Department of Biological Sciences, University of Southern California, Los Angeles, Los Angeles, United States.
Moscow Institute of Physics and Technology, Dolgoprudny, Russia.
Elife. 2018 Jun 21;7:e34729. doi: 10.7554/eLife.34729.
Engineering of GPCR constructs with improved thermostability is a key for successful structural and biochemical studies of this transmembrane protein family, targeted by 40% of all therapeutic drugs. Here we introduce a comprehensive computational approach to effective prediction of stabilizing mutations in GPCRs, named CompoMug, which employs sequence-based analysis, structural information, and a derived machine learning predictor. Tested experimentally on the serotonin 5-HT receptor target, CompoMug predictions resulted in 10 new stabilizing mutations, with an apparent thermostability gain ~8.8°C for the best single mutation and ~13°C for a triple mutant. Binding of antagonists confers further stabilization for the triple mutant receptor, with total gains of ~21°C as compared to wild type apo 5-HT. The predicted mutations enabled crystallization and structure determination for the 5-HT receptor complexes in inactive and active-like states. While CompoMug already shows high 25% hit rate and utility in GPCR structural studies, further improvements are expected with accumulation of structural and mutation data.
工程化具有更好热稳定性的 GPCR 构建体是成功研究该跨膜蛋白家族的结构和生化特性的关键，该家族是 40%的治疗药物的靶点。在这里，我们引入了一种全面的计算方法来有效预测 GPCR 的稳定突变，称为 CompoMug，它结合了基于序列的分析、结构信息和衍生的机器学习预测器。在血清素 5-HT 受体靶标上进行了实验测试，CompoMug 预测产生了 10 种新的稳定突变，最佳单突变的表观热稳定性增益约为 8.8°C，三突变体的增益约为 13°C。拮抗剂的结合进一步赋予三突变体受体稳定性，与野生型 apo 5-HT 相比，总增益约为 21°C。预测的突变使 5-HT 受体复合物在非活性和类似活性状态下能够结晶和结构确定。虽然 CompoMug 在 GPCR 结构研究中已经显示出 25%的高命中率和实用性，但随着结构和突变数据的积累，预计还会有进一步的改进。
