Cryo-EM Structures of SARS-CoV-2 Spike without and with ACE2 Reveal a pH-Dependent Switch to Mediate Endosomal Positioning of Receptor-Binding Domains

Abstract

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 impact spike conformation, we determined cryo-EM 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, cryo-EM structures revealed single-RBD-up conformations to dominate 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 findings provide insight into how receptor interactions and endosomal pH alter RBD positioning and potentially facilitate immune evasion from RBD-up binding antibody.

Paired heavy and light chain signatures contribute to potent SARS-CoV-2 neutralization in public antibody responses

“…Conversely, a sustained RBD 'up' also could make the virus more sensitive to neutralization, as the exposed 'up' RBD enhances exposure of vulnerable epitopes (Mansbach et al, 2020;Zhou et al, 2020c). We outlined differences in RBD display caused by the D614G mutation that enhance antibody class recognition of spike across a broad pH range, and we show that D614G had no detrimental impact on IGHV3-53/3-66 antibody class neutralization, which agrees with prior reports (Plante et al, 2020;Weisblum et al, 2020;Weissman et al, 2020).…”

“…Negative-staining electron microscopy revealed particles of 910-30 Fab bound to SARS-CoV-2 S2P at pH 5.5 ( Fig. 1B) (Zhou et al, 2020c), and also across a broader pH range of 4.0-7.4 ( Fig. S1).…”

“…We investigated class binding at three pH values related to known RBD 'up' versus 'down' states for the D614 and D614G mutational variants (Fig. 4A) (Benton et al, 2020;Wrapp et al, 2020b;Yurkovetskiy et al, 2020;Zhou et al, 2020c). dhACE2 competition ELISA assays at pH 5.5 and 4.5 showed that IGHV3-53/3-66 class members compete in a concentration-dependent manner with dimeric human ACE2 for binding to SARS-CoV-2 S2P spike, and to the D614G S2P spike ( Fig.…”

Structural Analysis of Neutralizing Epitopes of the SARS-CoV-2 Spike to Guide Therapy and Vaccine Design Strategies

“…Heparan sulfate has been suggested to interact with the RBD to enhance its open state for ACE2 binding to occur ( Figure 2 A) [ 24 ]. On the other hand, the closed state is favored at endosomal pH (~5.5) [ 25 ]. S interacts with its receptor with high affinity, with both the SARS-CoV-2 S RBD and trimer binding to monomeric ACE2 with low nanomolar dissociation constants (K D ) (subnanomolar in the case of dimeric ACE2) [ 15 , 22 ].…”

“…Thus, certain S mAbs and nanobodies might be useful for engineering bispecific antibodies against SARS-CoV-2 and other coronaviruses. Of note, it has been shown that at lower pH, representative of what is observed in the endosome, S is predominantly in the closed state [ 25 ]. As a result, mAbs that bind to a cryptic epitope or require the RBD to be in the up state would be less likely to bind and neutralize viruses fusing via the endosomal pathway.…”