Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis, Athens 15771, Greece.
Department of Biology, National and Kapodistrian University of Athens, Panepistimiopolis, Athens 15781, Greece.
J Mol Biol. 2023 Oct 1;435(19):168226. doi: 10.1016/j.jmb.2023.168226. Epub 2023 Aug 5.
Transporters mediate the uptake of solutes, metabolites and drugs across the cell membrane. The eukaryotic FurE nucleobase/H symporter of Aspergillus nidulans has been used as a model protein to address structure-function relationships in the APC transporter superfamily, members of which are characterized by the LeuT-fold and seem to operate by the so-called 'rocking-bundle' mechanism. In this study, we reveal the binding mode, translocation and release pathway of uracil/H by FurE using path collective variable, funnel metadynamics and rational mutational analysis. Our study reveals a stepwise, induced-fit, mechanism of ordered sequential transport of proton and uracil, which in turn suggests that FurE, functions as a multi-step gated pore, rather than employing 'rocking' of compact domains, as often proposed for APC transporters. Finally, our work supports that specific residues of the cytoplasmic N-tail are involved in substrate translocation, in line with their essentiality for FurE function.
转运蛋白介导溶质、代谢物和药物穿过细胞膜的摄取。产黄青霉 FurE 核苷/H 同型转运蛋白已被用作模型蛋白，用于解决 APC 转运蛋白超家族的结构-功能关系，该家族的成员具有 LeuT 折叠结构，似乎通过所谓的“摆动束”机制发挥作用。在这项研究中，我们使用路径总体变量、漏斗元动力学和合理的突变分析揭示了 FurE 结合尿嘧啶/H 的模式、易位和释放途径。我们的研究揭示了质子和尿嘧啶有序顺序转运的逐步诱导契合机制，这反过来表明 FurE 作为多步门控孔发挥作用，而不是像 APC 转运蛋白那样经常提出的紧凑结构域的“摆动”。最后，我们的工作支持细胞质 N 端的特定残基参与底物易位，这与它们对 FurE 功能的重要性一致。
