Vergara-Jaque Ariela, Fenollar-Ferrer Cristina, Kaufmann Desirée, Forrest Lucy R
Computational Structural Biology Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke - National Institutes of Health, Bethesda, MD USA.
Front Pharmacol. 2015 Sep 4;6:183. doi: 10.3389/fphar.2015.00183. eCollection 2015.
Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to one or other side of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (a)symmetry of these systems has been successfully used as a bioinformatic tool, called "repeat-swap modeling" to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeat-swap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that nucleoside transport also occurs via an elevator-like mechanism.
次级主动转运蛋白对于突触传递过程中神经递质的清除和再循环以及营养物质的摄取至关重要。这些蛋白质通过利用离子顺着预先存在的浓度梯度移动时释放的能量，介导溶质逆着其浓度梯度移动。为实现这一点，转运蛋白符合所谓的交替访问假说，即蛋白质采用至少两种构象，其中底物结合位点暴露于膜的一侧或另一侧，但不会同时暴露于两侧。神经元谷氨酸转运蛋白GltPh的细菌同源物在几种不同构象状态下的结构表明，该蛋白质结构在向外开放和向内开放状态下是不对称的，并且连接它们的构象变化涉及底物结合结构域跨膜的类似电梯的移动。结构不对称是由反向拓扑重复产生的，即具有相似整体折叠的结构重复，其跨膜拓扑通过围绕平行于膜平面的轴的双重伪对称相互关联。在大约四分之三的次级转运蛋白折叠中发现了反向重复。此外，这些系统的（非）对称性已成功用作一种生物信息学工具，称为“重复交换建模”，以使用互补构象中转运蛋白的已知结构作为模板来预测一种构象中转运蛋白的结构模型。在这里，我们描述了一种更新的重复交换同源建模方案，并使用已知向内和向外构象的GltPh校准该方法的准确性。然后，我们将这种重复交换同源建模程序应用于一种浓缩核苷转运蛋白VcCNT，其三维排列与GltPh相关。VcCNT的重复交换模型预测核苷转运也通过类似电梯的机制发生。
