Computational Studies of Membrane Transport Proteins

膜转运蛋白是一大类完整的膜蛋白，参与小分子跨细胞膜的运动。这些蛋白质中的许多被称为次级活性转运蛋白，它们利用预先存在的底物浓度梯度作为能量来源，相对于其浓度梯度转移另一种底物。每个生物体都表达数十种不同的次级转运蛋白，具有不同的结构折叠，每种蛋白都针对不同的底物，从离子到神经递质。要详细了解每种膜转运蛋白的机制，需要了解其在多种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。我们小组在过去一年的研究提供了对一些生物医学上重要的转运蛋白的见解，如下所述。 2014年，我们通过确定与钠偶联二元酸转运蛋白VcINDY的已知结构的进化关系，确定了一个负责肾脏摄取钠偶联磷酸盐的二级转运蛋白的结构折叠，称为Napi-IIa，然后将其用作同源建模的模板。在该模型中，我们预测了所需的三个钠离子中的两个(Na2和Na3)和磷酸基团的结合位置(Fenollar-Ferrer等人，生物物理杂志，2014年)。然而，Na1是第一个结合的钠离子，其位置尚未确定。我们使用我们的结构模型，结合来自Werner和Forster实验室的生化和电生理测量，来开发一个详细的预测，以配合该钠离子(参考。1)。这些结果为确定NaPI-II同系物中钠的化学计量比差异的分子来源提供了重要的一步。同源建模也被用来产生短杆菌BbMAT的囊泡单胺转运体的同系物的结构模型。功能未知的蛋白质YajR被用作BbMAT的同源建模的模板，从而能够识别潜在的有趣的途径内可电离残基。Schuldiner和Singh实验室的合作者对这些残基的修饰表明，它们在质子依赖的运输中发挥了关键作用，并为理解神经递质运输的机制向前迈出了重要的一步。2)。类似的分子建模策略在确定哺乳动物渗透压转运蛋白BGT1中糖基化位点的结构位置和行为方面很有用，推动了Ziegler实验室对糖基化在肾脏中这种蛋白质的运输和质膜插入中的作用的实验研究(参考文献.3)。 我们使用结构建模来探索寡肽转运体PepT1的构象机制，PepT1负责质子驱动的多肽和药物进入肠道的摄取，以及最大的次级转运体组的成员，主要的促进器超家族(MFS)。我们在Newstead实验室的合作者报告了来自尼氏希瓦氏杆菌和嗜热链球菌(分别为PepTSo和PepTST)的PepT同源物在面向细胞质的构象中的结构。基于这些结构的重复互换模型是面向外部的。这些结构模型与晶体结构进行了比较，并与Newstead/Fowler小组进行的分子动力学模拟、光谱分析和功能分析的结果进行了比较。总之，这些结果被用来推导出一种MFS蛋白质交替访问的分子机制，它比先前提出的简单的“摇杆开关”或“摇摆束”机制更详细(参考文献)。4)。另外，我们在探索次级转运蛋白构象机制的多样性方面也取得了进展。具体地说，我们研究了之前仅针对天冬氨酸转运蛋白GltPh所描述的电梯样构象机制是否被其他具有不同结构折叠的转运蛋白家族所使用。使用一种更新的-更准确的-重复交换建模的协议，我们预测来自霍乱弧菌VcCNT的浓缩核苷转运体同源基因也使用升降型构象机制(参考文献。5)。最后，我们使用系统的生物信息学分析来证明两个看似不相关的转运蛋白家族--对氨基苯甲酰谷氨酸转运蛋白(AbgT)和二价阴离子钠转运蛋白(DASS)家族的结构实际上具有相同的一般结构。6)，为研究这两个蛋白质家族的底物结合方式和构象变化提供了新的途径。

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.