{"publication_status":"published","date_published":"2020-09-09T00:00:00Z","abstract":[{"text":"Mrp (Multi resistance and pH adaptation) are broadly distributed secondary active antiporters that catalyze the transport of monovalent ions such as sodium and potassium outside of the cell coupled to the inward translocation of protons. Mrp antiporters are unique in a way that they are composed of seven subunits (MrpABCDEFG) encoded in a single operon, whereas other antiporters catalyzing the same reaction are mostly encoded by a single gene. Mrp exchangers are crucial for intracellular pH homeostasis and Na+ efflux, essential mechanisms for H+ uptake under alkaline environments and for reduction of the intracellular concentration of toxic cations. Mrp displays no homology to any other monovalent Na+(K+)/H+ antiporters but Mrp subunits have primary sequence similarity to essential redox-driven proton pumps, such as respiratory complex I and membrane-bound hydrogenases. This similarity reinforces the hypothesis that these present day redox-driven proton pumps are descended from the Mrp antiporter. The Mrp structure serves as a model to understand the yet obscure coupling mechanism between ion or electron transfer and proton translocation in this large group of proteins. In the thesis, I am presenting the purification, biochemical analysis, cryo-EM analysis and molecular structure of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å resolution. Numerous conditions were screened to purify Mrp to high homogeneity and to obtain an appropriate distribution of single particles on cryo-EM grids covered with a continuous layer of ultrathin carbon. A preferred particle orientation problem was solved by performing a tilted data collection. The activity assays showed the specific pH-dependent\r\nprofile of secondary active antiporters. The molecular structure shows that Mrp is a dimer of seven-subunit protomers with 50 trans-membrane helices each. The dimer interface is built by many short and tilted transmembrane helices, probably causing a thinning of the bacterial membrane. The surface charge distribution shows an extraordinary asymmetry within each monomer, revealing presumable proton and sodium translocation pathways. The two largest\r\nand homologous Mrp subunits MrpA and MrpD probably translocate one proton each into the cell. The sodium ion is likely being translocated in the opposite direction within the small subunits along a ladder of charged and conserved residues. Based on the structure, we propose a mechanism were the antiport activity is accomplished via electrostatic interactions between the charged cations and key charged residues. The flexible key TM helices coordinate these\r\nelectrostatic interactions, while the membrane thinning between the monomers enables the translocation of sodium across the charged membrane. The entire family of redox-driven proton pumps is likely to perform their mechanism in a likewise manner.","lang":"eng"}],"department":[{"_id":"LeSa"}],"degree_awarded":"PhD","day":"09","file":[{"access_level":"open_access","creator":"jsteiner","file_name":"Thesis_Julia_Steiner_pdfA.pdf","content_type":"application/pdf","checksum":"2388d7e6e7a4d364c096fa89f305c3de","relation":"main_file","date_created":"2020-09-09T14:22:35Z","file_size":117547589,"file_id":"8354","date_updated":"2021-09-16T12:40:56Z"},{"file_name":"Thesis_Julia_Steiner.docx","access_level":"closed","creator":"jsteiner","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","relation":"source_file","checksum":"ba112f957b7145462d0ab79044873ee9","file_size":223328668,"date_created":"2020-09-09T14:23:25Z","file_id":"8355","date_updated":"2020-09-15T08:48:37Z"}],"type":"dissertation","date_updated":"2023-09-07T13:14:09Z","article_processing_charge":"No","supervisor":[{"orcid":"0000-0002-0977-7989","first_name":"Leonid A","last_name":"Sazanov","full_name":"Sazanov, Leonid A","id":"338D39FE-F248-11E8-B48F-1D18A9856A87"}],"month":"09","related_material":{"record":[{"status":"public","id":"8284","relation":"part_of_dissertation"}]},"status":"public","title":"Biochemical and structural investigation of the Mrp antiporter, an ancestor of complex I","alternative_title":["ISTA Thesis"],"date_created":"2020-09-09T14:27:01Z","user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","oa":1,"oa_version":"None","publisher":"Institute of Science and Technology Austria","citation":{"mla":"Steiner, Julia. <i>Biochemical and Structural Investigation of the Mrp Antiporter, an Ancestor of Complex I</i>. Institute of Science and Technology Austria, 2020, doi:<a href=\"https://doi.org/10.15479/AT:ISTA:8353\">10.15479/AT:ISTA:8353</a>.","ieee":"J. Steiner, “Biochemical and structural investigation of the Mrp antiporter, an ancestor of complex I,” Institute of Science and Technology Austria, 2020.","ama":"Steiner J. Biochemical and structural investigation of the Mrp antiporter, an ancestor of complex I. 2020. doi:<a href=\"https://doi.org/10.15479/AT:ISTA:8353\">10.15479/AT:ISTA:8353</a>","chicago":"Steiner, Julia. “Biochemical and Structural Investigation of the Mrp Antiporter, an Ancestor of Complex I.” Institute of Science and Technology Austria, 2020. <a href=\"https://doi.org/10.15479/AT:ISTA:8353\">https://doi.org/10.15479/AT:ISTA:8353</a>.","ista":"Steiner J. 2020. Biochemical and structural investigation of the Mrp antiporter, an ancestor of complex I. Institute of Science and Technology Austria.","apa":"Steiner, J. (2020). <i>Biochemical and structural investigation of the Mrp antiporter, an ancestor of complex I</i>. Institute of Science and Technology Austria. <a href=\"https://doi.org/10.15479/AT:ISTA:8353\">https://doi.org/10.15479/AT:ISTA:8353</a>","short":"J. Steiner, Biochemical and Structural Investigation of the Mrp Antiporter, an Ancestor of Complex I, Institute of Science and Technology Austria, 2020."},"language":[{"iso":"eng"}],"_id":"8353","doi":"10.15479/AT:ISTA:8353","page":"191","author":[{"orcid":"0000-0003-0493-3775","last_name":"Steiner","first_name":"Julia","id":"3BB67EB0-F248-11E8-B48F-1D18A9856A87","full_name":"Steiner, Julia"}],"has_accepted_license":"1","publication_identifier":{"issn":["2663-337X"]},"acknowledgement":"I acknowledge the scientific service units of the IST Austria for providing resources by the Life Science Facility, the Electron Microscopy Facility and the high-performance computer cluster. Special thanks to the cryo-EM specialists Valentin Hodirnau and Daniel Johann Gütl for spending many hours with me in front of the microscope and for supporting me to collect the data presented here. I also want to thank Professor Masahiro Ito for providing plasmid DNA\r\nencoding Mrp from Anoxybacillus flavithermus WK1. I am a recipient of a DOC Fellowship of the Austrian Academy of Sciences.","file_date_updated":"2021-09-16T12:40:56Z","ddc":["572"],"project":[{"grant_number":"24741","_id":"26169496-B435-11E9-9278-68D0E5697425","name":"Revealing the functional mechanism of Mrp antiporter, an ancestor of complex I"}],"acknowledged_ssus":[{"_id":"LifeSc"},{"_id":"EM-Fac"},{"_id":"ScienComp"}],"year":"2020"}