G protein–coupled olfactory receptors (ORs) enable us to detect innumerous odorants. They are also ectopically expressed in nonolfactory tissues and emerging as attractive drug targets. ORs can be promiscuous or highly specific, which is part of a larger mechanism for odor discrimination. Here, we demonstrate that the OR extracellular loop 2 (ECL2) plays critical roles in OR promiscuity and specificity. Using site-directed mutagenesis and molecular modeling, we constructed 3D OR models in which ECL2 forms a lid over the orthosteric pocket. We demonstrate using molecular dynamics simulations that ECL2 controls the shape and volume of the odorant-binding pocket, maintains the pocket hydrophobicity, and acts as a gatekeeper of odorant binding. Therefore, we propose the interplay between the specific orthosteric pocket and the variable, less specific ECL2 controls OR specificity and promiscuity. Furthermore, the 3D models created here enabled virtual screening of new OR agonists and antagonists, which exhibited a 70% hit rate in cell assays. Our approach can potentially be generalized to structure-based ligand screening for other G protein–coupled receptors that lack high-resolution 3D structures.

G 蛋白偶联嗅觉受体 (OR) 使我们能够检测到无数的气味。它们还在非嗅觉组织中异位表达，并成为有吸引力的药物靶标。OR 可以是混杂的或高度特异性的，这是更大的气味辨别机制的一部分。在这里，我们证明 OR 细胞外环 2 (ECL2) 在 OR 滥交和特异性中起关键作用。使用定点诱变和分子建模，我们构建了 3D OR 模型，其中 ECL2 在正构袋上形成一个盖子。我们使用分子动力学模拟证明 ECL2 控制气味结合袋的形状和体积，保持袋的疏水性，并充当气味结合的看门人。因此，我们提出了特定正畸口袋和变量之间的相互作用，不太具体的 ECL2 控制或特异性和滥交。此外，此处创建的 3D 模型能够虚拟筛选新的 OR 激动剂和拮抗剂，在细胞测定中显示出 70% 的命中率。我们的方法可以潜在地推广到基于结构的配体筛选，以筛选其他缺乏高分辨率 3D 结构的 G 蛋白偶联受体。