Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232.
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232
Proc Natl Acad Sci U S A. 2020 Mar 3;117(9):4732-4740. doi: 10.1073/pnas.1917139117. Epub 2020 Feb 19.
Multidrug and toxic compound extrusion (MATE) transporters are ubiquitous ion-coupled antiporters that extrude structurally and chemically dissimilar cytotoxic compounds and have been implicated in conferring multidrug resistance. Here, we integrate double electron-electron resonance (DEER) with functional assays and site-directed mutagenesis of conserved residues to illuminate principles of ligand-dependent alternating access of PfMATE, a proton-coupled MATE from the hyperthermophilic archaeon Pairs of spin labels monitoring the two sides of the transporter reconstituted into nanodiscs reveal large-amplitude movement of helices that alter the orientation of a putative substrate binding cavity. We found that acidic pH favors formation of an inward-facing (IF) conformation, whereas elevated pH (>7) and the substrate rhodamine 6G stabilizes an outward-facing (OF) conformation. The lipid-dependent PfMATE isomerization between OF and IF conformation is driven by protonation of a previously unidentified intracellular glutamate residue that is critical for drug resistance. Our results can be framed in a mechanistic model of transport that addresses central aspects of ligand coupling and alternating access.
多药和毒性化合物外排（MATE）转运蛋白是普遍存在的离子偶联反向转运体，可将结构和化学性质不同的细胞毒性化合物排出体外，并与多药耐药性的产生有关。在这里，我们将双电子-电子共振（DEER）与保守残基的功能测定和定点突变相结合，阐明 PfMATE（一种来自嗜热古菌 Pairs 的质子偶联 MATE）配体依赖性交替访问的原理，PfMATE 是一种从嗜热古菌 Pairs 中分离出来的质子偶联 MATE。监测转运体两侧的双自旋标记对在纳米盘中重建后揭示了螺旋的大幅度运动，改变了假定的底物结合腔的方向。我们发现酸性 pH 值有利于形成内向（IF）构象，而升高的 pH 值（>7）和底物罗丹明 6G 稳定外向（OF）构象。脂依赖性 PfMATE 在 OF 和 IF 构象之间的异构化由先前未鉴定的细胞内谷氨酸残基的质子化驱动，该残基对药物耐药性至关重要。我们的结果可以在运输的机制模型中进行阐述，该模型解决了配体偶联和交替访问的核心方面。
