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

Mrp型钠/质子逆向转运蛋白的结构和功能及其与呼吸复合物I的关系

• 批准号: 405943872
• 负责人: Professor Dr. Volker Zickermann
• 金额: --
• 依托单位: Arbeitsgruppe Strukturelle Bioenergetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/405943872?language=en
• 关键词: Structure; function; Mrp; type; sodium

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.

钠/质子反向转运蛋白控制细胞内pH和Na+浓度，对所有活细胞都是必需的。与阳离子/质子反向转运蛋白（CPA）家族1和2的成员相比，对归类为CPA 3家族的Mrp（多重抗性和pH适应）反向转运蛋白的了解要少得多。Mrp反向转运蛋白广泛存在于细菌和古细菌中。它们是通常包含七个亚基（MrpA-G）的大的异源寡聚膜蛋白复合物。在几个物种中发现了Mrp操纵子的非经典版本。Mrp反向转运蛋白的结构尚不清楚，但已确定MrpA、MrpD和MrpC与呼吸复合物I的疏水亚基相关，呼吸复合物I是一种在有氧能量代谢中起核心作用的大型膜蛋白复合物。我们已经解决了复杂的结构从好氧酵母解脂耶氏酵母和建议质子转运途径在MrpA和MrpD直向同源亚基。基于同源性模型，我们假设，核心结构的MRP反向转运显着偏离其他研究的反向转运蛋白和运输蛋白，这表明一个概念上不同的运输机制。我们的目标是了解CPA 3家族成员的Na+/H+反向转运，以及古老的Mrp反向转运蛋白如何转化为复合物I的质子泵模块。我们克隆、表达并纯化了汉赛巴尔通体（Bartonella henselae）的经典Mrp反向转运蛋白和热自养甲烷杆菌（Methanobacterium thermoautotrophicum）的Mrp类反向转运蛋白。我们的目标是通过冷冻电镜或X射线晶体学来解决它们的结构。最初的定点诱变实验，以确定离子结合和运输的重要残基将设计的基础上提出的质子易位途径在复杂的I和同源模型。我们将在实验室建立功能分析方法，分析野生型和突变体的反向转运活性。Mrp反向转运蛋白的结构和功能的分析将有助于我们理解一般的生物膜运输过程，特别是将揭示新的光在呼吸复合物I的氧化还原连接的质子易位的机制仍然悬而未决。