Department of Plant Pathology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, China; Department of Chemistry and Molecular Biology, Gothenburg University, Box 462, 405 30 Göteborg, Sweden.
Department of Chemistry and Molecular Biology, Gothenburg University, Box 462, 405 30 Göteborg, Sweden.
Biochim Biophys Acta Biomembr. 2020 Feb 1;1862(2):183065. doi: 10.1016/j.bbamem.2019.183065. Epub 2019 Sep 12.
Aquaporins are membrane-intrinsic proteins initially defined as water (HO) channels in all organisms and subsequently found to have multiple substrate specificities, such as hydrogen peroxide (HO). HO is a signaling molecule that partakes in immune responses where its transport is mediated by aquaporins. To shed further light on the molecular basis of the aquaporin function in HO transport, we have characterized an Arabidopsis thaliana aquaporin, AtPIP2;4, recombinantly produced to high yields in Pichia pastoris. Here, we present a newly established assay that allows detection of HO transport by purified aquaporins reconstituted into liposomes, enabling us to compare aquaporin homologues with respect to substrate specificity. To get additional insight into the structural determinants for aquaporin-mediated HO transport, we solved the 3D-structure of AtPIP2;4 to 3.7 Å resolution and found structural identity to the water channel from spinach (SoPIP2;1), with the difference that Cd cation is not required to retain the closed conformation. The transport specificities of the two plant aquaporins were compared to a human homologue, AQP1. Overall, we conclude that AtPIP2;4, SoPIP2;1 and hAQP1 are all transporters of both HO and HO, but have different efficiencies for various specificities. Notably, all three homologues expedite HO transport equally well while the plant aquaporins are more permeable to HO than hAQP1. Comparison of the structures indicates that the observed variations in HO and HO transport cannot be explained by differences in the monomeric pore. Possibly, the determinants for transport specificities reside in the flexible domains outside the membrane core of these channels.
水通道蛋白最初被定义为所有生物中的水 (HO) 通道，随后发现其具有多种底物特异性，如过氧化氢 (HO)。HO 是一种信号分子，参与免疫反应，其运输由水通道蛋白介导。为了进一步阐明水通道蛋白在 HO 运输中的分子基础，我们对拟南芥水通道蛋白 AtPIP2;4 进行了表征，该蛋白在巴斯德毕赤酵母中以高产量重组表达。在这里，我们提出了一种新的测定方法，该方法允许通过重建到脂质体中的纯化水通道蛋白检测 HO 的运输，使我们能够比较水通道蛋白同源物的底物特异性。为了更深入地了解水通道蛋白介导的 HO 运输的结构决定因素，我们以 3.7 Å 的分辨率解决了 AtPIP2;4 的 3D 结构，并发现与菠菜水通道蛋白 (SoPIP2;1) 具有结构同一性，不同之处在于不需要 Cd 阳离子来保留关闭构象。比较了这两种植物水通道蛋白的转运特性与人类同源物 AQP1。总的来说，我们得出结论，AtPIP2;4、SoPIP2;1 和 hAQP1 都是 HO 和 HO 的转运体，但对各种特异性的效率不同。值得注意的是，所有三种同源物都同样有效地促进 HO 的运输，而植物水通道蛋白对 HO 的通透性比 hAQP1 更高。结构比较表明，HO 和 HO 转运的观察到的差异不能用单体孔的差异来解释。可能，运输特异性的决定因素存在于这些通道膜核心外的柔性结构域中。
