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。每个膜转运蛋白的机制的详细了解需要在许多不同的构象状态和识别的底物或底物的结合区域的三维结构的知识。 我们基于对其蛋白质序列中重复元素的分析，并通过与已知蛋白质结构的比较，确定了一种负责肾脏钠偶联磷酸盐摄取的二级转运蛋白（称为NaPi-IIa）的新折叠（1）。我们确定了一个模板内的一个远亲相关的蛋白质的亚结构，并使用国家的最先进的序列比对策略和同源建模构建的三维模型的折叠NaPi-IIa。该模型进行了比较，其跨膜拓扑结构与现有的实验数据，发现极好的协议。然后，我们预测了三个必需钠离子中的两个和磷酸根基团的结合位点;通过在预测的NaPi-IIa结合位点的适当位置进行位点特异性突变，并通过电生理学和摄取测量（由苏黎世的Forster实验室进行），对这些预测进行了测试，为预测提供了强有力的支持（1）。 二级转运蛋白在转运过程中通过许多不同的构象状态循环，因此理解底物或共底物结合到哪些特定状态是至关重要的。我们的合作者（齐格勒实验室，MPI法兰克福/里根斯堡大学）解决了钠偶联甜菜碱转运蛋白（BetP）的结构，其结构折叠与神经递质：钠转运蛋白（NSS）相似-其中蛋白质明显采用不同的状态。在两种不同的外向构象中，可以清楚地区分底物（胆碱，在这种情况下）的存在和不存在，但钠的存在尚不清楚（2）。我们使用分子动力学模拟来表征这些状态的钠占据，发现胆碱结合和无胆碱的外向构象都可能被钠占据（2）。此外，结合位点表现出不同程度的溶剂化，表明在胆碱结合时以及在朝向封闭和闭合状态的构象变化时存在渐进的离子脱水机制（2）。 神经元突触中神经递质的水平由存在于神经元膜中的神经递质转运蛋白的量调节，神经递质转运蛋白的量又由与细胞质蛋白如突触融合蛋白1A的相互作用调节。例如，5-羟色胺转运蛋白（SERT）的5-羟色胺摄取是通过涉及其N-和C-末端结构域的相互作用来调节的，其结构难以使用晶体学来表征。我们进行了一项大规模的建模研究（3），以使用基于片段的结构预测技术Rosetta预测人SERT的（83个残基）N-末端结构域和（60个残基）C-末端结构域的折叠。具体来说，我们构建了每个末端结构域一百万个候选折叠，然后通过结构相似性对其进行聚类，并基于来自我们的合作者（Sitte实验室，维也纳大学）的FRET测量结果进行过滤。SERT末端结构域模型的二级结构组成与质子NMR测量结果（Konrat实验室，维也纳大学）以及N-末端结构域构建体的圆二色光谱（Konrat实验室，维也纳大学; Singh实验室，耶鲁大学）（3）一致。我们的结论是，SERT末端域包含结构化的元素，虽然有时分开的非结构化（本质上无序）的片段。我们将候选折叠与基于模板的跨膜结构域模型相结合，构建了SERT的全长模型，并分析了两个结构域在两种不同构象状态下的可能位置和分离。使用这些低分辨率全长模型，推导出SERT与调节蛋白相互作用的假设（3）。

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 and identification of the binding regions for the substrate or substrates. We have identified a novel fold for a secondary transporter responsible for sodium-coupled phosphate uptake in the kidney, called NaPi-IIa, based on an analysis of repeated elements in its protein sequence, and by comparison with structures of known proteins (1). We identified a template for NaPi-IIa within a substructure of a distantly-related protein, and used state-of-the-art sequence alignment strategies and homology modeling to construct a three-dimensional model of the fold of NaPi-IIa. The model was compared with available experimental data on its transmembrane topology, finding excellent agreement. We then predicted binding sites for two of the three required sodium ions and for the phosphate group; these predictions were tested by site-specific mutagenesis of judicious positions in the predicted binding site of NaPi-IIa that was mutated at judicious positions in the predicted sites, by electrophysiology and uptake measurements (carried out by the Forster lab, Zurich), providing strong support for the predictions (1). Secondary transporters cycle through a number of different conformational states during transport, and therefore it is critical to understand to which particular states the substrates or co-substrates are bound. Our collaborators (Ziegler lab, MPI Frankfurt/University Regensburg) solved structures of a sodium-coupled betaine transporter (BetP) whose structural fold is similar to the neurotransmitter:sodium transporters (NSS) - where the protein clearly adopts different states. In two distinct outward-facing conformations, the presence and absence of the substrate (choline, in this case) could be clearly distinguished, but the presence of sodium was unclear (2). We used molecular dynamics simulations to characterize the sodium occupancy of these states, finding that both choline-bound and choline-free outward-facing conformations are likely to be occupied by sodium (2). Moreover, the binding sites exhibited differing degrees of solvation, indicating a progressive ion dehydration mechanism upon choline binding, and upon the conformational change toward the occluded and closed states (2). The levels of neurotransmitter in neuronal synapses is regulated by the amount of neurotransmitter transporter that is present in the neuronal membrane, which in turn is modulated by interactions with cytoplasmic proteins such as syntaxin 1A. Serotonin uptake by serotonin transporter (SERT), for example, is regulated through interactions involving its N- and C-terminal domains, whose structures have been difficult to characterize using crystallography. We carried out a large-scale modeling study (3) to predict the folds of the (83-residue) N-terminal domain and the (60-residue) C-terminal domain of human SERT using the fragment-based structure prediction technique, Rosetta. Specifically, we constructed one million candidate folds per terminal domain, which were then clustered by structural similarity, and filtered based on results from FRET measurements from our collaborators (Sitte lab, University of Vienna). The secondary structure composition of the SERT terminal domain models agreed well with results from proton NMR measurements (Konrat lab, University of Vienna), as well as circular dichroism spectroscopy on constructs of the N-terminal domain (Konrat lab, University of Vienna; Singh lab, Yale University) (3). We conclude that the SERT terminal domains contain structured elements, albeit sometimes separated by unstructured (intrinsically disordered) segments. We combined candidate folds with template-based models of the transmembrane domains to construct full-length models of SERT and analyzed the probable locations and separation of the two domains in two different conformational states. Using these low-resolution, full-length models, hypotheses for the interactions of SERT with regulatory proteins were derived (3).