Prediction of the 3D structure of FMRF-amide neuropeptides bound to the mouse MrgC11 GPCR and experimental validation
Prediction of the 3D structure of FMRF-amide neuropeptides bound to the mouse MrgC11 GPCR and experimental validation
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DOI：
10.1002/cbic.200700188
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发表时间：
2007-09-03
期刊：
影响因子：
3.2
通讯作者：
Goddard, William A., III
中科院分区：
文献类型：
--
作者：
Heo, Jiyoung;Han, Sang-Kyou;Goddard, William A., III
我们报道了用MembStruk计算程序预测小鼠mrgC11(MmmgC11)受体的三维结构，以及预测的F-M-R-F-NH2神经肽与四个单手性修饰配体的结合位置。我们预测，该四肽的R-F-NH2部分粘在跨膜结构域3、4、5和6之间的蛋白质中。Phe(F-NH2)与Tyr110(TM3)有良好的相互作用，而Arg则与Asp161(TM4)和Asp179(TMS)建立盐桥。我们预测Met从结合位点延伸，但末端Phe残基粘回到Tyr237、Leu238、Leu240和Tyr256(TM6)和Trp162(TM4)两侧的芳香/疏水位点。我们随后进行了突变实验，随后进行了细胞内钙释放试验，结果表明，根据我们的模型预测，Tyr110Als、Asp161Ala和Asp179Ala替换的活性显著降低。这些实验提供了强有力的证据，证明我们预测的G蛋白偶联受体(GPCR)结构足够准确，可以识别选择性配体的结合位点。对不与R-F-NH2二肽结合的mMRg-A1受体也进行了类似的研究，我们解释这是由于mMRgA1结合口袋的疏水性增加所致。
We report the 3D structure predicted for the mouse MrgC11 (mMrgC11) receptor by using the MembStruk computational protocol, and the predicted binding site for the F-M-R-F-NH2 neuro-peptide together with four singly chirally modified ligands. We predicted that the R-F-NH2 part of the tetrapeptide sticks down into the protein between the transmembrane (TM) domains 3, 4, 5, and 6. The Phe (F-NH2) interacted favorably with Tyr110 (TM3) while the Arg makes salt bridges to Asp161 (TM4) and Asp179 (TMS). We predicted that the Met extends from the binding site, but the terminal Phe residue sticks back into an aromatic/hydrophobic site flanked by Tyr237, Leu238, Leu240, and Tyr256 (TM6), and Trp162 (TM4). We carried out subsequent mutagenesis experiments followed by intracellular calcium-release assays that demonstrated the dramatic decrease in activity for the Tyr110Als, Asp161Ala, and Asp179Ala substitutions, which was predicted by our model. These experiments provide strong evidence that our predicted G protein-coupled receptor (GPCR) structure is sufficiently accurate to identify binding sites for selective ligands. Similar studies were made with the mMrg-A1 receptor, which did not bind the R-F-NH2 dipeptide, we explain this to be due to the increased hydrophobic character of the binding pocket in mMrgA1.