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

钠/质子反转运蛋白控制细胞内pH和Na+浓度，是所有活细胞所必需的。与被充分研究的阳离子/质子反转运蛋白（CPA）家族1和2成员相比，被归类为CPA3家族的Mrp（多重抗性和pH适应）反转运蛋白所知甚少。Mrp反转运蛋白广泛存在于细菌和古细菌中。它们是大型异聚膜蛋白复合物，通常由7个亚基（MrpA-G）组成。在一些物种中发现了Mrp操纵子的非规范版本。Mrp反转运蛋白的结构尚不清楚，但已经确定MrpA， MrpD和MrpC与呼吸复合物I的疏水亚基有关，呼吸复合物I是一种大型膜蛋白复合物，在有氧能量代谢中起核心作用。我们已经从需氧酵母解脂耶氏菌中解出了复合物I的结构，并提出了MrpA和MrpD同源亚基中的质子易位途径。基于同源性模型，我们假设Mrp反转运蛋白的核心结构与其他已被充分研究的反转运蛋白和转运蛋白明显不同，这表明在概念上存在不同的转运机制。我们的目标是了解CPA3家族成员的Na+/H+反转运，以及一个古老的Mrp反转运蛋白是如何转化为复合体i的质子泵模块的。我们已经克隆、表达和纯化了来自亨塞巴尔通体的典型Mrp反转运蛋白和来自热自养甲烷杆菌的Mrp样反转运蛋白。我们的目标是通过低温电镜或x射线晶体学来解决它们的结构。初步的位点导向诱变实验，以确定离子结合和运输的重要残基，将基于复合物I和同源模型中提出的质子易位途径设计。我们将在我们的实验室建立功能测定来分析野生型和突变体的抗port活性。Mrp反转运蛋白的结构和功能分析将有助于我们对生物膜运输过程的理解，特别是对呼吸复合物I氧化还原连接质子易位的尚未解决的机制有新的认识。

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.