Computational Studies of Membrane Transport Proteins

膜转运蛋白是一大类参与小分子跨细胞膜运动的整合膜蛋白。这些蛋白质中的许多被称为次级主动转运蛋白，使用预先存在的底物浓度梯度作为能量来源，用于逆着其浓度梯度转移另一种底物。每种生物体都表达数十种不同的二级转运蛋白，这些蛋白具有不同的结构折叠阵列，并且每种蛋白质对不同的底物具有特异性，这些底物的范围从离子到神经递质。详细了解每种膜转运蛋白的机制需要了解其在许多不同构象状态下的三维结构，以及识别底物或底物的结合区域。我们小组在过去一年的研究为一些生物医学上重要的转运蛋白提供了见解，如下所述。 在2014年，我们确定了负责肾脏钠偶联磷酸盐摄取的二级转运蛋白（称为NaPi-IIa）的结构折叠，通过确定与钠偶联二羧酸转运蛋白（称为VcINDY）的已知结构的进化关系，然后将其用作同源性建模的模板。在该模型中，我们预测了三种所需钠离子中的两种（Na 2和Na 3）和磷酸基团的结合位点（Fenollar-Ferrer等人，Biophysical Journal，2014）。然而，第一个结合的钠离子Na 1的位置尚未得到解决。我们使用我们的结构模型，结合沃纳和福斯特实验室的生化和电生理测量，对与钠离子配位的残基进行了详细的预测（参考文献1）。这些结果提供了一个重要的一步，在确定的分子来源的钠化学计量差异NaPi-II同系物。同源性建模也被用来产生一个同源的囊泡单胺转运蛋白的结构模型从短芽孢杆菌，BbMAT。未知功能的蛋白质，YajR，被用作BbMAT的同源性建模的模板，允许识别潜在的感兴趣的路径衬里电离残基。Schuldiner和Singh实验室的合作者对这些残基进行的修饰表明，它们在质子依赖性转运中发挥关键作用，并为理解神经递质转运机制提供了重要步骤（参考文献2）。类似的分子建模策略可用于确定哺乳动物渗透压转运蛋白BGT 1中糖基化位点的结构位置和行为，从而推动了齐格勒实验室对糖基化在该蛋白质在肾脏中的运输和质膜插入中的作用进行实验研究（参考文献3）。 我们已经使用结构建模来探索寡肽同向转运体PepT 1的构象机制，PepT 1负责肽和药物进入肠道的质子驱动摄取，并且是二级转运体的最大组的成员，主要促进剂超家族（MFS）。我们在Newstead实验室的合作者报告了Shewanella oniedensis和Streptococcus thermopiles（分别为PepTSo和PepTSt）的PepT同系物在面向细胞质构象中的结构。基于这些结构的重复交换模型是面向外部的。这些结构模型进行了比较的晶体结构，并与分子动力学模拟，光谱分析，和功能测定的Newstead/Fowler集团进行的结果。这些结果一起被用来推导出一个分子机制，交替访问MFS蛋白，这是更详细的比简单的“摇杆开关”或“摇摆束”的机制，以前提出的（参考文献4）。另外，我们在探索二级转运蛋白构象机制的多样性方面取得了进展。具体而言，我们已经研究了是否电梯样构象机制，如前所述，仅为天冬氨酸转运蛋白GltPh，使用其他转运蛋白家族具有不同的结构折叠。使用更新且更准确的重复交换建模方案，我们预测来自霍乱弧菌的浓缩核苷转运蛋白同系物VcCNT也使用升降机型构象机制（参考文献5）。最后，我们使用了系统的生物信息学分析，以证明两个明显不相关的转运蛋白家族的结构，对氨基苯甲酰谷氨酸转运蛋白（AbgT）和二价阴离子钠同向转运蛋白（DASS）家族，实际上有相同的一般结构的共同点（参考文献6），提供了新的途径，研究模式的底物结合和构象变化，在这两个蛋白质家族。

Membrane transport proteins are a large class of integral membrane proteins involved in the movement of small molecules across cellular membranes. Many of these proteins, known as secondary active transporters, use pre-existing substrate concentration gradients as an energy source for translocating another substrate against its concentration gradient. Every organism expresses dozens of different secondary transporter proteins with a diverse array of structural folds, and each protein is specific for a different substrate, which range from ions to neurotransmitters. A detailed understanding of the mechanism of each membrane transport protein requires knowledge of its three-dimensional structure in a number of different conformational states, as well as identification of the binding regions for the substrate or substrates. Studies from our group over the last year have provided insights into a number of biomedically important transporters, as detailed below. In 2014, we identified the structural fold of a secondary transporter responsible for sodium-coupled phosphate uptake in the kidney, called NaPi-IIa, by identifying an evolutionary relationship with a known structure of a sodium-coupled dicarboxylate transporter called VcINDY, which was then used as a template for homology modeling. Within that model we had predicted binding sites for two of the three required sodium ions (Na2 and Na3) and for the phosphate group (Fenollar-Ferrer et al, Biophysical Journal, 2014). However, the location of Na1, which is the first sodium ion to bind, was unresolved. We used our structural model, combined with biochemical and electrophysiological measurements from the Werner and Forster laboratories, to develop a detailed prediction for the residues that coordinate this sodium ion (ref. 1). These results provide an important step forward in identifying the molecular origins of sodium stoichiometry differences in NaPi-II homologs. Homology modeling was also used to generate a structural model of a homolog of vesicular monoamine transporters from Bacillobrevis brevis, BbMAT. A protein of unknown function, YajR, was used as a template for the homology modeling of BbMAT, allowing the identification of potentially interesting pathway-lining ionizable residues. Modification of those residues by our collaborators from the Schuldiner and Singh laboratories demonstrated that they play a key role in proton-dependence of transport, and providing important steps forward in understanding the mechanisms of neurotransmitter transport (ref. 2). A similar molecular modeling strategy was useful in identifying the structural location and behavior of glycosylation sites in a mammalian osmolyte transporter, BGT1, driving experimental studies by the Ziegler lab of the role of glycosylation in trafficking and plasma membrane insertion of this protein in the kidney (ref. 3). We have used structural modeling to explore the conformational mechanism of the oligopeptide symporter, PepT1, responsible for proton-driven uptake of peptides and drugs into the intestine, and a member of the largest group of secondary transporters, the major facilitator superfamily (MFS). Our collaborators in the Newstead laboratory have reported structures of PepT homologs from Shewanella oniedensis and Streptococcus thermopiles (PepTSo and PepTSt, respectively), in cytoplasm-facing conformations. Repeat-swap models based on these structures were outward-facing. These structural models were compared with the crystal structures, and with the results of molecular dynamics simulations, spectroscopic analysis, and functional assays carried out by the Newstead/Fowler groups. Together these results were used to derive a molecular mechanism for alternating access for MFS proteins that is more detailed than the simplistic "rocker-switch" or "rocking bundle" mechanisms proposed previously (ref. 4). Separately, we have made progress in exploring the diversity of conformational mechanisms in secondary transporters. Specifically, we have examined whether elevator-like conformational mechanisms, as described previously only for the aspartate transporter GltPh, are used by other transporter families with different structural folds. Using an updated - and more accurate - protocol for repeat-swap modeling, we predicted that the concentrative nucleoside transporter homolog from Vibrio cholerae, VcCNT also uses an elevator-type conformational mechanism (ref. 5). Finally, we used a systematic bioinformatic analysis to demonstrate that structures of two apparently unrelated families of transporters, the paminobenzoyl-glutamate transporter (AbgT) and the divalent anion sodium symporter (DASS) families, in fact have the same general architecture in common (ref. 6), providing novel avenues for studying the modes of substrate binding and conformational change in both these protein families.