The interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among co-factor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.

中文翻译：

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脂质塑造外围膜蛋白二氢乳清酸酯脱氢酶的电子受体结合位点

蛋白质和生物膜之间的相互作用对于药物开发很重要，但众所周知，它对结构研究仍然是不利的。我们将非变性质谱（MS）与分子动力学（MD）模拟相结合，以揭示辅因子，脂质和外周膜蛋白二氢乳清酸脱氢酶（DHODH）（关键的抗癌靶标）中抑制剂的结合。MS对完整的DHODH复合物的询问表明，磷脂通过其带电头基团在有限的位点结合，而抑制剂布雷奎纳的结合则涉及与去污剂分子的同时缔合。MD模拟显示，脂质在膜结合结构域中支持柔性链段，并使抑制剂和电子受体结合位点远离膜表面，