Computational studies of membrane transport proteins

次级活性转运蛋白是一类膜蛋白，它利用预先存在的分子浓度梯度作为能量源来转运另一种底物，如营养物质或神经递质，以对抗其浓度梯度。这个过程需要蛋白质改变构象，从而暴露出一条通往膜一侧或另一侧底物结合位点(S)的途径，这一循环被称为交替访问。每个生物体都表达几十种不同的次级转运蛋白，这些蛋白表现出一套不同的结构，尽管它们总是具有某种形式的内部结构对称性。在过去的十年中，人们从三维结构中获得了前所未有的开创性见解。然而，要详细了解每种膜转运蛋白的机制，需要了解其在更多构象状态下的结构，包括识别底物或底物的结合区。此外，这些结构需要被置于由动能障碍隔开的热力学景观上的动态集合的背景下。在过去的一年里，我们小组的研究继续研究许多膜蛋白中的这些问题。 转运蛋白和其他膜蛋白可以影响其周围膜的形态，这一点越来越被认识到。不太被认识到的是，这些变形的程度和自由能成本可能在蛋白质的不同功能状态之间有所不同，因此，它们可能对定义其机制做出重大贡献。我们考虑了三聚体钠-天冬氨酸转运体GltPh，它是一类重要的神经元转运体EAATs的同系物，其机制导致了已知最剧烈的结构变化之一。在与Faraldo-Gomez实验室(NHLBI)的合作下，我们对GltPh进行了分子模拟，结果表明，当原型变得向内时，它们会引起深的、远程的、但又相互独立的膜变形。使用一种新的模拟方法，我们估计这种膜扰动的自由能成本是巨大的，这提出了关于膜在神经元谷氨酸摄取中的作用的重要新问题，特别是在拥挤的环境中，如视网膜EAAT蛋白(参考文献)。1)。 膜相互作用对水溶性蛋白质也是至关重要的，例如，通过钙依赖的静电与带电脂质的相互作用。在与Ward实验室(NINDS)的合作中，我们使用结构建模来预测Annexin A11的羧基末端结构域中的钙结合位置和所产生的静电电位的变化(参考文献。2)，这是一种与神经退行性疾病额颞部痴呆有关的蛋白质。膜联蛋白A11的氨基末端片段与RNA颗粒有关，其围绕神经元的长距离运动机制尚不清楚。这些结构模型提供了一个可测试的机制预测，即通过膜联蛋白A11与溶酶体进行钙依赖的结合，这一过程将使RNA颗粒能够搭便车到局部转录位点。 对于许多转运蛋白和其他膜蛋白，如通道，近年来显示了前所未有的大量结构数据，部分原因是使用冷冻电子显微镜进行的研究。一个特别令人感兴趣的例子是离子通道的大而多样的瞬时受体电位(Trp)家族，其中100多个结构在短短几年内已被报道。这些过多的数据需要一种系统的方法来分析共同的特征，如途径和结合位点。我们之前已经开发了结构比对程序，可以对大量的膜蛋白结构进行比较。在与斯沃茨实验室(NINDS)的合作下，我们针对Trp通道结构的具体情况调整了这些程序，从而能够进行全面、系统的调查，从而产生多种可测试的假说(参考文献3)，为系统分析已有大量结构的膜蛋白家族奠定了基础。 综上所述，我们今年的出版物反映了与实验实验室密切合作利用计算方法的持续努力，并推动了对神经过程中生物医学重要蛋白质机制的理解，包括转运体、通道和其他膜相关蛋白质。

Secondary active transporters are a class of membrane proteins that utilize pre-existing molecular concentration gradients as an energy source for translocating another substrate, such as a nutrient or a neurotransmitter, against its concentration gradient. This process requires the protein to change conformations so as to expose a pathway to the substrate binding site(s) on one or other side of the membrane, in a cycle known as alternating access. Every organism expresses dozens of different secondary transporter proteins, and these exhibit a diverse set of architectures, albeit always with some form of internal structural symmetry. Unprecedented, ground-breaking insights have been garnered from three-dimensional structures obtained in the last decade. Nevertheless, a detailed understanding of the mechanism of each membrane transport protein requires knowledge of its structure in many more conformational states, including identification of the binding regions for the substrate or substrates. Moreover, those structures need to be placed into a context of dynamic ensembles on a thermodynamic landscape separated by kinetic barriers. Studies from our group over the last year have continued to investigate these issues in many membrane proteins. That transporters and other membrane proteins can influence the morphology of their surrounding membrane is increasingly recognized. Less appreciated is that the extent and free-energy cost of these deformations likely varies among different functional states of a protein, and thus, that they might contribute significantly to defining its mechanism. We considered the trimeric sodium-aspartate symporter GltPh, a homolog of an important class of neuronal transporters, the EAATs, whose mechanism entails one of the most drastic structural changes known. In collaboration with the Faraldo-Gomez lab (NHLBI), we carried out molecular simulations of GltPh which indicated that when the protomers become inward-facing, they cause deep, long-ranged, and yet mutually-independent membrane deformations. Using a novel simulation methodology, we estimated that the free-energy cost of this membrane perturbation is substantial, raising important new questions about the role of the membrane in neuronal glutamate uptake, especially in crowded environments such as that experienced by retinal EAAT proteins (Ref. 1). Membrane interactions are also critical for water-soluble proteins, for example, through calcium-dependent electrostatic interactions with charged lipids. In collaboration with the Ward lab (NINDS), we used structural modeling to predict calcium binding sites and resultant changes in electrostatic potential in the carboxy-terminal domain of annexin A11 (Ref. 2), a protein implicated in the neurodegenerative disease Frontotemporal Dementia. The amino-terminal segment of annexin A11, is associated with RNA granules, whose mechanism of long-distance movement around neurons remained to be identified. The structural models provided a testable mechanistic prediction of calcium-dependent attachment with lysosomes through annexin A11, a process that would enable to RNA granules to hitchhike to local sites of transcription. For many transporters and other membrane proteins such as channels, recent years have shown an unprecedented amount of structural data, in part due to studies using cryo-electron microscopy. One case of particular interest is the large and diverse transient receptor potential (TRP) family of ion channels, of which over one hundred structures have been reported in just a few years. This plethora of data requires a systematic approach to enable analysis of common features such as pathways and binding sites. We have previously developed structure alignment procedures that allow comparison of large numbers of membrane protein structures. In collaboration with the Swartz lab (NINDS), we adapted these procedures for the specific case of TRP channel structures, enabling a comprehensive, systematic survey that led to multiple, testable hypotheses (Ref. 3), and laid the groundwork for systematic analyses of other membrane protein families for which large numbers of structures become available. In summary, our publications this year reflect ongoing efforts to utilize computational approaches in close collaboration with experimental laboratories, and drive understanding of the mechanism of biomedically-important proteins in neuronal processes, including transporters, channels and other membrane-associated proteins.