{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T18:54:50Z","timestamp":1791485690888,"version":"4.3.4"},"reference-count":68,"publisher":"National Academy of Sciences","issue":"6","license":[{"start":{"date-parts":[[2025,2,6]],"date-time":"2025-02-06T00:00:00Z","timestamp":1738800000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000060","name":"HHS | NIH | National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["Al155473"],"award-info":[{"award-number":["Al155473"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"HHS | NIH | National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["GM61576"],"award-info":[{"award-number":["GM61576"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"HHS | NIH | National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["AI132422"],"award-info":[{"award-number":["AI132422"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2025,2,11]]},"abstract":"<jats:p>HflX is a highly conserved ribosome-associated GTPase implicated in rescuing stalled ribosomes and mediating antibiotic resistance in several bacteria, including macrolide-lincosamide antibiotic resistance in mycobacteria. Mycobacterial HflXs carry a distinct N-terminal extension (NTE) and a small insertion, as compared to their eubacterial homologs. Here, we present several high-resolution cryo-EM structures of mycobacterial HflX in complex with the 70S ribosome and its 50S subunit, with and without antibiotics. These structures reveal a distinct mechanism for HflX-mediated ribosome splitting and antibiotic resistance in mycobacteria. Our findings indicate that the NTE of mycobacterial HflX induces persistent disordering of multiple 23S rRNA helices, facilitating the dissociation of the 70S ribosome and generating an inactive pool of 50S subunits. During this process, HflX undergoes a large conformational change that stabilizes its NTE. Mycobacterial HflX also acts as an anti-association factor by binding to predissociated 50S subunits. Our structures show that a mycobacteria-specific insertion in HflX reaches far into the peptidyl transferase center (PTC), such that it would overlap with the ribosome-bound macrolide antibiotics. However, in the presence of antibiotics, this insertion retracts, adjusts around, and interacts with the antibiotic molecules. These results suggest that mycobacterial HflX is agnostic to antibiotic presence in the PTC. It mediates antibiotic resistance by splitting antibiotic-stalled 70S ribosomes and inactivating the resulting 50S subunits.<\/jats:p>","DOI":"10.1073\/pnas.2419826122","type":"journal-article","created":{"date-parts":[[2025,2,6]],"date-time":"2025-02-06T11:56:21Z","timestamp":1738842981000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":13,"title":["HflX-mediated drug resistance through ribosome splitting and rRNA disordering in mycobacteria"],"prefix":"10.1073","volume":"122","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6518-9057","authenticated-orcid":false,"given":"Soneya","family":"Majumdar","sequence":"first","affiliation":[{"name":"Division of Translational Medicine, Wadsworth Center, New York State Department of Health","place":["Albany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amuliya","family":"Kashyap","sequence":"additional","affiliation":[{"name":"Division of Genetics, Wadsworth Center, New York State Department of Health","place":["Albany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ravi K.","family":"Koripella","sequence":"additional","affiliation":[{"name":"Division of Translational Medicine, Wadsworth Center, New York State Department of Health","place":["Albany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4821-748X","authenticated-orcid":false,"given":"Manjuli R.","family":"Sharma","sequence":"additional","affiliation":[{"name":"Division of Translational Medicine, Wadsworth Center, New York State Department of Health","place":["Albany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kelley","family":"Hurst-Hess","sequence":"additional","affiliation":[{"name":"Division of Genetics, Wadsworth Center, New York State Department of Health","place":["Albany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Swati R.","family":"Manjari","sequence":"additional","affiliation":[{"name":"Division of Translational Medicine, Wadsworth Center, New York State Department of Health","place":["Albany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2206-7049","authenticated-orcid":false,"given":"Nilesh K.","family":"Banavali","sequence":"additional","affiliation":[{"name":"Division of Translational Medicine, Wadsworth Center, New York State Department of Health","place":["Albany"]},{"name":"Department of Biomedical Sciences, University at Albany","place":["Albany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pallavi","family":"Ghosh","sequence":"additional","affiliation":[{"name":"Division of Genetics, Wadsworth Center, New York State Department of Health","place":["Albany"]},{"name":"Department of Biomedical Sciences, University at Albany","place":["Albany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9392-0065","authenticated-orcid":false,"given":"Rajendra K.","family":"Agrawal","sequence":"additional","affiliation":[{"name":"Division of Translational Medicine, Wadsworth Center, New York State Department of Health","place":["Albany"]},{"name":"Department of Biomedical Sciences, University at Albany","place":["Albany"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2025,2,6]]},"reference":[{"key":"e_1_3_4_1_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1906748117"},{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkt705"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1038\/nsmb.3103"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2006717118"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac934"},{"key":"e_1_3_4_6_2","doi-asserted-by":"crossref","unstructured":"D. Wu Y. Dai N. Gao Cryo-EM structure of the 50S-HflX complex reveals a novel mechanism of antibiotic resistance in E. coli. bioRxiv [Preprint] (2022) https:\/\/doi.org\/10.1101\/2022.11.25.517942 (Accessed 1 August 2023).","DOI":"10.1101\/2022.11.25.517942"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.01213-06"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1709588114"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1810555115"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.str.2024.06.016"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2023.12.027"},{"key":"e_1_3_4_12_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkae128"},{"key":"e_1_3_4_13_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2017.06.029"},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1804555115"},{"key":"e_1_3_4_15_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkx785"},{"key":"e_1_3_4_16_2","first-page":"384","article-title":"Cryo-EM structure of Mycobacterium smegmatis ribosome reveals two unidentified ribosomal proteins close to the functional centers","volume":"9","author":"Li Z.","year":"2018","unstructured":"Z. Li , Cryo-EM structure of Mycobacterium smegmatis ribosome reveals two unidentified ribosomal proteins close to the functional centers. Protein Cell 9, 384\u2013388 (2018), 10.1007\/s13238-017-0456-9.","journal-title":"Protein Cell"},{"key":"e_1_3_4_17_2","doi-asserted-by":"publisher","DOI":"10.1038\/10695"},{"key":"e_1_3_4_18_2","doi-asserted-by":"publisher","DOI":"10.1093\/emboj\/cdf326"},{"key":"e_1_3_4_19_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2005.10.028"},{"key":"e_1_3_4_20_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1179709"},{"key":"e_1_3_4_21_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1179700"},{"key":"e_1_3_4_22_2","doi-asserted-by":"publisher","DOI":"10.1038\/emboj.2012.22"},{"key":"e_1_3_4_23_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-17715-2"},{"key":"e_1_3_4_24_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2016.02.009"},{"key":"e_1_3_4_25_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)80690-X"},{"key":"e_1_3_4_26_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1060089"},{"key":"e_1_3_4_27_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkw678"},{"key":"e_1_3_4_28_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2016.11.020"},{"key":"e_1_3_4_29_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-42266-7"},{"key":"e_1_3_4_30_2","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms4501"},{"key":"e_1_3_4_31_2","doi-asserted-by":"publisher","DOI":"10.1080\/22221751.2021.2022439"},{"key":"e_1_3_4_32_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmolb.2018.00048"},{"key":"e_1_3_4_33_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1007988107"},{"key":"e_1_3_4_34_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0022-2836(03)00662-4"},{"key":"e_1_3_4_35_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.37.9.2027"},{"key":"e_1_3_4_36_2","volume-title":"eLS","author":"D\u00f6nh\u00f6fer A.","year":"2009","unstructured":"A. D\u00f6nh\u00f6fer , \"Factor-mediated ribosome assembly in bacteria\" in eLS (John Wiley & Sons, Ltd., 2009), 10.1002\/9780470015902.a0021836."},{"key":"e_1_3_4_37_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-23617-8"},{"key":"e_1_3_4_38_2","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.68806"},{"key":"e_1_3_4_39_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijbiomac.2023.126876"},{"key":"e_1_3_4_40_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01347-17"},{"key":"e_1_3_4_41_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01184-21"},{"key":"e_1_3_4_42_2","doi-asserted-by":"publisher","DOI":"10.1093\/femsre\/fuab050"},{"key":"e_1_3_4_43_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkg050"},{"key":"e_1_3_4_44_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41594-022-00720-y"},{"key":"e_1_3_4_45_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-29274-9"},{"key":"e_1_3_4_46_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-020-0386-z"},{"key":"e_1_3_4_47_2","doi-asserted-by":"publisher","DOI":"10.1002\/1873-3468.13984"},{"key":"e_1_3_4_48_2","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.4169"},{"key":"e_1_3_4_49_2","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.20084"},{"key":"e_1_3_4_50_2","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444910007493"},{"key":"e_1_3_4_51_2","doi-asserted-by":"publisher","DOI":"10.1107\/S2059798319011471"},{"key":"e_1_3_4_52_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkab1061"},{"key":"e_1_3_4_53_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad1011"},{"key":"e_1_3_4_54_2","doi-asserted-by":"publisher","DOI":"10.1002\/pro.4792"},{"key":"e_1_3_4_55_2","doi-asserted-by":"publisher","DOI":"10.1002\/pro.3943"},{"key":"e_1_3_4_56_2","doi-asserted-by":"publisher","DOI":"10.1002\/pro.3235"},{"key":"e_1_3_4_57_2","unstructured":"S. Majumdar Structure of Mycobacterium smegmatis HflX bound to a 70S ribosome. Protein Data Bank. https:\/\/www.rcsb.org\/structure\/8VIO. Deposited 3 January 2024."},{"key":"e_1_3_4_58_2","doi-asserted-by":"crossref","unstructured":"S. Majumdar Structure of Mycobacterium smegmatis 50S ribosomal subunit bound to HflX:50S-HflX-A. Protein Data Bank. https:\/\/www.rcsb.org\/structure\/8VK0. Deposited 5 January 2024.","DOI":"10.2210\/pdb8why\/pdb"},{"key":"e_1_3_4_59_2","doi-asserted-by":"crossref","unstructured":"S. Majumdar Structure of Mycobacterium smegmatis 50S ribosomal subunit bound to HflX:50S-HflX-B. Protein Data Bank. https:\/\/www.rcsb.org\/structure\/8VK7. Deposited 8 January 2024.","DOI":"10.2210\/pdb8why\/pdb"},{"key":"e_1_3_4_60_2","doi-asserted-by":"crossref","unstructured":"S. Majumdar Structure of Mycobacterium smegmatis 50S ribosomal subunit bound to HflX:50S-HflX-C. Protein Data Bank. https:\/\/www.rcsb.org\/structure\/8VKI. Deposited 8 January 2024.","DOI":"10.2210\/pdb8why\/pdb"},{"key":"e_1_3_4_61_2","unstructured":"S. Majumdar Structure of Mycobacterium smegmatis 50S ribosomal subunit bound to delNTE-HflX. Protein Data Bank. https:\/\/www.rcsb.org\/structure\/8VKW. Deposited 10 January 2024."},{"key":"e_1_3_4_62_2","unstructured":"S. Majumdar Structure of Mycobacterium smegmatis 50S ribosomal subunit bound to HflX and erythromycin:50S-HflX-A-Ery. Protein Data Bank. https:\/\/www.rcsb.org\/structure\/8VR4. Deposited 18 January 2024."},{"key":"e_1_3_4_63_2","unstructured":"S. Majumdar Structure of Mycobacterium smegmatis 50S ribosomal subunit bound to HflX and erythromycin:50S-HflX-B-Ery. Protein Data Bank. https:\/\/www.rcsb.org\/structure\/8VPK. Deposited 15 January 2024."},{"key":"e_1_3_4_64_2","unstructured":"S. Majumdar Structure of Mycobacterium smegmatis 50S ribosomal subunit bound to HflX and chloramphenicol:50S-HflX-A-Clm. Protein Data Bank. https:\/\/www.rcsb.org\/structure\/8VRL. Deposited 20 January 2024."},{"key":"e_1_3_4_65_2","unstructured":"S. Majumdar Structure of Mycobacterium smegmatis 50S ribosomal subunit bound to HflX and chloramphenicol:50S-HflX-B-Clm. Protein Data Bank. https:\/\/www.rcsb.org\/structure\/8VR8. Deposited 20 January 2024."},{"key":"e_1_3_4_66_2","unstructured":"S. Majumdar Mycobacterium smegmatis 70S ribosome bound to P-tRNA. Protein Data Bank. https:\/\/www.emdataresource.org\/EMD-43791. Deposited 23 February 2024."},{"key":"e_1_3_4_67_2","unstructured":"S. Majumdar Structure of HflX mediated inactive Mycobacterium smegmatis 50S ribosomal subunit. Protein Data Bank. https:\/\/www.emdataresource.org\/EMD-43778. Deposited 22 February 2024."},{"key":"e_1_3_4_68_2","unstructured":"S. Majumdar Pre-dissociated Mycobacterium smegmatis 50S ribosomal subunit-HflX-GMPPCP complex. Protein Data Bank. https:\/\/www.emdataresource.org\/EMD-44044. Deposited 23 February 2024."}],"container-title":["Proceedings of the National Academy of Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pnas.org\/doi\/pdf\/10.1073\/pnas.2419826122","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,2,6]],"date-time":"2025-02-06T11:56:54Z","timestamp":1738843014000},"score":1,"resource":{"primary":{"URL":"https:\/\/pnas.org\/doi\/10.1073\/pnas.2419826122"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,2,6]]},"references-count":68,"journal-issue":{"issue":"6","published-print":{"date-parts":[[2025,2,11]]}},"alternative-id":["10.1073\/pnas.2419826122"],"URL":"https:\/\/doi.org\/10.1073\/pnas.2419826122","relation":{},"ISSN":["0027-8424","1091-6490"],"issn-type":[{"value":"0027-8424","type":"print"},{"value":"1091-6490","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,2,6]]},"assertion":[{"value":"2024-09-30","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-01-10","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-02-06","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e2419826122"}}