Basic residues R260 and K357 affect the conformational dynamics of the major facilitator superfamily multidrug transporter LmrP.
Basic residues R260 and K357 affect the conformational dynamics of the major facilitator superfamily multidrug transporter LmrP.
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DOI：
10.1371/journal.pone.0038715
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
van Veen HW
中科院分区：
文献类型：
--
作者：
Wang W;van Veen HW
次级活性多药转运体可以通过底物/H+(Na+)逆向转运介导细胞从细胞内靶点排出，从而增强细胞对药物的耐药性。虽然这些转运体中的催化羧基与偶联离子和底物(药物)的相互作用已被详细研究，但碱性残基的功能重要性却很少受到关注。乳酸乳球菌主要促进剂超家族转运蛋白LmrP中跨膜螺旋中仅有的两个碱性残基R260和K357存在于蛋白质的外表面，在那里它们暴露在细胞膜的外叶(R260)和内叶(K357)的磷脂头基区。虽然我们对底物质子转运的质子动力依赖性和动力学以及底物依赖性质子转运的观察表明，与野生型LmrP相比，K357A和R260A突变体在乙锭-质子和苯扎溴-质子反向转运中受到影响，但我们的发现表明，R260和K357并不直接参与底物的结合或质子的转运。二次活性的多药转运体被认为是通过底物的结合部位交替暴露在膜的每一面的机制来运作的。用LmrP的双半胱氨酸突变体进行了二硫键交联实验，该突变体报告了底物刺激下从外向状态向内向状态的转变，具有高底物结合亲和力。在实验中，R260A和K357A突变被发现影响运输周期中这些主要蛋白质构象的动态，可能是通过消除R260和K357与磷脂和/或LmrP中其他残基的相互作用。因此，R260A和K357A突变改变了运输周期的最大运行速度，由于构象状态之间的转变受到质子动力分量的不同影响，这些突变也影响了运输的能量学。
Secondary-active multidrug transporters can confer resistance on cells to pharmaceuticals by mediating their extrusion away from intracellular targets via substrate/H+(Na+) antiport. While the interactions of catalytic carboxylates in these transporters with coupling ions and substrates (drugs) have been studied in some detail, the functional importance of basic residues has received much less attention. The only two basic residues R260 and K357 in transmembrane helices in the Major Facilitator Superfamily transporter LmrP from Lactococcus lactis are present on the outer surface of the protein, where they are exposed to the phospholipid head group region of the outer leaflet (R260) and inner leaflet (K357) of the cytoplasmic membrane. Although our observations on the proton-motive force dependence and kinetics of substrate transport, and substrate-dependent proton transport demonstrate that K357A and R260A mutants are affected in ethidium-proton and benzalkonium-proton antiport compared to wildtype LmrP, our findings suggest that R260 and K357 are not directly involved in the binding of substrates or the translocation of protons. Secondary-active multidrug transporters are thought to operate by a mechanism in which binding sites for substrates are alternately exposed to each face of the membrane. Disulfide crosslinking experiments were performed with a double cysteine mutant of LmrP that reports the substrate-stimulated transition from the outward-facing state to the inward-facing state with high substrate-binding affinity. In the experiments, the R260A and K357A mutations were found to influence the dynamics of these major protein conformations in the transport cycle, potentially by removing the interactions of R260 and K357 with phospholipids and/or other residues in LmrP. The R260A and K357A mutations therefore modify the maximum rate at which the transport cycle can operate and, as the transitions between conformational states are differently affected by components of the proton-motive force, the mutations also influence the energetics of transport.
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影响因子：
64.8
作者：
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通讯作者：
MacKinnon, Roderick
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作者：
Gbaguidi, B;Mazurkiewicz, P;Vigano, C
通讯作者：
Vigano, C
影响因子：
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作者：
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通讯作者：
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影响因子：
16.6
作者：
Dowhan W;Bogdanov M
通讯作者：
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