Suppr超能文献

ATP驱动的MalK二聚体闭合与重新打开以及“EAA”基序的构象变化对于麦芽糖ATP结合盒转运蛋白(MalFGK2)的功能至关重要。

ATP-driven MalK dimer closure and reopening and conformational changes of the "EAA" motifs are crucial for function of the maltose ATP-binding cassette transporter (MalFGK2).

作者信息

Daus Martin L, Grote Mathias, Müller Peter, Doebber Meike, Herrmann Andreas, Steinhoff Heinz-Jürgen, Dassa Elie, Schneider Erwin

机构信息

Institut für Biologie/Bakterienphysiologie, Humboldt Universität zu Berlin, Chausseestrasse 117, D-10115 Berlin, Germany.

出版信息

J Biol Chem. 2007 Aug 3;282(31):22387-96. doi: 10.1074/jbc.M701979200. Epub 2007 Jun 1.

Abstract

We have investigated conformational changes of the purified maltose ATP-binding cassette transporter (MalFGK(2)) upon binding of ATP. The transport complex is composed of a heterodimer of the hydrophobic subunits MalF and MalG constituting the translocation pore and of a homodimer of MalK, representing the ATP-hydrolyzing subunit. Substrate is delivered to the transporter in complex with periplasmic maltose-binding protein (MalE). Cross-linking experiments with a variant containing an A85C mutation within the Q-loop of each MalK monomer indicated an ATP-induced shortening of the distance between both monomers. Cross-linking caused a substantial inhibition of MalE-maltose-stimulated ATPase activity. We further demonstrated that a mutation affecting the "catalytic carboxylate" (E159Q) locks the MalK dimer in the closed state, whereas a transporter containing the "ABC signature" mutation Q140K permanently resides in the resting state. Cross-linking experiments with variants containing the A85C mutation combined with cysteine substitutions in the conserved EAA motifs of MalF and MalG, respectively, revealed close proximity of these residues in the resting state. The formation of a MalK-MalG heterodimer remained unchanged upon the addition of ATP, indicating that MalG-EAA moves along with MalK during dimer closure. In contrast, the yield of MalK-MalF dimers was substantially reduced. This might be taken as further evidence for asymmetric functions of both EAA motifs. Cross-linking also caused inhibition of ATPase activity, suggesting that transporter function requires conformational changes of both EAA motifs. Together, our data support ATP-driven MalK dimer closure and reopening as crucial steps in the translocation cycle of the intact maltose transporter and are discussed with respect to a current model.

摘要

我们研究了纯化的麦芽糖ATP结合盒转运蛋白(MalFGK(2))在结合ATP时的构象变化。转运复合物由构成转运孔的疏水亚基MalF和MalG的异二聚体以及代表ATP水解亚基的MalK同二聚体组成。底物与周质麦芽糖结合蛋白(MalE)形成复合物后被递送至转运蛋白。对每个MalK单体的Q环内含有A85C突变的变体进行交联实验表明,ATP诱导两个单体之间的距离缩短。交联导致MalE-麦芽糖刺激的ATP酶活性受到显著抑制。我们进一步证明,影响“催化羧酸盐”(E159Q)的突变将MalK二聚体锁定在关闭状态,而含有“ABC特征”突变Q140K的转运蛋白则永久处于静止状态。分别对含有A85C突变并在MalF和MalG的保守EAA基序中进行半胱氨酸取代的变体进行交联实验,结果显示这些残基在静止状态下距离很近。添加ATP后,MalK-MalG异二聚体的形成保持不变,这表明在二聚体关闭过程中,MalG-EAA与MalK一起移动。相比之下,MalK-MalF二聚体的产量大幅降低。这可能进一步证明了两个EAA基序具有不对称功能。交联还导致ATP酶活性受到抑制,这表明转运蛋白功能需要两个EAA基序的构象变化。总之,我们的数据支持ATP驱动的MalK二聚体关闭和重新打开是完整麦芽糖转运蛋白转运循环中的关键步骤,并结合当前模型进行了讨论。

文献AI研究员

20分钟写一篇综述,助力文献阅读效率提升50倍。

立即体验

用中文搜PubMed

大模型驱动的PubMed中文搜索引擎

马上搜索

文档翻译

学术文献翻译模型,支持多种主流文档格式。

立即体验