Center for Structural Biology, Vanderbilt University, Nashville, TN, 37240, USA.
Department of Chemistry, Vanderbilt University, Nashville, TN, 37235, USA.
J Biomol NMR. 2019 Jul;73(6-7):347-364. doi: 10.1007/s10858-019-00257-1. Epub 2019 Jun 26.
The translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is a membrane protein located on the outer mitochondrial membrane. Experimentally-derived structures of mouse TSPO (mTSPO) and its homologs from bacterial species have been determined by NMR spectroscopy and X-ray crystallography, respectively. These structures and ligand interactions within the TSPO binding pocket display distinct differences. Here, we leverage experimental and computational studies to derive a unified structural model of mTSPO in the presence and absence of the TSPO ligand, PK11195, and study the effects of DPC detergent micelles on the TSPO structure and ligand binding. From this work, we conclude that that the lipid-mimetic system used to solubilize mTSPO for NMR studies thermodynamically destabilizes the protein, introduces structural perturbations, and alters the characteristics of ligand binding. Furthermore, we used Rosetta to construct a unified mTSPO model that reconciles deviating features of the mammalian and bacterial TSPO. These deviating features are likely a consequence of the detergent system used for structure determination of mTSPO by NMR. The unified mTSPO model agrees with available experimental NMR data, appears to be physically realistic (i.e. thermodynamically not frustrated as judged by the Rosetta energy function), and simultaneously shares the structural features observed in sequence-conserved regions of the bacterial proteins. Finally, we identified the binding site for an imaging ligand VUIIS8310 that is currently positioned for clinical translation using NMR spectroscopy and propose a computational model of the VUIIS8310-mTSPO complex.
转位蛋白（TSPO），以前称为外周苯二氮䓬受体（PBR），是一种位于线粒体外膜的膜蛋白。通过 NMR 光谱和 X 射线晶体学分别确定了实验衍生的小鼠 TSPO（mTSPO）及其来自细菌物种的同源物的结构。这些结构和 TSPO 结合口袋内的配体相互作用显示出明显的差异。在这里，我们利用实验和计算研究来推导出存在和不存在 TSPO 配体 PK11195 时 mTSPO 的统一结构模型，并研究 DPC 去污剂胶束对 TSPO 结构和配体结合的影响。从这项工作中，我们得出结论，用于 NMR 研究的溶解 mTSPO 的脂质模拟系统在热力学上使蛋白质不稳定，引入结构扰动，并改变配体结合的特性。此外，我们使用 Rosetta 构建了一个统一的 mTSPO 模型，该模型协调了哺乳动物和细菌 TSPO 的差异特征。这些差异特征可能是由于 NMR 确定 mTSPO 结构时使用的去污剂系统所致。统一的 mTSPO 模型与现有的实验 NMR 数据一致，似乎具有物理现实性（即根据 Rosetta 能量函数判断，热力学上不会受阻），并且同时共享在细菌蛋白序列保守区域观察到的结构特征。最后，我们确定了目前正在使用 NMR 光谱学进行临床转化的成像配体 VUIIS8310 的结合位点，并提出了 VUIIS8310-mTSPO 复合物的计算模型。
