--- _id: '12757' abstract: - lang: eng text: My group and myself have studied respiratory complex I for almost 30 years, starting in 1994 when it was known as a L-shaped giant ‘black box' of bioenergetics. First breakthrough was the X-ray structure of the peripheral arm, followed by structures of the membrane arm and finally the entire complex from Thermus thermophilus. The developments in cryo-EM technology allowed us to solve the first complete structure of the twice larger, ∼1 MDa mammalian enzyme in 2016. However, the mechanism coupling, over large distances, the transfer of two electrons to pumping of four protons across the membrane remained an enigma. Recently we have solved high-resolution structures of mammalian and bacterial complex I under a range of redox conditions, including catalytic turnover. This allowed us to propose a robust and universal mechanism for complex I and related protein families. Redox reactions initially drive conformational changes around the quinone cavity and a long-distance transfer of substrate protons. These set up a stage for a series of electrostatically driven proton transfers along the membrane arm (‘domino effect'), eventually resulting in proton expulsion from the distal antiporter-like subunit. The mechanism radically differs from previous suggestions, however, it naturally explains all the unusual structural features of complex I. In this review I discuss the state of knowledge on complex I, including the current most controversial issues. article_processing_charge: No article_type: review author: - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 citation: ama: 'Sazanov LA. From the “black box” to “domino effect” mechanism: What have we learned from the structures of respiratory complex I. The Biochemical Journal. 2023;480(5):319-333. doi:10.1042/BCJ20210285' apa: 'Sazanov, L. A. (2023). From the “black box” to “domino effect” mechanism: What have we learned from the structures of respiratory complex I. The Biochemical Journal. Portland Press. https://doi.org/10.1042/BCJ20210285' chicago: 'Sazanov, Leonid A. “From the ‘black Box’ to ‘Domino Effect’ Mechanism: What Have We Learned from the Structures of Respiratory Complex I.” The Biochemical Journal. Portland Press, 2023. https://doi.org/10.1042/BCJ20210285.' ieee: 'L. A. Sazanov, “From the ‘black box’ to ‘domino effect’ mechanism: What have we learned from the structures of respiratory complex I,” The Biochemical Journal, vol. 480, no. 5. Portland Press, pp. 319–333, 2023.' ista: 'Sazanov LA. 2023. From the ‘black box’ to ‘domino effect’ mechanism: What have we learned from the structures of respiratory complex I. The Biochemical Journal. 480(5), 319–333.' mla: 'Sazanov, Leonid A. “From the ‘black Box’ to ‘Domino Effect’ Mechanism: What Have We Learned from the Structures of Respiratory Complex I.” The Biochemical Journal, vol. 480, no. 5, Portland Press, 2023, pp. 319–33, doi:10.1042/BCJ20210285.' short: L.A. Sazanov, The Biochemical Journal 480 (2023) 319–333. date_created: 2023-03-26T22:01:06Z date_published: 2023-03-15T00:00:00Z date_updated: 2023-08-01T13:45:12Z day: '15' ddc: - '570' department: - _id: LeSa doi: 10.1042/BCJ20210285 external_id: isi: - '000957065700001' pmid: - '36920092' has_accepted_license: '1' intvolume: ' 480' isi: 1 issue: '5' language: - iso: eng main_file_link: - open_access: '1' url: https://doi.org/10.1042/BCJ20210285 month: '03' oa: 1 oa_version: Published Version page: 319-333 pmid: 1 publication: The Biochemical Journal publication_identifier: eissn: - 1470-8728 issn: - 0264-6021 publication_status: published publisher: Portland Press quality_controlled: '1' scopus_import: '1' status: public title: 'From the ''black box'' to ''domino effect'' mechanism: What have we learned from the structures of respiratory complex I' tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 480 year: '2023' ... --- _id: '13232' abstract: - lang: eng text: The potential of immune-evasive mutation accumulation in the SARS-CoV-2 virus has led to its rapid spread, causing over 600 million confirmed cases and more than 6.5 million confirmed deaths. The huge demand for the rapid development and deployment of low-cost and effective vaccines against emerging variants has renewed interest in DNA vaccine technology. Here, we report the rapid generation and immunological evaluation of novel DNA vaccine candidates against the Wuhan-Hu-1 and Omicron variants based on the RBD protein fused with the Potato virus X coat protein (PVXCP). The delivery of DNA vaccines using electroporation in a two-dose regimen induced high-antibody titers and profound cellular responses in mice. The antibody titers induced against the Omicron variant of the vaccine were sufficient for effective protection against both Omicron and Wuhan-Hu-1 virus infections. The PVXCP protein in the vaccine construct shifted the immune response to the favorable Th1-like type and provided the oligomerization of RBD-PVXCP protein. Naked DNA delivery by needle-free injection allowed us to achieve antibody titers comparable with mRNA-LNP delivery in rabbits. These data identify the RBD-PVXCP DNA vaccine platform as a promising solution for robust and effective SARS-CoV-2 protection, supporting further translational study. acknowledgement: The authors declare that this study received funding from Immunofusion. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. The authors express their gratitude to the Institute of Physiology of the National Academy of Sciences of Belarus for providing assistance in keeping laboratory animals. article_number: '1014' article_processing_charge: No article_type: original author: - first_name: Dmitri full_name: Dormeshkin, Dmitri last_name: Dormeshkin - first_name: Mikalai full_name: Katsin, Mikalai last_name: Katsin - first_name: Maria full_name: Stegantseva, Maria last_name: Stegantseva - first_name: Sergey full_name: Golenchenko, Sergey last_name: Golenchenko - first_name: Michail full_name: Shapira, Michail last_name: Shapira - first_name: Simon full_name: Dubovik, Simon last_name: Dubovik - first_name: Dzmitry full_name: Lutskovich, Dzmitry last_name: Lutskovich - first_name: Anton full_name: Kavaleuski, Anton id: 62304f89-eb97-11eb-a6c2-8903dd183976 last_name: Kavaleuski orcid: 0000-0003-2091-526X - first_name: Alexander full_name: Meleshko, Alexander last_name: Meleshko citation: ama: Dormeshkin D, Katsin M, Stegantseva M, et al. Design and immunogenicity of SARS-CoV-2 DNA vaccine encoding RBD-PVXCP fusion protein. Vaccines. 2023;11(6). doi:10.3390/vaccines11061014 apa: Dormeshkin, D., Katsin, M., Stegantseva, M., Golenchenko, S., Shapira, M., Dubovik, S., … Meleshko, A. (2023). Design and immunogenicity of SARS-CoV-2 DNA vaccine encoding RBD-PVXCP fusion protein. Vaccines. MDPI. https://doi.org/10.3390/vaccines11061014 chicago: Dormeshkin, Dmitri, Mikalai Katsin, Maria Stegantseva, Sergey Golenchenko, Michail Shapira, Simon Dubovik, Dzmitry Lutskovich, Anton Kavaleuski, and Alexander Meleshko. “Design and Immunogenicity of SARS-CoV-2 DNA Vaccine Encoding RBD-PVXCP Fusion Protein.” Vaccines. MDPI, 2023. https://doi.org/10.3390/vaccines11061014. ieee: D. Dormeshkin et al., “Design and immunogenicity of SARS-CoV-2 DNA vaccine encoding RBD-PVXCP fusion protein,” Vaccines, vol. 11, no. 6. MDPI, 2023. ista: Dormeshkin D, Katsin M, Stegantseva M, Golenchenko S, Shapira M, Dubovik S, Lutskovich D, Kavaleuski A, Meleshko A. 2023. Design and immunogenicity of SARS-CoV-2 DNA vaccine encoding RBD-PVXCP fusion protein. Vaccines. 11(6), 1014. mla: Dormeshkin, Dmitri, et al. “Design and Immunogenicity of SARS-CoV-2 DNA Vaccine Encoding RBD-PVXCP Fusion Protein.” Vaccines, vol. 11, no. 6, 1014, MDPI, 2023, doi:10.3390/vaccines11061014. short: D. Dormeshkin, M. Katsin, M. Stegantseva, S. Golenchenko, M. Shapira, S. Dubovik, D. Lutskovich, A. Kavaleuski, A. Meleshko, Vaccines 11 (2023). date_created: 2023-07-16T22:01:10Z date_published: 2023-06-01T00:00:00Z date_updated: 2023-08-02T06:31:19Z day: '01' ddc: - '570' department: - _id: LeSa doi: 10.3390/vaccines11061014 external_id: isi: - '001017740000001' file: - access_level: open_access checksum: 8f484c0f30f8699c589b1c29a0fd7d7f content_type: application/pdf creator: dernst date_created: 2023-07-18T07:25:43Z date_updated: 2023-07-18T07:25:43Z file_id: '13244' file_name: 2023_Vaccines_Dormeshkin.pdf file_size: 2339746 relation: main_file success: 1 file_date_updated: 2023-07-18T07:25:43Z has_accepted_license: '1' intvolume: ' 11' isi: 1 issue: '6' language: - iso: eng month: '06' oa: 1 oa_version: Published Version publication: Vaccines publication_identifier: eissn: - 2076-393X publication_status: published publisher: MDPI quality_controlled: '1' scopus_import: '1' status: public title: Design and immunogenicity of SARS-CoV-2 DNA vaccine encoding RBD-PVXCP fusion protein tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 11 year: '2023' ... --- _id: '12781' 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" acknowledged_ssus: - _id: EM-Fac alternative_title: - ISTA Thesis article_processing_charge: No author: - first_name: Vladyslav full_name: Kravchuk, Vladyslav id: 4D62F2A6-F248-11E8-B48F-1D18A9856A87 last_name: Kravchuk 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 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. 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. 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. short: V. Kravchuk, Structural and Mechanistic Study of Bacterial Complex I and Its Cyanobacterial Ortholog, Institute of Science and Technology Austria, 2023. date_created: 2023-03-31T12:24:42Z date_published: 2023-03-23T00:00:00Z date_updated: 2023-08-04T08:54:51Z day: '23' ddc: - '570' - '572' degree_awarded: PhD department: - _id: GradSch - _id: LeSa doi: 10.15479/at:ista:12781 ec_funded: 1 file: - access_level: closed checksum: 5ebb6345cb4119f93460c81310265a6d content_type: application/pdf creator: vkravchu date_created: 2023-04-19T14:33:41Z date_updated: 2023-04-19T14:33:41Z embargo: 2024-04-20 embargo_to: local file_id: '12852' file_name: VladyslavKravchuk_PhD_Thesis_PostSub_Final_1.pdf file_size: 6071553 relation: main_file - access_level: closed checksum: c12055c48411d030d2afa51de2166221 content_type: application/vnd.openxmlformats-officedocument.wordprocessingml.document creator: vkravchu date_created: 2023-04-19T14:33:52Z date_updated: 2023-04-20T07:02:59Z embargo: 2024-04-20 embargo_to: local file_id: '12853' file_name: VladyslavKravchuk_PhD_Thesis_PostSub_Final.docx file_size: 19468766 relation: source_file file_date_updated: 2023-04-20T07:02:59Z has_accepted_license: '1' language: - iso: eng month: '03' oa_version: Published Version page: '127' project: - _id: 238A0A5A-32DE-11EA-91FC-C7463DDC885E grant_number: '25541' name: 'Structural characterization of E. coli complex I: an important mechanistic model' - _id: 627abdeb-2b32-11ec-9570-ec31a97243d3 call_identifier: H2020 grant_number: '101020697' name: Structure and mechanism of respiratory chain molecular machines publication_identifier: isbn: - 978-3-99078-029-9 issn: - 2663-337X publication_status: published publisher: Institute of Science and Technology Austria related_material: record: - id: '12138' relation: part_of_dissertation status: public status: public supervisor: - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 title: Structural and mechanistic study of bacterial complex I and its cyanobacterial ortholog type: dissertation user_id: 8b945eb4-e2f2-11eb-945a-df72226e66a9 year: '2023' ... --- _id: '14040' abstract: - lang: eng text: Robust oxygenic photosynthesis requires a suite of accessory factors to ensure efficient assembly and repair of the oxygen-evolving photosystem two (PSII) complex. The highly conserved Ycf48 assembly factor binds to the newly synthesized D1 reaction center polypeptide and promotes the initial steps of PSII assembly, but its binding site is unclear. Here we use cryo-electron microscopy to determine the structure of a cyanobacterial PSII D1/D2 reaction center assembly complex with Ycf48 attached. Ycf48, a 7-bladed beta propeller, binds to the amino-acid residues of D1 that ultimately ligate the water-oxidising Mn4CaO5 cluster, thereby preventing the premature binding of Mn2+ and Ca2+ ions and protecting the site from damage. Interactions with D2 help explain how Ycf48 promotes assembly of the D1/D2 complex. Overall, our work provides valuable insights into the early stages of PSII assembly and the structural changes that create the binding site for the Mn4CaO5 cluster. acknowledged_ssus: - _id: EM-Fac - _id: LifeSc - _id: ScienComp acknowledgement: P.J.N. and J.W.M. are grateful for the support of the Biotechnology & Biological Sciences Research Council (awards BB/L003260/1 and BB/P00931X/1). J. Knoppová, R.S. and J. Komenda were supported by the Czech Science Foundation (project 19-29225X) and by ERC project Photoredesign (no. 854126) and L.A.S. 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. article_number: '4681' article_processing_charge: Yes article_type: original author: - first_name: Ziyu full_name: Zhao, Ziyu last_name: Zhao - first_name: Irene full_name: Vercellino, Irene id: 3ED6AF16-F248-11E8-B48F-1D18A9856A87 last_name: Vercellino orcid: 0000-0001-5618-3449 - first_name: Jana full_name: Knoppová, Jana last_name: Knoppová - first_name: Roman full_name: Sobotka, Roman last_name: Sobotka - first_name: James W. full_name: Murray, James W. last_name: Murray - first_name: Peter J. full_name: Nixon, Peter J. last_name: Nixon - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 - first_name: Josef full_name: Komenda, Josef last_name: Komenda citation: ama: Zhao Z, Vercellino I, Knoppová J, et al. The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis. Nature Communications. 2023;14. doi:10.1038/s41467-023-40388-6 apa: Zhao, Z., Vercellino, I., Knoppová, J., Sobotka, R., Murray, J. W., Nixon, P. J., … Komenda, J. (2023). The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis. Nature Communications. Springer Nature. https://doi.org/10.1038/s41467-023-40388-6 chicago: Zhao, Ziyu, Irene Vercellino, Jana Knoppová, Roman Sobotka, James W. Murray, Peter J. Nixon, Leonid A Sazanov, and Josef Komenda. “The Ycf48 Accessory Factor Occupies the Site of the Oxygen-Evolving Manganese Cluster during Photosystem II Biogenesis.” Nature Communications. Springer Nature, 2023. https://doi.org/10.1038/s41467-023-40388-6. ieee: Z. Zhao et al., “The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis,” Nature Communications, vol. 14. Springer Nature, 2023. ista: Zhao Z, Vercellino I, Knoppová J, Sobotka R, Murray JW, Nixon PJ, Sazanov LA, Komenda J. 2023. The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis. Nature Communications. 14, 4681. mla: Zhao, Ziyu, et al. “The Ycf48 Accessory Factor Occupies the Site of the Oxygen-Evolving Manganese Cluster during Photosystem II Biogenesis.” Nature Communications, vol. 14, 4681, Springer Nature, 2023, doi:10.1038/s41467-023-40388-6. short: Z. Zhao, I. Vercellino, J. Knoppová, R. Sobotka, J.W. Murray, P.J. Nixon, L.A. Sazanov, J. Komenda, Nature Communications 14 (2023). date_created: 2023-08-13T22:01:13Z date_published: 2023-08-04T00:00:00Z date_updated: 2023-12-13T12:06:56Z day: '04' ddc: - '570' department: - _id: LeSa doi: 10.1038/s41467-023-40388-6 external_id: isi: - '001042606700004' file: - access_level: open_access checksum: 3b9043df3d51c300f9be95eac3ff9d0b content_type: application/pdf creator: dernst date_created: 2023-08-14T07:01:12Z date_updated: 2023-08-14T07:01:12Z file_id: '14044' file_name: 2023_NatureComm_Zhao.pdf file_size: 2315325 relation: main_file success: 1 file_date_updated: 2023-08-14T07:01:12Z has_accepted_license: '1' intvolume: ' 14' isi: 1 language: - iso: eng month: '08' oa: 1 oa_version: Published Version publication: Nature Communications publication_identifier: eissn: - 2041-1723 publication_status: published publisher: Springer Nature quality_controlled: '1' scopus_import: '1' status: public title: The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 14 year: '2023' ... --- _id: '10182' abstract: - lang: eng text: The mitochondrial oxidative phosphorylation system is central to cellular metabolism. It comprises five enzymatic complexes and two mobile electron carriers that work in a mitochondrial respiratory chain. By coupling the oxidation of reducing equivalents coming into mitochondria to the generation and subsequent dissipation of a proton gradient across the inner mitochondrial membrane, this electron transport chain drives the production of ATP, which is then used as a primary energy carrier in virtually all cellular processes. Minimal perturbations of the respiratory chain activity are linked to diseases; therefore, it is necessary to understand how these complexes are assembled and regulated and how they function. In this Review, we outline the latest assembly models for each individual complex, and we also highlight the recent discoveries indicating that the formation of larger assemblies, known as respiratory supercomplexes, originates from the association of the intermediates of individual complexes. We then discuss how recent cryo-electron microscopy structures have been key to answering open questions on the function of the electron transport chain in mitochondrial respiration and how supercomplexes and other factors, including metabolites, can regulate the activity of the single complexes. When relevant, we discuss how these mechanisms contribute to physiology and outline their deregulation in human diseases. article_processing_charge: No article_type: original author: - first_name: Irene full_name: Vercellino, Irene id: 3ED6AF16-F248-11E8-B48F-1D18A9856A87 last_name: Vercellino orcid: ' 0000-0001-5618-3449' - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 citation: ama: Vercellino I, Sazanov LA. The assembly, regulation and function of the mitochondrial respiratory chain. Nature Reviews Molecular Cell Biology. 2022;23:141–161. doi:10.1038/s41580-021-00415-0 apa: Vercellino, I., & Sazanov, L. A. (2022). The assembly, regulation and function of the mitochondrial respiratory chain. Nature Reviews Molecular Cell Biology. Springer Nature. https://doi.org/10.1038/s41580-021-00415-0 chicago: Vercellino, Irene, and Leonid A Sazanov. “The Assembly, Regulation and Function of the Mitochondrial Respiratory Chain.” Nature Reviews Molecular Cell Biology. Springer Nature, 2022. https://doi.org/10.1038/s41580-021-00415-0. ieee: I. Vercellino and L. A. Sazanov, “The assembly, regulation and function of the mitochondrial respiratory chain,” Nature Reviews Molecular Cell Biology, vol. 23. Springer Nature, pp. 141–161, 2022. ista: Vercellino I, Sazanov LA. 2022. The assembly, regulation and function of the mitochondrial respiratory chain. Nature Reviews Molecular Cell Biology. 23, 141–161. mla: Vercellino, Irene, and Leonid A. Sazanov. “The Assembly, Regulation and Function of the Mitochondrial Respiratory Chain.” Nature Reviews Molecular Cell Biology, vol. 23, Springer Nature, 2022, pp. 141–161, doi:10.1038/s41580-021-00415-0. short: I. Vercellino, L.A. Sazanov, Nature Reviews Molecular Cell Biology 23 (2022) 141–161. date_created: 2021-10-24T22:01:35Z date_published: 2022-02-01T00:00:00Z date_updated: 2023-08-02T06:55:42Z day: '01' department: - _id: LeSa doi: 10.1038/s41580-021-00415-0 external_id: isi: - '000705697100001' pmid: - '34621061' intvolume: ' 23' isi: 1 language: - iso: eng month: '02' oa_version: None page: 141–161 pmid: 1 publication: Nature Reviews Molecular Cell Biology publication_identifier: eissn: - 1471-0080 issn: - 1471-0072 publication_status: published publisher: Springer Nature quality_controlled: '1' scopus_import: '1' status: public title: The assembly, regulation and function of the mitochondrial respiratory chain type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 23 year: '2022' ... --- _id: '11167' abstract: - lang: eng text: Complex I is one of the major respiratory complexes, conserved from bacteria to mammals. It oxidises NADH, reduces quinone and pumps protons across the membrane, thus playing a central role in the oxidative energy metabolism. In this review we discuss our current state of understanding the structure of complex I from various species of mammals, plants, fungi, and bacteria, as well as of several complex I-related proteins. By comparing the structural evidence from these systems in different redox states and data from mutagenesis and molecular simulations, we formulate the mechanisms of electron transfer and proton pumping and explain how they are conformationally and electrostatically coupled. Finally, we discuss the structural basis of the deactivation phenomenon in mammalian complex I. article_number: '102350' article_processing_charge: Yes (via OA deal) article_type: original author: - first_name: Domen full_name: Kampjut, Domen id: 37233050-F248-11E8-B48F-1D18A9856A87 last_name: Kampjut - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 citation: ama: Kampjut D, Sazanov LA. Structure of respiratory complex I – An emerging blueprint for the mechanism. Current Opinion in Structural Biology. 2022;74. doi:10.1016/j.sbi.2022.102350 apa: Kampjut, D., & Sazanov, L. A. (2022). Structure of respiratory complex I – An emerging blueprint for the mechanism. Current Opinion in Structural Biology. Elsevier. https://doi.org/10.1016/j.sbi.2022.102350 chicago: Kampjut, Domen, and Leonid A Sazanov. “Structure of Respiratory Complex I – An Emerging Blueprint for the Mechanism.” Current Opinion in Structural Biology. Elsevier, 2022. https://doi.org/10.1016/j.sbi.2022.102350. ieee: D. Kampjut and L. A. Sazanov, “Structure of respiratory complex I – An emerging blueprint for the mechanism,” Current Opinion in Structural Biology, vol. 74. Elsevier, 2022. ista: Kampjut D, Sazanov LA. 2022. Structure of respiratory complex I – An emerging blueprint for the mechanism. Current Opinion in Structural Biology. 74, 102350. mla: Kampjut, Domen, and Leonid A. Sazanov. “Structure of Respiratory Complex I – An Emerging Blueprint for the Mechanism.” Current Opinion in Structural Biology, vol. 74, 102350, Elsevier, 2022, doi:10.1016/j.sbi.2022.102350. short: D. Kampjut, L.A. Sazanov, Current Opinion in Structural Biology 74 (2022). date_created: 2022-04-15T09:32:35Z date_published: 2022-06-01T00:00:00Z date_updated: 2023-08-03T06:31:06Z day: '01' ddc: - '570' department: - _id: LeSa doi: 10.1016/j.sbi.2022.102350 external_id: isi: - '000829029500020' pmid: - '35316665' file: - access_level: open_access checksum: 72bdde48853643a32d42b75f54965c44 content_type: application/pdf creator: dernst date_created: 2022-08-05T05:56:03Z date_updated: 2022-08-05T05:56:03Z file_id: '11725' file_name: 2022_CurrentOpStructBiology_Kampjut.pdf file_size: 815607 relation: main_file success: 1 file_date_updated: 2022-08-05T05:56:03Z has_accepted_license: '1' intvolume: ' 74' isi: 1 keyword: - Molecular Biology - Structural Biology language: - iso: eng month: '06' oa: 1 oa_version: Published Version pmid: 1 publication: Current Opinion in Structural Biology publication_identifier: issn: - 0959-440X publication_status: published publisher: Elsevier quality_controlled: '1' scopus_import: '1' status: public title: Structure of respiratory complex I – An emerging blueprint for the mechanism tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 74 year: '2022' ... --- _id: '11551' abstract: - lang: eng text: Imbalanced mitochondrial dNTP pools are known players in the pathogenesis of multiple human diseases. Here we show that, even under physiological conditions, dGTP is largely overrepresented among other dNTPs in mitochondria of mouse tissues and human cultured cells. In addition, a vast majority of mitochondrial dGTP is tightly bound to NDUFA10, an accessory subunit of complex I of the mitochondrial respiratory chain. NDUFA10 shares a deoxyribonucleoside kinase (dNK) domain with deoxyribonucleoside kinases in the nucleotide salvage pathway, though no specific function beyond stabilizing the complex I holoenzyme has been described for this subunit. We mutated the dNK domain of NDUFA10 in human HEK-293T cells while preserving complex I assembly and activity. The NDUFA10E160A/R161A shows reduced dGTP binding capacity in vitro and leads to a 50% reduction in mitochondrial dGTP content, proving that most dGTP is directly bound to the dNK domain of NDUFA10. This interaction may represent a hitherto unknown mechanism regulating mitochondrial dNTP availability and linking oxidative metabolism to DNA maintenance. acknowledgement: "We thank Dr, Luke Formosa (Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia) for his valuable advice and assistance on NDUFA10 molecular studies and Dr. Francesc Canals and his team (Proteomics Laboratory, Vall d’Hebron Institute of Oncology [VHIO], Universitat Autònoma de Barcelona, Barcelona, Spain) for their assistance with LC-MS/MS analyses. This work was supported by the Spanish Ministry of Industry, Economy and Competitiveness [grants BFU2014-52618-R, SAF2017-87506, and PID2020-112929RB-I00 to Y.C.], by the Spanish Instituto de Salud Carlos III [grants PI21/00554 and PMP15/00025 to R.M.], co-financed by the European Regional Development Fund (ERDF), and by an NHMRC Project grant to M.R. (GNT1164459).\r\n" article_number: '620' article_processing_charge: No author: - first_name: David full_name: Molina-Granada, David last_name: Molina-Granada - first_name: Emiliano full_name: González-Vioque, Emiliano last_name: González-Vioque - first_name: Marris G. full_name: Dibley, Marris G. last_name: Dibley - first_name: Raquel full_name: Cabrera-Pérez, Raquel last_name: Cabrera-Pérez - first_name: Antoni full_name: Vallbona-Garcia, Antoni last_name: Vallbona-Garcia - first_name: Javier full_name: Torres-Torronteras, Javier last_name: Torres-Torronteras - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 - first_name: Michael T. full_name: Ryan, Michael T. last_name: Ryan - first_name: Yolanda full_name: Cámara, Yolanda last_name: Cámara - first_name: Ramon full_name: Martí, Ramon last_name: Martí citation: ama: Molina-Granada D, González-Vioque E, Dibley MG, et al. Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit. Communications Biology. 2022;5(1). doi:10.1038/s42003-022-03568-6 apa: Molina-Granada, D., González-Vioque, E., Dibley, M. G., Cabrera-Pérez, R., Vallbona-Garcia, A., Torres-Torronteras, J., … Martí, R. (2022). Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit. Communications Biology. Springer Nature. https://doi.org/10.1038/s42003-022-03568-6 chicago: Molina-Granada, David, Emiliano González-Vioque, Marris G. Dibley, Raquel Cabrera-Pérez, Antoni Vallbona-Garcia, Javier Torres-Torronteras, Leonid A Sazanov, Michael T. Ryan, Yolanda Cámara, and Ramon Martí. “Most Mitochondrial DGTP Is Tightly Bound to Respiratory Complex I through the NDUFA10 Subunit.” Communications Biology. Springer Nature, 2022. https://doi.org/10.1038/s42003-022-03568-6. ieee: D. Molina-Granada et al., “Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit,” Communications Biology, vol. 5, no. 1. Springer Nature, 2022. ista: Molina-Granada D, González-Vioque E, Dibley MG, Cabrera-Pérez R, Vallbona-Garcia A, Torres-Torronteras J, Sazanov LA, Ryan MT, Cámara Y, Martí R. 2022. Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit. Communications Biology. 5(1), 620. mla: Molina-Granada, David, et al. “Most Mitochondrial DGTP Is Tightly Bound to Respiratory Complex I through the NDUFA10 Subunit.” Communications Biology, vol. 5, no. 1, 620, Springer Nature, 2022, doi:10.1038/s42003-022-03568-6. short: D. Molina-Granada, E. González-Vioque, M.G. Dibley, R. Cabrera-Pérez, A. Vallbona-Garcia, J. Torres-Torronteras, L.A. Sazanov, M.T. Ryan, Y. Cámara, R. Martí, Communications Biology 5 (2022). date_created: 2022-07-10T22:01:52Z date_published: 2022-06-23T00:00:00Z date_updated: 2023-08-03T11:51:58Z day: '23' ddc: - '570' department: - _id: LeSa doi: 10.1038/s42003-022-03568-6 external_id: isi: - '000815098500002' pmid: - ' 35739187' file: - access_level: open_access checksum: 965f88bbcef3fd0c3e121340555c4467 content_type: application/pdf creator: kschuh date_created: 2022-07-13T07:44:58Z date_updated: 2022-07-13T07:44:58Z file_id: '11571' file_name: 2022_communicationsbiology_Molina-Granada.pdf file_size: 2335369 relation: main_file success: 1 file_date_updated: 2022-07-13T07:44:58Z has_accepted_license: '1' intvolume: ' 5' isi: 1 issue: '1' language: - iso: eng month: '06' oa: 1 oa_version: Published Version pmid: 1 publication: Communications Biology publication_identifier: eissn: - '23993642' publication_status: published publisher: Springer Nature quality_controlled: '1' scopus_import: '1' status: public title: Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 5 year: '2022' ... --- _id: '11648' abstract: - lang: eng text: 'Progress in structural membrane biology has been significantly accelerated by the ongoing ''Resolution Revolution'' in cryo electron microscopy (cryo-EM). In particular, structure determination by single particle analysis has evolved into the most powerful method for atomic model building of multisubunit membrane protein complexes. This has created an ever increasing demand in cryo-EM machine time, which to satisfy is in need of new and affordable cryo electron microscopes. Here, we review our experience in using the JEOL CRYO ARM 200 prototype for the structure determination by single particle analysis of three different multisubunit membrane complexes: the Thermus thermophilus V-type ATPase VO complex, the Thermosynechococcus elongatus photosystem I monomer and the flagellar motor LP-ring from Salmonella enterica.' acknowledgement: "Cyclic Innovation for Clinical Empowerment (JP17pc0101020 from Japan Agency for Medical Research and Development (AMED) to K.N. and G.K.); Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research) from AMED (JP20am0101117 to K.N., JP16K07266 to Atsunori Oshima and C.G., JP22ama121001j0001 to Masaki Yamamoto, G.K., T.K. and C.G.); a JSPS KAHKENHI\r\ngrant (20K06514 to J.K.) and a Grant-in-aid for JSPS fellows (20J00162 to A.N.).\r\nWe are grateful for initiation and scientific support from Matthias Rogner, Marc M. Nowaczyk, Anna Frank and ̈Yuko Misumi for the PSI monomer project and also would like to thank Hideki Shigematsu for critical reading of the manuscript. And we are indebted to the two anonymous reviewers who helped us to improve our manuscript." article_processing_charge: No article_type: original author: - first_name: Christoph full_name: Gerle, Christoph last_name: Gerle - first_name: Jun-ichi full_name: Kishikawa, Jun-ichi last_name: Kishikawa - first_name: Tomoko full_name: Yamaguchi, Tomoko last_name: Yamaguchi - first_name: Atsuko full_name: Nakanishi, Atsuko last_name: Nakanishi - first_name: Mehmet Orkun full_name: Çoruh, Mehmet Orkun id: d25163e5-8d53-11eb-a251-e6dd8ea1b8ef last_name: Çoruh orcid: 0000-0002-3219-2022 - first_name: Fumiaki full_name: Makino, Fumiaki last_name: Makino - first_name: Tomoko full_name: Miyata, Tomoko last_name: Miyata - first_name: Akihiro full_name: Kawamoto, Akihiro last_name: Kawamoto - first_name: Ken full_name: Yokoyama, Ken last_name: Yokoyama - first_name: Keiichi full_name: Namba, Keiichi last_name: Namba - first_name: Genji full_name: Kurisu, Genji last_name: Kurisu - first_name: Takayuki full_name: Kato, Takayuki last_name: Kato citation: ama: Gerle C, Kishikawa J, Yamaguchi T, et al. Structures of multisubunit membrane complexes with the CRYO ARM 200. Microscopy. 2022;71(5):249-261. doi:10.1093/jmicro/dfac037 apa: Gerle, C., Kishikawa, J., Yamaguchi, T., Nakanishi, A., Çoruh, M. O., Makino, F., … Kato, T. (2022). Structures of multisubunit membrane complexes with the CRYO ARM 200. Microscopy. Oxford University Press. https://doi.org/10.1093/jmicro/dfac037 chicago: Gerle, Christoph, Jun-ichi Kishikawa, Tomoko Yamaguchi, Atsuko Nakanishi, Mehmet Orkun Çoruh, Fumiaki Makino, Tomoko Miyata, et al. “Structures of Multisubunit Membrane Complexes with the CRYO ARM 200.” Microscopy. Oxford University Press, 2022. https://doi.org/10.1093/jmicro/dfac037. ieee: C. Gerle et al., “Structures of multisubunit membrane complexes with the CRYO ARM 200,” Microscopy, vol. 71, no. 5. Oxford University Press, pp. 249–261, 2022. ista: Gerle C, Kishikawa J, Yamaguchi T, Nakanishi A, Çoruh MO, Makino F, Miyata T, Kawamoto A, Yokoyama K, Namba K, Kurisu G, Kato T. 2022. Structures of multisubunit membrane complexes with the CRYO ARM 200. Microscopy. 71(5), 249–261. mla: Gerle, Christoph, et al. “Structures of Multisubunit Membrane Complexes with the CRYO ARM 200.” Microscopy, vol. 71, no. 5, Oxford University Press, 2022, pp. 249–61, doi:10.1093/jmicro/dfac037. short: C. Gerle, J. Kishikawa, T. Yamaguchi, A. Nakanishi, M.O. Çoruh, F. Makino, T. Miyata, A. Kawamoto, K. Yokoyama, K. Namba, G. Kurisu, T. Kato, Microscopy 71 (2022) 249–261. date_created: 2022-07-25T10:04:58Z date_published: 2022-10-01T00:00:00Z date_updated: 2023-08-03T12:13:37Z day: '01' ddc: - '570' department: - _id: LeSa doi: 10.1093/jmicro/dfac037 external_id: isi: - '000837950900001' pmid: - '35861182' file: - access_level: open_access checksum: 23b51c163636bf9313f7f0818312e67e content_type: application/pdf creator: dernst date_created: 2023-02-03T08:34:48Z date_updated: 2023-02-03T08:34:48Z file_id: '12498' file_name: 2022_Microscopy_Gerle.pdf file_size: 7812696 relation: main_file success: 1 file_date_updated: 2023-02-03T08:34:48Z has_accepted_license: '1' intvolume: ' 71' isi: 1 issue: '5' keyword: - Radiology - Nuclear Medicine and imaging - Instrumentation - Structural Biology language: - iso: eng month: '10' oa: 1 oa_version: Published Version page: 249-261 pmid: 1 publication: Microscopy publication_identifier: eissn: - 2050-5701 issn: - 2050-5698 publication_status: published publisher: Oxford University Press quality_controlled: '1' scopus_import: '1' status: public title: Structures of multisubunit membrane complexes with the CRYO ARM 200 tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 71 year: '2022' ... --- _id: '12138' 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.' acknowledged_ssus: - _id: EM-Fac - _id: LifeSc - _id: ScienComp 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). article_processing_charge: No article_type: original author: - first_name: Vladyslav full_name: Kravchuk, Vladyslav id: 4D62F2A6-F248-11E8-B48F-1D18A9856A87 last_name: Kravchuk - first_name: Olga full_name: Petrova, Olga id: 5D8C9660-5D49-11EA-8188-567B3DDC885E last_name: Petrova - first_name: Domen full_name: Kampjut, Domen id: 37233050-F248-11E8-B48F-1D18A9856A87 last_name: Kampjut - first_name: Anna full_name: Wojciechowska-Bason, Anna last_name: Wojciechowska-Bason - first_name: Zara full_name: Breese, Zara last_name: Breese - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 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 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. 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. 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. short: V. Kravchuk, O. Petrova, D. Kampjut, A. Wojciechowska-Bason, Z. Breese, L.A. Sazanov, Nature 609 (2022) 808–814. date_created: 2023-01-12T12:04:33Z date_published: 2022-09-22T00:00:00Z date_updated: 2023-08-04T08:54:52Z day: '22' ddc: - '572' department: - _id: LeSa doi: 10.1038/s41586-022-05199-7 ec_funded: 1 external_id: isi: - '000854788200001' pmid: - '36104567' file: - access_level: open_access checksum: d42a93e24f59e883ef0b5429832391d0 content_type: application/pdf creator: lsazanov date_created: 2023-05-30T17:05:31Z date_updated: 2023-05-30T17:05:31Z file_id: '13104' file_name: EcCxI_manuscript_rev3_noSI_updated_withFigs_opt.pdf file_size: 1425655 relation: main_file success: 1 - access_level: open_access checksum: 5422bc0a73b3daadafa262c7ea6deae3 content_type: application/pdf creator: lsazanov date_created: 2023-05-30T17:07:05Z date_updated: 2023-05-30T17:07:05Z file_id: '13105' file_name: EcCxI_manuscript_rev3_SI_All_opt_upd.pdf file_size: 9842513 relation: main_file success: 1 file_date_updated: 2023-05-30T17:07:05Z has_accepted_license: '1' intvolume: ' 609' isi: 1 issue: '7928' keyword: - Multidisciplinary language: - iso: eng month: '09' oa: 1 oa_version: Submitted Version page: 808-814 pmid: 1 project: - _id: 238A0A5A-32DE-11EA-91FC-C7463DDC885E grant_number: '25541' name: 'Structural characterization of E. coli complex I: an important mechanistic model' - _id: 627abdeb-2b32-11ec-9570-ec31a97243d3 call_identifier: H2020 grant_number: '101020697' name: Structure and mechanism of respiratory chain molecular machines publication: Nature publication_identifier: eissn: - 1476-4687 issn: - 0028-0836 publication_status: published publisher: Springer Nature quality_controlled: '1' related_material: link: - relation: erratum url: https://doi.org/10.1038/s41586-022-05457-8 - description: News on ISTA website relation: press_release url: https://ista.ac.at/en/news/proton-dominos-kick-off-life/ record: - id: '12781' relation: dissertation_contains status: public scopus_import: '1' status: public title: A universal coupling mechanism of respiratory complex I type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 609 year: '2022' ... --- _id: '12252' abstract: - lang: eng text: The COVID−19 pandemic not only resulted in a global crisis, but also accelerated vaccine development and antibody discovery. Herein we report a synthetic humanized VHH library development pipeline for nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) isolation. Trinucleotide-based randomization of CDRs by Kunkel mutagenesis with the subsequent rolling-cycle amplification resulted in more than 1011 diverse phage display library in a manageable for a single person number of electroporation reactions. We identified a number of nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) by screening a novel synthetic humanized antibody library. In order to explore the most robust and fast method for affinity improvement, we performed affinity maturation by CDR1 and CDR2 shuffling and avidity engineering by multivalent trimeric VHH fusion protein construction. As a result, H7-Fc and G12x3-Fc binders were developed with the affinities in nM and pM range respectively. Importantly, these affinities are weakly influenced by most of SARS-CoV-2 VoC mutations and they retain moderate binding to BA.4\5. The plaque reduction neutralization test (PRNT) resulted in IC50 = 100 ng\ml and 9.6 ng\ml for H7-Fc and G12x3-Fc antibodies, respectively, for the emerging Omicron BA.1 variant. Therefore, these VHH could expand the present landscape of SARS-CoV-2 neutralization binders with the therapeutic potential for present and future SARS-CoV-2 variants. acknowledgement: The authors declare that this study received funding from Immunofusion. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. article_number: '965446' article_processing_charge: No article_type: original author: - first_name: Dmitri full_name: Dormeshkin, Dmitri last_name: Dormeshkin - first_name: Michail full_name: Shapira, Michail last_name: Shapira - first_name: Simon full_name: Dubovik, Simon last_name: Dubovik - first_name: Anton full_name: Kavaleuski, Anton id: 4968f7ad-eb97-11eb-a6c2-8ed382e8912c last_name: Kavaleuski orcid: 0000-0003-2091-526X - first_name: Mikalai full_name: Katsin, Mikalai last_name: Katsin - first_name: Alexandr full_name: Migas, Alexandr last_name: Migas - first_name: Alexander full_name: Meleshko, Alexander last_name: Meleshko - first_name: Sergei full_name: Semyonov, Sergei last_name: Semyonov citation: ama: Dormeshkin D, Shapira M, Dubovik S, et al. Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library. Frontiers in Immunology. 2022;13. doi:10.3389/fimmu.2022.965446 apa: Dormeshkin, D., Shapira, M., Dubovik, S., Kavaleuski, A., Katsin, M., Migas, A., … Semyonov, S. (2022). Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library. Frontiers in Immunology. Frontiers Media. https://doi.org/10.3389/fimmu.2022.965446 chicago: Dormeshkin, Dmitri, Michail Shapira, Simon Dubovik, Anton Kavaleuski, Mikalai Katsin, Alexandr Migas, Alexander Meleshko, and Sergei Semyonov. “Isolation of an Escape-Resistant SARS-CoV-2 Neutralizing Nanobody from a Novel Synthetic Nanobody Library.” Frontiers in Immunology. Frontiers Media, 2022. https://doi.org/10.3389/fimmu.2022.965446. ieee: D. Dormeshkin et al., “Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library,” Frontiers in Immunology, vol. 13. Frontiers Media, 2022. ista: Dormeshkin D, Shapira M, Dubovik S, Kavaleuski A, Katsin M, Migas A, Meleshko A, Semyonov S. 2022. Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library. Frontiers in Immunology. 13, 965446. mla: Dormeshkin, Dmitri, et al. “Isolation of an Escape-Resistant SARS-CoV-2 Neutralizing Nanobody from a Novel Synthetic Nanobody Library.” Frontiers in Immunology, vol. 13, 965446, Frontiers Media, 2022, doi:10.3389/fimmu.2022.965446. short: D. Dormeshkin, M. Shapira, S. Dubovik, A. Kavaleuski, M. Katsin, A. Migas, A. Meleshko, S. Semyonov, Frontiers in Immunology 13 (2022). date_created: 2023-01-16T09:56:57Z date_published: 2022-09-16T00:00:00Z date_updated: 2023-08-04T09:49:24Z day: '16' ddc: - '570' department: - _id: LeSa doi: 10.3389/fimmu.2022.965446 external_id: isi: - '000862479100001' file: - access_level: open_access checksum: f8f5d8110710033d0532e7e08bf9dad4 content_type: application/pdf creator: dernst date_created: 2023-01-30T09:22:26Z date_updated: 2023-01-30T09:22:26Z file_id: '12443' file_name: 2022_FrontiersImmunology_Dormeshkin.pdf file_size: 5695892 relation: main_file success: 1 file_date_updated: 2023-01-30T09:22:26Z has_accepted_license: '1' intvolume: ' 13' isi: 1 keyword: - Immunology - Immunology and Allergy - COVID-19 - SARS-CoV-2 - synthetic library - RBD - neutralization nanobody - VHH language: - iso: eng month: '09' oa: 1 oa_version: Published Version publication: Frontiers in Immunology publication_identifier: issn: - 1664-3224 publication_status: published publisher: Frontiers Media quality_controlled: '1' scopus_import: '1' status: public title: Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 13 year: '2022' ...