserved residues. In both EIAV and HIV-1, IP6 regulates immature assembly via conserved lysine residues within the CACTD and SP. Lastly, we demonstrate that IP6 stimulates in vitro assembly of immature particles of several other retroviruses in the lentivirus genus, suggesting a conserved role for IP6 in lentiviral assembly.","lang":"eng"}],"issue":"1","publication":"PLOS Pathogens","citation":{"ieee":"R. A. Dick et al., “Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly,” PLOS Pathogens, vol. 16, no. 1. Public Library of Science, 2020.","apa":"Dick, R. A., Xu, C., Morado, D. R., Kravchuk, V., Ricana, C. L., Lyddon, T. D., … Schur, F. K. (2020). Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly. PLOS Pathogens. Public Library of Science. https://doi.org/10.1371/journal.ppat.1008277","ista":"Dick RA, Xu C, Morado DR, Kravchuk V, Ricana CL, Lyddon TD, Broad AM, Feathers JR, Johnson MC, Vogt VM, Perilla JR, Briggs JAG, Schur FK. 2020. Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly. PLOS Pathogens. 16(1), e1008277.","ama":"Dick RA, Xu C, Morado DR, et al. Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly. PLOS Pathogens. 2020;16(1). doi:10.1371/journal.ppat.1008277","chicago":"Dick, Robert A., Chaoyi Xu, Dustin R. Morado, Vladyslav Kravchuk, Clifton L. Ricana, Terri D. Lyddon, Arianna M. Broad, et al. “Structures of Immature EIAV Gag Lattices Reveal a Conserved Role for IP6 in Lentivirus Assembly.” PLOS Pathogens. Public Library of Science, 2020. https://doi.org/10.1371/journal.ppat.1008277.","short":"R.A. Dick, C. Xu, D.R. Morado, V. Kravchuk, C.L. Ricana, T.D. Lyddon, A.M. Broad, J.R. Feathers, M.C. Johnson, V.M. Vogt, J.R. Perilla, J.A.G. Briggs, F.K. Schur, PLOS Pathogens 16 (2020).","mla":"Dick, Robert A., et al. “Structures of Immature EIAV Gag Lattices Reveal a Conserved Role for IP6 in Lentivirus Assembly.” PLOS Pathogens, vol. 16, no. 1, e1008277, Public Library of Science, 2020, doi:10.1371/journal.ppat.1008277."},"article_type":"original","date_published":"2020-01-27T00:00:00Z","scopus_import":"1","day":"27","has_accepted_license":"1","article_processing_charge":"No","year":"2020","pmid":1,"publication_status":"published","department":[{"_id":"FlSc"}],"publisher":"Public Library of Science","author":[{"last_name":"Dick","first_name":"Robert A.","full_name":"Dick, Robert A."},{"last_name":"Xu","first_name":"Chaoyi","full_name":"Xu, Chaoyi"},{"first_name":"Dustin R.","last_name":"Morado","full_name":"Morado, Dustin R."},{"full_name":"Kravchuk, Vladyslav","last_name":"Kravchuk","first_name":"Vladyslav","orcid":"0000-0001-9523-9089","id":"4D62F2A6-F248-11E8-B48F-1D18A9856A87"},{"full_name":"Ricana, Clifton L.","last_name":"Ricana","first_name":"Clifton L."},{"last_name":"Lyddon","first_name":"Terri D.","full_name":"Lyddon, Terri D."},{"full_name":"Broad, Arianna M.","last_name":"Broad","first_name":"Arianna M."},{"full_name":"Feathers, J. Ryan","last_name":"Feathers","first_name":"J. Ryan"},{"full_name":"Johnson, Marc C.","last_name":"Johnson","first_name":"Marc C."},{"full_name":"Vogt, Volker M.","last_name":"Vogt","first_name":"Volker M."},{"last_name":"Perilla","first_name":"Juan R.","full_name":"Perilla, Juan R."},{"full_name":"Briggs, John A. G.","last_name":"Briggs","first_name":"John A. G."},{"first_name":"Florian KM","last_name":"Schur","id":"48AD8942-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-4790-8078","full_name":"Schur, Florian KM"}],"related_material":{"record":[{"id":"9723","status":"deleted","relation":"research_data"}]},"date_created":"2020-02-06T18:47:17Z","date_updated":"2023-10-17T12:29:34Z","volume":16,"article_number":"e1008277","file_date_updated":"2020-07-14T12:47:59Z","external_id":{"isi":["000510746400010"],"pmid":["31986188"]},"tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","short":"CC BY (4.0)","image":"/images/cc_by.png"},"oa":1,"isi":1,"quality_controlled":"1","project":[{"_id":"26736D6A-B435-11E9-9278-68D0E5697425","grant_number":"P31445","name":"Structural conservation and diversity in retroviral capsid","call_identifier":"FWF"}],"doi":"10.1371/journal.ppat.1008277","acknowledged_ssus":[{"_id":"ScienComp"}],"language":[{"iso":"eng"}],"month":"01","publication_identifier":{"issn":["1553-7374"]}}]