Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli
Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli
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DOI：
10.1073/pnas.1317903111
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发表时间：
2014-02-11
影响因子：
11.1
通讯作者：
Plueckthun, Andreas
中科院分区：
文献类型：
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作者：
Egloff, Pascal;Hillenbrand, Matthias;Plueckthun, Andreas
结晶学促进了我们对G蛋白偶联受体的理解，但低表达水平和溶液中的不稳定性限制了对这个大蛋白家族中极少数选定成员的结构洞察。使用神经降压素受体1(NTR1)作为原理证明，我们最近开发的两种定向进化技术具有克服这些问题的潜力。我们从大肠杆菌中纯化了三个神经降压素结合的NTR1变异体，并通过气相扩散结晶实验确定了它们的X射线结构，分辨率高达2.75埃。结晶化的构建物具有药理学特征，表现出配体依赖的信号传递、内化和野生型激动剂和拮抗剂的亲和力。我们的结构与所有生物化学定义的配体接触残基完全一致，它们代表了胞液侧的非活性NTR1状态。它们与先前确定的配体结合口袋中的NTR1结构(蛋白质数据库ID代码4GRV)以及两亲性螺旋8的存在有显著差异。NTR1与其他结晶的G蛋白偶联受体之间的螺旋8稳定性决定因素的比较表明，在许多受体中，两亲性螺旋的正则位置的占有率在不同程度上减少了，我们已经阐明了稳定螺旋8的序列决定因素。我们的分析也为C末端棕榈酰化反应对G蛋白偶联受体信号转导、受体成熟和脱敏的影响提供了结构基础。
Crystallography has advanced our understanding of G protein-coupled receptors, but low expression levels and instability in solution have limited structural insights to very few selected members of this large protein family. Using neurotensin receptor 1 (NTR1) as a proof of principle, we show that two directed evolution technologies that we recently developed have the potential to overcome these problems. We purified three neurotensin-bound NTR1 variants from Escherichia coli and determined their X-ray structures at up to 2.75 angstrom resolution using vapor diffusion crystallization experiments. A crystallized construct was pharmacologically characterized and exhibited ligand-dependent signaling, internalization, and wild-type-like agonist and antagonist affinities. Our structures are fully consistent with all biochemically defined ligand-contacting residues, and they represent an inactive NTR1 state at the cytosolic side. They exhibit significant differences to a previously determined NTR1 structure (Protein Data Bank ID code 4GRV) in the ligand-binding pocket and by the presence of the amphipathic helix 8. A comparison of helix 8 stability determinants between NTR1 and other crystallized G protein-coupled receptors suggests that the occupancy of the canonical position of the amphipathic helix is reduced to various extents in many receptors, and we have elucidated the sequence determinants for a stable helix 8. Our analysis also provides a structural rationale for the long-known effects of C-terminal palmitoylation reactions on G protein-coupled receptor signaling, receptor maturation, and desensitization.