Membrane proteins interact with a myriad of lipid species in the biological membrane, leading to a bewildering number of possible protein−lipid assemblies. Despite this inherent complexity, the identification of specific protein−lipid interactions and the crucial role of lipids in the folding, structure, and function of membrane proteins is emerging from an increasing number of reports. Fundamental questions remain, however, regarding the ability of specific lipid binding events to membrane proteins to alter remote binding sites for lipids of a different type, a property referred to as allostery [Monod J, Wyman J, Changeux JP (1965) J Mol Biol 12:88–118]. Here, we use native mass spectrometry to determine the allosteric nature of heterogeneous lipid binding events to membrane proteins. We monitored individual lipid binding events to the ammonia channel (AmtB) from Escherichia coli, enabling determination of their equilibrium binding constants. We found that different lipid pairs display a range of allosteric modulation. In particular, the binding of phosphatidylethanolamine and cardiolipin-like molecules to AmtB exhibited the largest degree of allosteric modulation, inspiring us to determine the cocrystal structure of AmtB in this lipid environment. The 2.45-Å resolution structure reveals a cardiolipin-like molecule bound to each subunit of the trimeric complex. Mutation of a single residue in AmtB abolishes the positive allosteric modulation observed for binding phosphatidylethanolamine and cardiolipin-like molecules. Our results demonstrate that specific lipid−protein interactions can act as allosteric modulators for the binding of different lipid types to integral membrane proteins.

膜蛋白与生物膜中无数的脂质种类相互作用，导致数量惊人的可能的蛋白-脂质组装体。尽管存在这种固有的复杂性，但越来越多的报道显示，对特定的蛋白质-脂质相互作用的鉴定以及脂质在膜蛋白的折叠，结构和功能中的关键作用正在逐渐显现。然而，关于特定脂质与膜蛋白的结合事件改变不同类型脂质的远程结合位点的能力仍存在根本问题，该性质称为变构[Monod J，Wyman J，Changeux JP（1965）J Mol Biol12：88–118]。在这里，我们使用天然质谱测定异质脂质结合事件与膜蛋白的变构性质。我们监测了单个脂质与大肠杆菌氨通道（AmtB）的结合事件，可以确定它们的平衡结合常数。我们发现不同的脂质对显示一系列的变构调节。特别地，磷脂酰乙醇胺和心磷脂样分子与AmtB的结合表现出最大的变构调节度，这启发了我们确定这种脂质环境中AmtB的共晶体结构。2.45Å的拆分结构揭示了与三聚体复合物的每个亚基结合的类心磷脂分子。AmtB中单个残基的突变消除了结合磷脂酰乙醇胺和类心磷脂分子所观察到的正变构调节。我们的结果表明，特定的脂蛋白相互作用可以充当变构调节剂，以使不同的脂质类型与完整的膜蛋白结合。