The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the human ACE2 receptor and to facilitate virus entry, which can occur through low-pH-endosomal pathways. To understand how ACE2 binding and low pH affect spike conformation, we determined cryo-electron microscopy structures—at serological and endosomal pH—delineating spike recognition of up to three ACE2 molecules. RBDs freely adopted “up” conformations required for ACE2 interaction, primarily through RBD movement combined with smaller alterations in neighboring domains. In the absence of ACE2, single-RBD-up conformations dominated at pH 5.5, resolving into a solitary all-down conformation at lower pH. Notably, a pH-dependent refolding region (residues 824–858) at the spike-interdomain interface displayed dramatic structural rearrangements and mediated RBD positioning through coordinated movements of the entire trimer apex. These structures provide a foundation for understanding prefusion-spike mechanics governing endosomal entry; we suggest that the low pH all-down conformation potentially facilitates immune evasion from RBD-up binding antibody.

SARS-CoV-2 刺突利用移动受体结合域 (RBD) 与人类 ACE2 受体结合并促进病毒进入，这可以通过低 pH 内体途径发生。为了了解 ACE2 结合和低 pH 值如何影响尖峰构象，我们在血清学和内体 pH 条件下确定了冷冻电子显微镜结构，描绘了最多三个 ACE2 分子的尖峰识别。 RBD 自由地采用 ACE2 相互作用所需的“向上”构象，主要是通过 RBD 运动与相邻域中较小的改变相结合。在没有 ACE2 的情况下，单 RBD 向上构象在 pH 5.5 时占主导地位，在较低 pH 下分解为单独的全向下构象。值得注意的是，刺突域间界面处的 pH 依赖性重折叠区域（残基 824-858）显示出显着的结构重排，并通过整个三聚体顶端的协调运动介导 RBD 定位。这些结构为理解控制内体进入的预融合尖峰机制提供了基础；我们认为低 pH 值全向下构象可能有助于 RBD-up 结合抗体的免疫逃避。