Unique structural features in an Nramp metal transporter impart substrate-specific proton cotransport and a kinetic bias to favor import
Unique structural features in an Nramp metal transporter impart substrate-specific proton cotransport and a kinetic bias to favor import
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DOI：
10.1085/jgp.201912428
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发表时间：
2019-12-01
影响因子：
3.8
通讯作者：
Gaudet, Rachelle
中科院分区：
文献类型：
--
作者：
Bozzi, Aaron T.;Bane, Lukas B.;Gaudet, Rachelle
天然耐药相关巨噬细胞蛋白（Nramp）转运体能够在许多生物环境中摄取必需的过渡金属微量营养素。这些蛋白质被认为是二级转运体，通过“耦合”质子和金属运输来利用质子梯度的电化学能量。在这里，我们使用耐辐射球菌（Dra） Nramp同源物，我们已经确定了多种构象的晶体结构，来研究金属和质子运输的机理细节。我们将DraNramp的质子-金属耦合行为分解为两种不同的现象：δ pH刺激金属输运速率和金属刺激质子输运。令人惊讶的是，金属类型影响底物化学计量，导致锰-质子共输运，但镉单输运，而质子单输运也发生。此外，生理负膜电位是高亲和力金属摄取所必需的。为了开始了解Nramp的结构如何赋予这些特性，我们瞄准了一个保守的盐桥网络，该网络形成了从金属结合位点到细胞质的质子传输途径。该网络的突变降低了金属传输速率对电压和δ pH的依赖，改变了底物的选择性，干扰或消除了金属刺激的质子传输，并侵蚀了生理条件下有利于向外向内金属传输的方向偏差。因此，这种独特的盐桥网络可能有助于nramp家族转运蛋白最大限度地吸收金属，减少获得金属的有害回输。我们为Nramp质子-金属共输运提供了一个新的机制模型，并提出了与传统的同运模型不同的功能优势。
Natural resistance-associated macrophage protein (Nramp) transporters enable uptake of essential transition metal micronutrients in numerous biological contexts. These proteins are believed to function as secondary transporters that harness the electrochemical energy of proton gradients by "coupling" proton and metal transport. Here we use the Deinococcus radiodurans (Dra) Nramp homologue, for which we have determined crystal structures in multiple conformations, to investigate mechanistic details of metal and proton transport. We untangle the proton-metal coupling behavior of DraNramp into two distinct phenomena: Delta pH stimulation of metal transport rates and metal stimulation of proton transport. Surprisingly, metal type influences substrate stoichiometry, leading to manganese-proton cotransport but cadmium uniport, while proton uniport also occurs. Additionally, a physiological negative membrane potential is required for high-affinity metal uptake. To begin to understand how Nramp's structure imparts these properties, we target a conserved salt-bridge network that forms a proton-transport pathway from the metal-binding site to the cytosol. Mutations to this network diminish voltage and Delta pH dependence of metal transport rates, alter substrate selectivity, perturb or eliminate metal-stimulated proton transport, and erode the directional bias favoring outward-to-inward metal transport under physiological-like conditions. Thus, this unique salt-bridge network may help Nramp-family transporters maximize metal uptake and reduce deleterious back-transport of acquired metals. We provide a new mechanistic model for Nramp proton-metal cotransport and propose that functional advantages may arise from deviations from the traditional model of symport.