Identification of the hydrophobic ligand binding pocket of the S1P1 receptor
Identification of the hydrophobic ligand binding pocket of the S1P1 receptor
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DOI：
10.1074/jbc.m609648200
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发表时间：
2007-01-26
影响因子：
4.8
通讯作者：
Tigyi, Gabor
中科院分区：
文献类型：
--
作者：
Fujiwara, Yuko;Osborne, Daniel A.;Tigyi, Gabor
1-磷酸鞘氨醇(S1P)是一种天然存在的鞘磷脂介体，也是几乎所有细胞类型中具有生长因子样作用的第二信使，是内皮分化基因家族中五个G蛋白偶联受体(GPCRs)的内源性配体。GPCR晶体结构的缺乏严重限制了合理的药物设计和电子计算机寻找亚型选择性配体。在这里，我们报告了S1P(1)GPCR围绕S1P脂肪部分的配体结合口袋的计算模型的实验验证。广泛的基于突变的验证证实了位于疏水配体结合口袋中的18个残基，与之前验证的三个头部基团相互作用的残基相结合，现在完成了S1P配体识别位点的定位。我们鉴定了6个突变体(L3.43G/L3.44G、L3.43E/L3.44E、L5.52A、F5.48G、V6.40L和F6.44G)，它们保持了野生型[P-32]S1P的结合，但S1P取消了配体依赖的激活。这些数据表明，这些氨基酸在S1P(1)向其激活状态的构象转变中发挥了作用。三个芳香族突变(F5.48Y、F6.44G和W6.48A)导致S1P或SEW2871的差异激活，表明两种激动剂之间的结构差异可以部分补偿氨基酸侧链的差异。现在验证的配体结合口袋为我们提供了一个药效团模型，用于NCI、美国国立卫生研究院、发展治疗化学文库的电子筛选，从而鉴定出两个新的S1P(1)的非脂类激动剂。
Sphingosine 1-phosphate (S1P), a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type, is an endogenous ligand of five G protein-coupled receptors (GPCRs) in the endothelial differentiation gene family. The lack of GPCR crystal structures sets serious limitations to rational drug design and in silico searches for subtype-selective ligands. Here we report on the experimental validation of a computational model of the ligand binding pocket of the S1P(1) GPCR surrounding the aliphatic portion of S1P. The extensive mutagenesis-based validation confirmed 18 residues lining the hydrophobic ligand binding pocket, which, combined with the previously validated three head group-interacting residues, now complete the mapping of the S1P ligand recognition site. We identified six mutants (L3.43G/L3.44G, L3.43E/L3.44E, L5.52A, F5.48G, V6.40L, and F6.44G) that maintained wild type [P-32]S1P binding with abolished ligand-dependent activation by S1P. These data suggest a role for these amino acids in the conformational transition of S1P(1) to its activated state. Three aromatic mutations (F5.48Y, F6.44G, and W6.48A) result in differential activation, by S1P or SEW2871, indicating that structural differences between the two agonists can partially compensate for differences in the amino acid side chain. The now validated ligand binding pocket provided us with a pharmacophore model, which was used for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics chemical library, leading to the identification of two novel nonlipid agonists of S1P(1).