Interactions between phosphatidylethanolamine headgroup and LmrP, a multidrug transporter
Interactions between phosphatidylethanolamine headgroup and LmrP, a multidrug transporter
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DOI：
10.1074/jbc.m708427200
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发表时间：
2008-04-04
影响因子：
4.8
通讯作者：
Govaerts, Cedric
中科院分区：
文献类型：
--
作者：
Hakizimana, Pierre;Masureel, Matthieu;Govaerts, Cedric
在许多情况下，膜蛋白的功能似乎需要在双层中存在特定的脂质物质。我们已经表明，二级多药物转运蛋白LmrP需要磷脂酰乙醇胺（PE）的存在下，因为它的替代磷脂酰胆碱（PC）抑制运输活动，并直接影响其结构，虽然潜在的机制是未知的。在这里，我们表明，PE对LmrP的结构和功能的影响是介导的脂质头基和蛋白质之间的相互作用。我们使用PE的甲基-PE和二甲基-PE类似物来表明，只有三个氢被甲基部分取代才会导致重构蛋白质的生物化学和生物物理性质的变化。这表明LmrP不依赖于所测试的磷脂的本体性质，而仅依赖于头基的氢键结合能力。然后，我们表明，一个单一的点突变LmrP，D 68 C，是足以概括精确的每一个生化和生物物理效应时，PE被PC取代，包括蛋白质色氨酸残基和脂质头基之间的能量转移。我们得出结论，带负电荷的天冬氨酸-68很可能参与与PE的相互作用，这种相互作用是质子梯度传感，底物结合和运输所必需的。由于Asp-68属于主要易化因子超家族（包括LacY和EmrD）中的高度保守基序，因此这种相互作用可能是这些转运蛋白的一般特征，这些转运蛋白参与质子梯度传感和脂质依赖。
In a number of cases, the function of membrane proteins appears to require the presence of specific lipid species in the bilayer. We have shown that the secondary multidrug transporter LmrP requires the presence of phosphatidylethanolamine (PE), as its replacement by phosphatidylcholine (PC) inhibits transport activity and directly affects its structure, although the underlying mechanism was unknown. Here, we show that the effect of PE on the structure and the function of LmrP is mediated by interactions between the lipid headgroup and the protein. We used methyl-PE and dimethyl-PE analogs of PE to show that only replacement of the three hydrogens by methyl moieties leads to changes in the biochemical and biophysical properties of the reconstituted protein. This suggests that LmrP does not depend on the bulk properties of the phospholipids tested but solely on the hydrogen bonding ability of the headgroup. We then show that a single point mutation in LmrP, D68C, is sufficient to recapitulate precisely every biochemical and biophysical effect observed when PE is replaced by PC, including energy transfer between the protein tryptophan residues and the lipid headgroups. We conclude that the negatively charged Asp-68 is likely to participate in the interaction with PE and that such interaction is required for proton gradient sensing, substrate binding, and transport. Because Asp-68 belongs to a highly conserved motif in the Major Facilitator Superfamily (which includes LacY and EmrD), this interaction might be a general feature of these transporters that is involved in proton gradient sensing and lipid dependence.