A dual substrate-accessing mechanism of a major facilitator superfamily protein facilitates lysophospholipid flipping across the cell membrane
A dual substrate-accessing mechanism of a major facilitator superfamily protein facilitates lysophospholipid flipping across the cell membrane
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DOI：
10.1074/jbc.ra118.005548
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发表时间：
2018-12-21
影响因子：
4.8
通讯作者：
Zheng, Lei
中科院分区：
文献类型：
--
作者：
Lin, Yibin;Deepak, R. N. V. Krishna;Zheng, Lei
溶血磷脂转运蛋白（LplT）是存在于许多革兰氏阴性菌中的主要易化剂超家族的成员。LplT催化溶血磷脂（LPL）翻转穿过细菌内膜，在细菌膜稳态中起重要作用。我们先前报道了LplT促进外源性LPL的摄取和膜内LPL翻转穿过双层。为了获得对这种双重LPL翻转活动的机理性洞察，在这里，我们实施了计算方法和LPL转运分析的组合来研究LPL结合和LplT的易位。我们的研究结果表明，LplT易位LPL通过一个细长的腔表现出极不对称的极性。我们发现两个D（E）N基序形成了一个头基结合位点，其中Asp-30的羧酸基团对于LPL头基识别是重要的。头基结合位点中的残基的取代破坏了LPL摄取和翻转活动。然而，N-和C-末端结构域之间的界面上的疏水残基的改变损害LPL翻转具体，导致LPL在膜中的积累，但LPL摄取保持活跃。这些结果表明了一种双重底物进入机制，其中LplT通过两种途径将LPL招募到其底物结合位点，要么从其细胞外进入，要么通过跨膜螺旋之间的膜嵌入凹槽，然后将它们移向内膜小叶。这种LPL翻转机制可能在许多细菌物种中是保守的，我们的研究结果说明了LplT如何调节主要的促进剂超家族易位途径以执行其多功能的脂质稳态功能。
Lysophospholipid transporter (LplT) is a member of the major facilitator superfamily present in many Gram-negative bacteria. LplT catalyzes flipping of lysophospholipids (LPLs) across the bacterial inner membrane, playing an important role in bacterial membrane homeostasis. We previously reported that LplT promotes both uptake of exogenous LPLs and intramembranous LPL flipping across the bilayer. To gain mechanistic insight into this dual LPL-flipping activity, here we implemented a combination of computational approaches and LPL transport analyses to study LPL binding of and translocation by LplT. Our results suggest that LplT translocates LPLs through an elongated cavity exhibiting an extremely asymmetric polarity. We found that two D(E)N motifs form a head group-binding site, in which the carboxylate group of Asp-30 is important for LPL head group recognition. Substitutions of residues in the head group-binding site disrupted both LPL uptake and flipping activities. However, alteration of hydrophobic residues on the interface between the N- and C-terminal domains impaired LPL flipping specifically, resulting in LPLs accumulation in the membrane, but LPL uptake remained active. These results suggest a dual substrate-accessing mechanism, in which LplT recruits LPLs to its substrate-binding site via two routes, either from its extracellular entry or through a membrane-embedded groove between transmembrane helices, and then moves them toward the inner membrane leaflet. This LPL-flipping mechanism is likely conserved in many bacterial species, and our findings illustrate how LplT adjusts the major facilitator superfamily translocation pathway to perform its versatile lipid homeostatic functions.