Nguyen Thao Tran, Mitani-Ueno Namiki, Urano Ryo, Saitoh Yasunori, Wang Peitong, Yamaji Naoki, Shen Jian-Ren, Shinoda Wataru, Ma Jian Feng, Suga Michihiro
Degree Program in Interdisciplinary Sciences, Graduate School of Environmental, Life, Natural Science, and Technology, Okayama University, Okayama 700-8530, Japan.
Research Core for Plant Stress Science, Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan.
Proc Natl Acad Sci U S A. 2025 Aug 12;122(32):e2501933122. doi: 10.1073/pnas.2501933122. Epub 2025 Aug 5.
is a major aluminum (Al)-tolerance gene in barley, encoding a citrate transporter that belongs to the multidrug and toxic compound extrusion (MATE) family. This transporter facilitates citrate secretion from the roots, thereby detoxifying external Al ions-a major constraint of crop production on acidic soils. In this study, we present the outward-facing crystal structure of HvAACT1, providing insights into a citrate transport mechanism. The putative citrate binding site consists of three basic residues-K126 in transmembrane helix 2 (TM2), R358 in TM7, and R535 in TM12-creating substantial positive charges in the C-lobe cavity. Proton coupling for substrate transport may involve two pairs of aspartate residues in the N-lobe cavity, one of which corresponds to the essential Asp pair found in prokaryotic H-coupled MATE transporters belonging to the DinF subfamily. Structural coupling between proton uptake in the N-lobe and citrate extrusion in the C-lobe can be enabled by an extensive, unique hydrogen-bonding network at the extracellular half of the N-lobe. Mutation-based functional analysis, structural comparisons, molecular dynamics simulation, and phylogenic analysis suggest an evolutionary link between citrate MATE transporters and the DinF MATE subfamily. Our findings provide a solid structural basis for citrate transport by HvAACT1 in barley and contribute to a broader understanding of citrate transporter structures in other plant species.
HvAACT1是大麦中一个主要的耐铝基因，编码一种属于多药与有毒化合物外排（MATE）家族的柠檬酸转运蛋白。该转运蛋白促进柠檬酸从根部分泌，从而解除外部铝离子的毒性，铝离子是酸性土壤上作物生产的主要限制因素。在本研究中，我们展示了HvAACT1向外的晶体结构，为柠檬酸转运机制提供了见解。推测的柠檬酸结合位点由三个碱性残基组成，分别是跨膜螺旋2（TM2）中的K126、TM7中的R358和TM12中的R535，它们在C叶腔中产生大量正电荷。底物转运的质子偶联可能涉及N叶腔中的两对天冬氨酸残基，其中一对对应于属于DinF亚家族的原核H偶联MATE转运蛋白中发现的必需天冬氨酸对。N叶中质子摄取与C叶中柠檬酸外排之间的结构偶联可通过N叶细胞外一半广泛而独特的氢键网络实现。基于突变的功能分析、结构比较、分子动力学模拟和系统发育分析表明，柠檬酸MATE转运蛋白与DinF MATE亚家族之间存在进化联系。我们的研究结果为大麦中HvAACT1介导的柠檬酸转运提供了坚实的结构基础，并有助于更广泛地理解其他植物物种中柠檬酸转运蛋白的结构。
