Because of their prominent roles in development, cancer, and HIV, the chemokine receptor CXCR4 and its ligand CXCL12 have been the subject of numerous structural and functional studies, but the determinants of ligand binding, selectivity, and signaling are still poorly understood. Here, building on our latest structural model, we used a systematic mutagenesis strategy to dissect the functional anatomy of the CXCR4-CXCL12 complex. Key charge swap mutagenesis experiments provided evidence for pairwise interactions between oppositely charged residues in the receptor and chemokine, confirming the accuracy of the predicted orientation of the chemokine relative to the receptor and providing insight into ligand selectivity. Progressive deletion of N-terminal residues revealed an unexpected contribution of the receptor N terminus to chemokine signaling. This finding challenges a longstanding “two-site” hypothesis about the essential features of the receptor-chemokine interaction in which the N terminus contributes only to binding affinity. Our results suggest that although the interaction of the chemokine N terminus with the receptor-binding pocket is the key driver of signaling, the signaling amplitude depends on the extent to which the receptor N terminus binds the chemokine. Together with systematic characterization of other epitopes, these data enable us to propose an experimentally consistent structural model for how CXCL12 binds CXCR4 and initiates signal transmission through the receptor transmembrane domain.

由于趋化因子受体 CXCR4 及其配体 CXCL12 在发育、癌症和 HIV 中的突出作用，它们已成为众多结构和功能研究的主题，但配体结合、选择性和信号传导的决定因素仍知之甚少。在这里，基于我们最新的结构模型，我们使用系统诱变策略来剖析 CXCR4-CXCL12 复合物的功能解剖结构。关键的电荷交换诱变实验为受体和趋化因子中带相反电荷的残基之间的成对相互作用提供了证据，证实了趋化因子相对于受体的预测方向的准确性，并提供了对配体选择性的深入了解。N 端残基的逐步删除揭示了受体 N 端对趋化因子信号传导的意外贡献。这一发现挑战了长期以来关于受体-趋化因子相互作用的基本特征的“双位点”假说，其中 N 末端仅有助于结合亲和力。我们的结果表明，虽然趋化因子 N 末端与受体结合口袋的相互作用是信号传导的关键驱动因素，但信号传导幅度取决于受体 N 末端与趋化因子结合的程度。结合其他表位的系统表征，这些数据使我们能够提出一个实验一致的结构模型，用于解释 CXCL12 如何结合 CXCR4 并通过受体跨膜域启动信号传输。