A twin histidine motif is the core structure for high-affinity substrate selection in plant ammonium transporters
A twin histidine motif is the core structure for high-affinity substrate selection in plant ammonium transporters
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DOI：
10.1074/jbc.ra119.010891
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发表时间：
2020-03-06
影响因子：
4.8
通讯作者：
Neuhaeuser, Benjamin
中科院分区：
文献类型：
--
作者：
Ganz, Pascal;Ijato, Toyosi;Neuhaeuser, Benjamin
铵转运蛋白（AMT），甲胺渗透酶（Mep），和更远的相关恒河猴因子（Rh）是三聚体膜蛋白存在于所有领域的生活。AMT/Mep/Rhs是铵吸收或释放所需的高度选择性膜蛋白，它们有效地排除类似大小的K+离子。以前报道的晶体结构显示，每个转运蛋白亚基含有一个独特的疏水性，但封闭的中心孔，但目前还不清楚是否碱（NH3）或NH3与H+耦合进行运输。在这里，使用两种植物AMT（AtAMT 1;2和AMT 2）在芽殖酵母中的表达，我们发现，在保守的双组氨酸基序，大多数AMT/Mep/Rh的标志，改变底物识别，运输能力，N同位素选择性和对K+的选择性的系统替换。发现组氨酸变体存在AMT特异性差异。完全失去铵氮同位素选择的变体，一个功能可能与NH 4+去质子化过程中通过，基本上运输K+除了NH 4+。值得注意的是，双组氨酸基序不是必需的铵运输。然而，它赋予了关键的AMT功能，如高底物亲和力和通过NH 4+去质子化机制对碱金属阳离子的选择性。我们的研究结果表明，双组氨酸基序是负责底物去质子化和AMT孔中的同位素偏好的核心结构，并且去质子化能力降低与对K+的选择性降低相关。我们的结论是，在植物AMT铵运输的优化代表了最佳选择性和高底物亲和性和运输速率之间的底物去质子化的妥协。
Ammonium transporters (AMT), methylamine permeases (Mep), and the more distantly related rhesus factors (Rh) are trimeric membrane proteins present in all domains of life. AMT/Mep/Rhs are highly selective membrane proteins required for ammonium uptake or release, and they efficiently exclude the similarly sized K+ ion. Previously reported crystal structures have revealed that each transporter subunit contains a unique hydrophobic but occluded central pore, but it is unclear whether the base (NH3) or NH3 coupled with an H+ are transported. Here, using expression of two plant AMTs (AtAMT1;2 and AMT2) in budding yeast, we found that systematic replacements in the conserved twin-histidine motif, a hallmark of most AMT/Mep/Rh, alter substrate recognition, transport capacities, N isotope selection, and selectivity against K+. AMT-specific differences were found for histidine variants. Variants that completely lost ammonium N isotope selection, a feature likely associated with NH4+ deprotonation during passage, substantially transported K+ in addition to NH4+. Of note, the twin-histidine motif was not essential for ammonium transport. However, it conferred key AMT features, such as high substrate affinity and selectivity against alkali cations via an NH4 + deprotonation mechanism. Our findings indicate that the twin-His motif is the core structure responsible for substrate deprotonation and isotopic preferences in AMT pores and that decreased deprotonation capacity is associated with reduced selectivity against K+. We conclude that optimization for ammonium transport in plant AMT represents a compromise between substrate deprotonation for optimal selectivity and high substrate affinity and transport rates.