<p>Underlying data for S6A–S6F Fig.</p>

Sphingosine-1-phosphate (S1P), a key metabolite of sphingolipids, plays crucial roles in a wide range of physiological and pathological processes. S1P primarily exerts its functions by binding to G protein-coupled sphingosine-1-phosphate receptors (S1PRs), which comprise five subtypes (S1PR1-5) in humans, thereby activating these receptors and their downstream signaling pathways. Understanding the molecular determinants that govern agonist selectivity among different S1PR subtypes is vital for the rational and precise development of targeted therapeutic agents. Here, four cryo-electron microscopy structures of agonist-bound S1PR1-Gi1 complexes are reported. Through an integrated approach combining structural analysis, molecular dynamics simulations, and pharmacological assays, the molecular basis for the selectivity of CYM5442, HY-X-1011, Ponesimod, and SAR247799 toward S1PR1 over S1PR2-S1PR5 is uncovered. Nonconserved residues within the ligand-binding pocket and at the Gi1-protein interface contribute to S1PR1 selectivity by these agonists. A distinct agonist binding orientation toward transmembrane helices 5–7, combined with branched substituents that increase the agonist’s molecular width, results in steric clashes with residues in S1PR3. Additionally, branched moieties located at the tail portions of the agonist restrict its deep insertion into the binding pocket of both S1PR3 and S1PR5. These structural features collectively enhance selectivity for S1PR1 over S1PR3 and S1PR5. Furthermore, polar interactions with conserved polar residues in the top region of the binding pocket also influence agonist selectivity. Besides, the relatively broad molecular width of the agonist sterically hinders its binding into S1PR2 and S1PR4 pocket by nonconserved residue pairs bearing bulky side chains. These findings establish a structural framework for the rational design of next-generation S1PR1 highly selective agonists with improved therapeutic potential.

鞘氨醇-1-磷酸（Sphingosine-1-phosphate, S1P）是鞘脂类的关键代谢物，广泛参与众多生理及病理过程。S1P主要通过结合G蛋白偶联鞘氨醇-1-磷酸受体（G protein-coupled sphingosine-1-phosphate receptors, S1PRs）发挥功能，人类体内该受体家族包含5种亚型（S1PR1~S1PR5），结合后可激活受体及其下游信号通路。阐明调控不同S1PR亚型间激动剂选择性的分子决定机制，对于合理精准开发靶向治疗药物至关重要。本研究报道了4种激动剂结合状态下S1PR1-Gi1复合物的冷冻电子显微镜（cryo-electron microscopy）结构。本研究通过整合结构分析、分子动力学模拟（molecular dynamics simulations）与药理学实验的研究策略，阐明了CYM5442、HY-X-1011、Ponesimod及SAR247799相较于S1PR2~S1PR5对S1PR1的选择性分子基础。配体结合口袋与Gi1蛋白结合界面内的非保守残基，介导了上述四类激动剂对S1PR1的选择性偏好。激动剂针对跨膜螺旋5~7的独特结合取向，加之可增大激动剂分子宽度的分支取代基，会与S1PR3中的对应残基产生空间位阻冲突。此外，位于激动剂尾部区域的分支基团，会阻碍其深入嵌入S1PR3与S1PR5的配体结合口袋。上述结构特征共同提升了激动剂对S1PR1相较于S1PR3与S1PR5的选择性偏好。此外，与结合口袋顶部区域保守极性残基形成的极性相互作用，同样会影响激动剂的选择性偏好。不仅如此，激动剂相对较宽的分子宽度，结合携带庞大侧链的非保守残基对，共同产生空间位阻，阻碍其与S1PR2及S1PR4的结合口袋结合。本研究结果为合理设计下一代高选择性S1PR1激动剂、提升其治疗潜力提供了坚实的结构框架。