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Ligand Modulation of the Conformational Dynamics of the A2A Adenosine Receptor Revealed by Single-Molecule Fluorescence
bioRxiv - Biophysics Pub Date : 2020-09-20 , DOI: 10.1101/2020.09.20.305425
Dennis D. Fernandes , Chris Neale , Gregory-Neal W. Gomes , Yuchong Li , Aimen Malik , Aditya Pandey , Alexander Orazietti , Xudong Wang , Libin Ye , R. Scott Prosser , Claudiu C. Gradinaru

G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra- and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A Receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Forster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 229 on TM6 was measured using Fluorescence Correlation Spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150-350 ns), intermediate (50-60 μs) and slow (200-300 μs) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y288, and Y290) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 229 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.

中文翻译:

配体调制的单分子荧光揭示的A2A腺苷受体的构象动力学。

G蛋白偶联受体(GPCR)是跨膜蛋白中最大的一类,使其成为治疗的重要靶标。这些受体的激活由正构配体调节,该配体稳定了复杂构象系综中的一个或多个状态。但是,关于州内和州际动态的文献不多。在这里,我们使用单分子荧光在纳秒至毫秒的时间尺度上测量腺苷A2A受体(A2AR)的配体调制构象动力学。实验是在无配体(apo)状态或与反向,部分或全部激动剂配体结合时,用去污剂纯化的A2R进行的。在洗涤剂溶解的A2AR上进行单分子Forster共振能量转移(smFRET),以通过跨膜(TM)螺旋4和6之间的分离来解析活性和非活性状态。smFRET分布的配体依赖性变化与构象选择一致状态间交换寿命≥3 ms。使用荧光相关光谱法(FCS)测量了TM6残基229周围的局部构象动力学,该结构捕获了由于共价连接的染料与近端芳香族残基之间的光诱导电子转移(PET)而引起的动态猝灭。对PET-FCS数据的全局分析显示,A2AR的构象动力学快(150-350 ns),中等(50-60μs)和慢(200-300μs),其寿命和幅度受配体和G蛋白模拟物(迷你G)。最为显着地,激动剂的结合和与mini-G的结合加速并增加了亚微秒相的相对贡献。分子动力学模拟确定了三个酪氨酸残基(Y112,Y288和Y290)是PET-FCS观察到的动态淬灭的原因,并揭示了TM6残基229附近的相关螺旋运动。这项研究提供了A2AR中构象动力学的定量描述,并支持了配体不仅偏向GPCR构象而且偏向于受体不同构象状态之内和之间的动力学的想法。和Y290)负责PET-FCS观察到的动态淬灭,并揭示了TM6残基229附近的相关螺旋运动。这项研究提供了A2AR中构象动力学的定量描述,并支持了配体不仅偏向GPCR构象而且偏向于受体不同构象状态之内和之间的动力学的想法。和Y290)负责PET-FCS观察到的动态淬灭,并揭示了TM6残基229附近的相关螺旋运动。这项研究提供了A2AR中构象动力学的定量描述,并支持了配体不仅偏向GPCR构象而且偏向于受体不同构象状态之内和之间的动力学的想法。
更新日期:2020-09-21
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