Department of Biology & Lund Protein Production Platform, Lund University, Lund, Sweden.
Department of Chemistry, Division of Physical Chemistry, Lund University, Lund, Sweden.
Nucleosides Nucleotides Nucleic Acids. 2020;39(10-12):1306-1319. doi: 10.1080/15257770.2019.1708100. Epub 2020 Jan 30.
Human dihydroorotate dehydrogenase (DHODH) is an integral protein of the inner mitochondrial membrane (IMM) that catalyzes the fourth step of the pyrimidine biosynthesis and is functionally connected to the respiratory chain via its lipophilic co-substrate, ubiquinone Q. DHODH is the target for drugs approved for the treatment of rheumatoid arthritis and multiple sclerosis, and mutations in its sequence have been identified as the cause of Miller syndrome, a rare genetic disorder. The N-terminus of DHODH consists of a signal peptide for mitochondrial import (MS), a transmembrane domain (TM), followed by a microdomain which interacts with the lipids of the IMM and has been proposed to form the binding site for ubiquinone Q. However, the mechanism by which DHODH interacts with the membrane-embedded Q and the lipids of the IMM remains unknown. We present the preparation and characterization of proteins necessary for investigating the structural interactions of DHODH with the lipids of the IMM, including expression and purification of full-length and N-terminally truncated (without MS and TM) DHODH. We characterized the interaction of truncated DHODH with lipid bilayers containing some key lipids of the IMM using Quartz Crystal Microbalance with Dissipation monitoring and compared it to the DHODH from , a DHODH that naturally lacks a TM. Our results suggest that although cardiolipin enhances the interaction of truncated DHODH with lipid bilayers, the presence of the TM in human DHODH is necessary for stable binding to and securing its location at the outer surface of the IMM.
人二氢乳清酸脱氢酶（DHODH）是线粒体内膜（IMM）的整合蛋白，它催化嘧啶生物合成的第四步，通过其亲脂性辅酶 Q 与呼吸链在功能上相连。DHODH 是已批准用于治疗类风湿关节炎和多发性硬化症的药物的靶标，其序列中的突变已被确定为米勒综合征的病因，米勒综合征是一种罕见的遗传疾病。DHODH 的 N 端由用于线粒体导入的信号肽（MS）、跨膜结构域（TM）组成，其后是一个微区，与 IMM 的脂质相互作用，并且据推测形成与辅酶 Q 的结合位点。然而，DHODH 与膜嵌入的 Q 和 IMM 的脂质相互作用的机制尚不清楚。我们介绍了制备和表征用于研究 DHODH 与 IMM 脂质结构相互作用所需的蛋白质，包括全长和 N 端截断（无 MS 和 TM）DHODH 的表达和纯化。我们使用石英晶体微天平耗散监测法表征了截断 DHODH 与含有 IMM 一些关键脂质的脂质双层的相互作用，并将其与天然缺乏 TM 的 DHODH 进行了比较。我们的结果表明，尽管心磷脂增强了截断 DHODH 与脂质双层的相互作用，但 TM 的存在对于人 DHODH 与脂质双层的稳定结合并确保其位于 IMM 的外表面是必要的。
