Constitutively active mutants of the alpha 1B‐adrenergic receptor: role of highly conserved polar amino acids in receptor activation.
Constitutively active mutants of the alpha 1B‐adrenergic receptor: role of highly conserved polar amino acids in receptor activation.
复制标题
α1B-肾上腺素能受体的组成型活性突变体：高度保守的极性氨基酸在受体激活中的作用。
作者：
Alexander Scheer;Francesca Fanelli;Tommaso Costa;P. G. D. Benedetti;Susanna Cotecchia
将 α1B 肾上腺素受体 (AR) 的定点诱变和分子动力学模拟相结合，探索与从 R（非活性状态）到 R（活性状态）转变相关的潜在分子变化。使用分子动力学分析，我们将组成型活性突变体的结构/动力学特征与野生型和非活性α1B-AR的结构/动力学特征进行比较，以建立定义R和R的基本特征的理论模型。定点诱变的结果与支持以下假设的模型的预测惊人地一致。 (i) R 和 R 之间的平衡分别取决于 DRY 基序 D142 的去质子化和质子化形式之间的平衡。事实上，用丙氨酸替代 D142 赋予 α1B-AR 高组成活性。 (ii) DRY 序列的 R143 从 N63、D91、N344 和 Y348 形成的保守“极性口袋”中移出是所有活性结构的共同特征，表明 R143 的作用对于介导受体激活至关重要。通过用丙氨酸取代 N63 来破坏这些分子内相互作用，从而持续激活 α1B-AR。我们的发现可能为 G 蛋白偶联受体的激活过程提供有趣的一般性。
Site‐directed mutagenesis and molecular dynamics simulations of the alpha 1B‐adrenergic receptor (AR) were combined to explore the potential molecular changes correlated with the transition from R (inactive state) to R (active state). Using molecular dynamics analysis we compared the structural/dynamic features of constitutively active mutants with those of the wild type and of an inactive alpha 1B‐AR to build a theoretical model which defines the essential features of R and R. The results of site‐directed mutagenesis were in striking agreement with the predictions of the model supporting the following hypothesis. (i) The equilibrium between R and R depends on the equilibrium between the deprotonated and protonated forms, respectively, of D142 of the DRY motif. In fact, replacement of D142 with alanine confers high constitutive activity to the alpha 1B‐AR. (ii) The shift of R143 of the DRY sequence out of a conserved ‘polar pocket’ formed by N63, D91, N344 and Y348 is a feature common to all the active structures, suggesting that the role of R143 is fundamental for mediating receptor activation. Disruption of these intramolecular interactions by replacing N63 with alanine constitutively activates the alpha 1B‐AR. Our findings might provide interesting generalities about the activation process of G protein‐coupled receptors.