Quantifying chemical substituent contributions to ligand-binding free energies is challenging due to nonadditive effects. Protein allostery is a frequent cause of nonadditivity, but the underlying allosteric mechanisms often remain elusive. Here, we propose a general NMR-based approach to elucidate such mechanisms and we apply it to the HCN4 ion channel, whose cAMP-binding domain is an archetypal conformational switch. Using NMR, we show that nonadditivity arises not only from concerted conformational transitions, but also from conformer-specific effects, such as steric frustration. Our results explain how affinity-reducing functional groups may lead to affinity gains if combined. Surprisingly, our approach also reveals that nonadditivity depends markedly on the receptor conformation. It is negligible for the inhibited state but highly significant for the active state, opening new opportunities to tune potency and agonism of allosteric effectors.

中文翻译：

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非加性取代基对蛋白质-配体结合的贡献的变构机制

由于非加性效应，量化化学取代基对配体结合自由能的贡献具有挑战性。蛋白质变构是非可加性的常见原因，但潜在的变构机制往往难以捉摸。在这里，我们提出了一种基于 NMR 的通用方法来阐明此类机制，并将其应用于 HCN4 离子通道，其 cAMP 结合域是原型构象开关。使用 NMR，我们表明非可加性不仅来自协调的构象转变，还来自构象异构体特定的效应，例如空间受阻。我们的结果解释了降低亲和力的官能团如何在组合时导致亲和力增加。令人惊讶的是，我们的方法还揭示了非可加性显着取决于受体构象。