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首页> 外文期刊>Biophysical Chemistry: An International Journal Devoted to the Physical Chemistry of Biological Phenomena >Molecular interaction of an antagonistic amylin analog with the extracellular domain of receptor activity-modifying protein 2 assessed by fluorescence polarization
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Molecular interaction of an antagonistic amylin analog with the extracellular domain of receptor activity-modifying protein 2 assessed by fluorescence polarization

机译：拮抗淀粉蛋白类似物与荧光极化评估的受体活性改性蛋白2细胞外结构域的分子相互作用

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The peptide hormone amylin receptor is a complex of the calcitonin receptor (CTR) and an accessory protein called receptor activity-modifying proteins (RAMPs). The soluble extracellular domain (ECD) of CTR is an important binding site of peptide hormone calcitonin. RAMPs also have an ECD and the association of CTR ECD with RAMP ECD enhances the affinity of peptide hormone amylin. However, the mechanism of how RAMP ECD association enhances amylin affinity remains elusive. Here, we report evidence supporting direct molecular interaction between an antagonistic amylin analog AC413 and RAMP2 ECD. We measured FITC-labeled peptide affinity for purified receptor ECD using fluorescence polarization (FP). We first found that RAMP2 ECD addition to maltose-binding protein (MBP)-tagged CTR ECD and an engineered MBP-tagged RAMP2 ECD-CTR ECD fusion protein (MBP-RAMP2-CTR ECD fusion) enhanced AC413 affinity. This suggests that these recombinant ECD systems represent functional amylin receptors. Interestingly, AC413 C-terminal residue Tyr25 (Y25) to Pro mutation eliminated its selective affinity for the MBP-RAMP2-CTR ECD fusion suggesting the critical role of the AC413 C-terminal residue in amylin receptor selectivity. Our structural model of the RAMP2 ECD:CTR ECD complex predicted molecular interaction of AC413 C-terminal residue Y25 with RAMP2 Glu101 (E101). Our FP peptide-binding assay showed that the RAMP2 E1 01A mutation of MBP-RAMP2-CTR ECD fusion decreased AC413 affinity by 7-fold, while the affinity of AC413 with the Y25P mutation was minimally changed. Consistently, AC413 binding affinity for the MBP-free RAMP2-CTR ECD fusion protein was also markedly decreased by the RAMP2 E101A mutation, while the affinity of AC413 with the Y25P mutation was moderately decreased. Together, our results support the molecular interaction between the AC413 C-terminal residue Y25 and RAMP2 E101 expanding our understanding of how the accessory protein RAMP2 enhances affinity of peptide hormone amylin for its receptor.
机译：肽激素淀粉蛋白受体是降钙素受体（CTR）的复合物和称为受体活性改性蛋白（斜坡）的辅助蛋白质。 CTR的可溶性细胞外结构域（ECD）是肽激素降钙素的重要结合位点。斜坡还具有ECD，CTR ECD与RAMP ECD的关联增强了肽激素淀粉蛋白的亲和力。然而，RAMP ECD协会如何增强氨纶亲和力的机制仍然难以捉摸。在这里，我们报告了支持拮抗淀粉蛋白模拟AC413和RAMP2 ECD之间的直接分子相互作用的证据。使用荧光极化（FP）测量用于纯化受体ECD的FITC标记的肽亲和力。我们首先发现ramp2 ECD添加到麦芽糖结合蛋白（MBP）-GGed CTR ECD和工程化MBP标记的RAMP2 ECD-CTR ECUC融合蛋白（MBP-RAMP2-CTR ECUC融合）增强了AC413亲和力。这表明这些重组ECD系统代表功能淀粉蛋白受体。有趣的是，AC413 C-末端残留TYR25（Y25）对PRO突变消除了对MBP-RAMP2-CTR ECD融合的选择性亲和力，表明AC413 C-末端残基在淀粉蛋白受体选择性中的关键作用。我们的ramp2 ECD的结构模型：CTR ECD复合物预测AC413 C末端残基Y25与RAMP2 GLU101（E101）的分子相互作用。我们的FP肽结合测定显示MBP-RAMP2-CTR ECD融合的斜坡2e1 01a突变通过7倍降低AC413亲和力，而AC413与Y25P突变的亲和力最小地变化。一致性地，通过斜坡2e101A突变也显着降低了对无MBP ramp2-CTR ECUC融合蛋白的AC413结合亲和力，而AC413与Y25P突变的亲和力是适度的降低。我们的结果支持AC413 C-末端残留Y25和RAMP2 E101之间的分子相互作用扩张我们对辅助蛋白斜面的理解如何增强肽激素淀粉蛋白的受体的亲和力。

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