Ligand-induced conformational changes in a SMALP-encapsulated GPCR
Ligand-induced conformational changes in a SMALP-encapsulated GPCR
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DOI：
10.1016/j.bbamem.2020.183235
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发表时间：
2020-06-01
影响因子：
3.4
通讯作者：
Wheatley, Mark
中科院分区：
文献类型：
--
作者：
Routledge, Sarah J.;Jamshad, Mohammed;Wheatley, Mark
腺苷2A受体（A（2A）R），G-蛋白偶联受体（GPCR），溶解和纯化封装在苯乙烯马来酸脂质颗粒（SMALP）。纯化的A（2A）R-SMALP与用于其表达的宿主巴斯德毕赤酵母（Pichia pastoris）的质膜的磷脂特征结合，证实A（2A）R-SMALP包封天然脂质。A（2A）R-SMALP的荧光光谱在330 nm处显示由内源性Trp残基产生的特征性宽发射峰。反向激动剂ZM 241385引起荧光发射增加30%，不寻常地伴随着发射波长的红移。发射光谱还在321 nm、335 nm和350 nm处显示出亚峰，表明在加入ZM 241385后，单个Trp存在于不同的环境中。激动剂NECA对A（2A）R-SMALP荧光光谱无影响。用Tyr取代两个Trp残基表明，ZM 241385影响了TM 6中Trp 246（6.48）和TM 7细胞外表面Trp 268（7.33）的环境和迁移率，导致向更疏水环境的转变。将荧光部分IAEDANS位点特异性引入TM 6的细胞内末端（残基231（6.33）），以报告A（2A）R的动态细胞质面。反向激动剂ZM 241385引起浓度依赖性的荧光发射增加，因为IAEDANS移动到更疏水的环境，与关闭G蛋白结合裂缝一致。NECA仅产生ZM 241385的30%的效果。这项研究提供了对SMALP环境的深入了解;封装支持A（2A）R和ZM 241385诱导的构象转变的组成型活性，但激动剂NECA仅产生很小的影响。
The adenosine 2A receptor (A(2A)R), a G-protein-coupled receptor (GPCR), was solubilised and purified encapsulated in styrene maleic acid lipid particles (SMALPs). The purified A(2A)R-SMALP was associated with phospholipids characteristic of the plasma membrane of Pichia pastoris, the host used for its expression, confirming that the A(2A)R-SMALP encapsulated native lipids. The fluorescence spectrum of the A(2A)R-SMALP showed a characteristic broad emission peak at 330 nm, produced by endogenous Trp residues. The inverse agonist ZM241385 caused 30% increase in fluorescence emission, unusually accompanied by a red-shift in the emission wavelength. The emission spectrum also showed sub-peaks at 321 nm, 335 nm and 350 nm, indicating that individual Trp inhabited different environments following ZM241385 addition. There was no effect of the agonist NECA on the A(2A)R-SMALP fluorescence spectrum. Substitution of two Trp residues by Tyr suggested that ZM241385 affected the environment and mobility of Trp246(6.48) in TM6 and Trp268(7.33) at the extracellular face of TM7, causing transition to a more hydrophobic environment. The fluorescent moiety IAEDANS was site-specifically introduced at the intracellular end of TM6 (residue 231(6.33)) to report on the dynamic cytoplasmic face of the A(2A)R. The inverse agonist ZM241385 caused a concentration-dependent increase in fluorescence emission as the IAEDANS moved to a more hydrophobic environment, consistent with closing the G-protein binding crevice. NECA generated only 30% of the effect of ZM241385. This study provides insight into the SMALP environment; encapsulation supported constitutive activity of the A(2A)R and ZM241385-induced conformational transitions but the agonist NECA generated only small effects.