Structure and function of Mrp type sodium/proton antiporters and their relation to respiratory complex I

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Sodium/proton antiporters control intracellular pH and Na+ concentration and are essential for all living cells. In contrast to the well studied members of the cation/proton antiporter (CPA) families 1 and 2 much less is known about the Mrp (multiple resistance and pH adaptation) antiporters classified as the CPA3 family. The Mrp antiporters are wide spread in bacteria and archea. They are large hetero-oligomeric membrane protein complexes typically comprising seven subunits (MrpA-G). In several species non-canonical versions of the Mrp operon are found. The structure of Mrp antiporters is unknown but it was established that MrpA, MrpD, and MrpC are related to hydrophobic subunits of respiratory complex I, a large membrane protein complex with a central role in aerobic energy metabolism. We have solved the structure of complex I from the aerobic yeast Yarrowia lipolytica and suggested proton translocation pathways in MrpA and MrpD orthologuous subunits. Based on homology models we hypothesize that the core structure of Mrp antiporters significantly deviates from other well studied antiporters and transport proteins suggesting a conceptually different transport mechanism. Our goal is to understand Na+/H+ antiport by members of the CPA3 family and how an ancient Mrp antiporter was converted into the proton pump module of complex I. We have cloned, expressed and purified the canonical Mrp antiporter from Bartonella henselae and the Mrp like antiporter from Methanobacterium thermoautotrophicum. Our aim is to solve their structures by cryo-EM or X-ray crystallography. Initial site-directed mutagenesis experiments to identify important residues for ion binding and transport will be designed based on proposed proton translocation pathways in complex I and homology models. We will establish functional assays in our laboratory to analyze antiport activity of wild type and mutants. Analysis of structure and function of Mrp antiporters will contribute to our understanding of biological membrane transport processes in general and will in particular shed new light on the still unresolved mechanism of redox-linked proton translocation by respiratory complex I.