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The paper details structural biology research investigating the mechanism of SARS-CoV-2 entry and subsequent viral escape from the immune system. Utilizing cryo-electron microscopy, the researchers analysed the trimeric spike glycoprotein across a range of acidic conditions simulating the endosomal pathway. A critical finding reveals the existence of a pH-dependent switch domain that undergoes significant internal rearrangement and protonation as the pH decreases. This structural transition coordinates large inter-protomer domain movements, causing the receptor-binding domains (RBDs) to be locked into an all-down conformation. This retraction acts as a form of conformational masking, dramatically lowering the spike's affinity for neutralising antibodies like CR3022, thereby suggesting an important new mechanism of immune evasion.

PDB DOI: https://doi.org/10.2210/pdb7KMB/pdbEM Map

EMD-22922: EMDB EMDataResource

Classification: Hydrolase/Viral Protein

Organism(s): Homo sapiens, Severe acute respiratory syndrome coronavirus 2

Expression System: Homo sapiens

Membrane Protein: Yes

Deposited: 2020-11-02

Released: 2020-12-09

Deposition Author(s): Gorman, J., Kwong, P.D., Shapiro, L.

Experimental Data Snapshot

Method: ELECTRON MICROSCOPY

Resolution: 3.39 Å

Aggregation State: PARTICLE

Reconstruction Method: SINGLE PARTICLE

Introduction: The SARS-CoV-2 Spike Glycoprotein

• The SARS-CoV-2 Spike Glycoprotein is a trimeric protein responsible for mediating viral entry into host cells.

• The structure undergoes large-scale conformational changes between its prefusion and postfusion conformations to facilitate membrane fusion.

• The key finding of the study (Zhou et al., 2020) is the discovery of a pH-dependent switch that mediates the positioning of the Receptor-Binding Domains (RBDs).

• This mechanism is crucial for the virus's ability to enter cells through low-pH endosomal pathways and may facilitate immune evasion

Function and Biological Context

• Primary Function: The spike glycoprotein engages the human ACE2 receptor to facilitate virus entry.

• Entry Pathway: SARS-CoV-2 entry can occur through low-pH endosomal pathways.

• The Receptor-Binding Domains (RBDs) must adopt an "up" conformation to interact with the ACE2 receptor. The RBD can also exist in an "all-down" conformation, which is related to the epitope availability for RBD-directed antibodies.

Cryo-EM Structures and Conformational States

• Total Structures Determined: Twelve cryo-EM structures of the spike were determined, either alone or complexed with the ACE2 receptor, at both serological (pH 7.4) and endosomal (pH 5.5, 4.5, 4.0) pHs.

• Experimental Setup: The researchers focused on the prefusion-stabilized spike trimeric ectodomain ("2P").

• ACE2-Bound States (pH 7.4 & 5.5): At serological pH (7.4), spike bound ACE2 at stoichiometries of 1:1, 1:2, and 1:3. Similar stoichiometries were observed at endosomal pH 5.5 (1:1, 1:2, 1:3). ACE2 binding introduced trimer asymmetry.

• Ligand-Free States (Low pH):

   At pH 5.5, structural analysis revealed conformational heterogeneity, dominated by single-RBD-up conformations, with an all-RBD-down conformation appearing in 10% of particles.

   At pH 4.5 and pH 4.0, the spike adopted an almost exclusive all-RBD-down conformation. The pH 4.0 structure achieved a high resolution of 2.4 Å, allowing individual water molecules to be observed.

• RBD Movement: The large movement of the RBD (from down to up) required for ACE2 binding was accompanied by more subtle movements in neighboring domains.

The pH-Dependent Switch Region: Structural Details

• Identification: A critical pH-dependent refolding region (residues 824–858), named the "switch" region, was identified as mediating RBD positioning. This region displays dramatic structural rearrangements. • Location and Composition: The switch domain resides at the nexus of the SD1 and SD2 domains of one protomer, and the HR1 (in S2 subunit) and NTD domains of the neighboring protomer. It contains four aspartic acid residues (830, 839, 843, and 848) and a disulfide linkage (Cys840-Cys851).

• Conformational Segregation: The switch region segregates into two conformations based on the cryo-EM data: "unprotonated-switches" and "protonated-switches".

Mechanism of the pH-Dependent Switch

• Unprotonated State (Higher pH, single-RBD-up): The unprotonated switches (e.g., in protomers B and C) interact with the neighbouring protomer's SD2 domain to transmit lateral displacements of domains, allowing the RBD to move to the up position. In this state, the aspartic acids face the solvent and appear negatively charged.

• Protonated State (Lower pH, all-down): As the pH drops (e.g., to pH 4.0), the switch region protonates, undergoing a conformational change (the protonated-switch conformation). This change causes the switch to reorient and interact with the NTD domain (within the same protomer), thereby breaking the coordinated interprotomer interactions. This action locks the RBDs in the down position.

Relevance to Viral Entry and Immune Evasion

• Immune Evasion: The all-RBD-down conformation adopted at low pH suggests a potential mechanism for immune evasion from antibodies that only recognize the RBD-up state.

• Antibody Shedding Example: The affinity of the antibody CR3022 for the soluble spike protein was dramatically reduced at pH 4.5 (KD > 1,000 nM) compared to serological pH (0.49 nM). Since CR3022 still bound strongly to the isolated RBD, this reduction in binding to the spike trimer is attributed to the conformational constraints of the spike structure at low pH (the all-RBD-down state).

• Endosomal Positioning: The low-pH induced switch locks the RBDs down, causing the shedding of RBD-up-binding antibodies (like CR3022) as the virus moves through the endosome to the lysosome (pH 4.5).

Conclusion

The central conclusion drawn from the cryo-EM analysis is the identification and structural definition of a pH-dependent switch mechanism (residues 824–858) within the SARS-CoV-2 spike protein that dictates the positioning of its Receptor-Binding Domains (RBDs) during endosomal entry. While the spike is conformationally heterogeneous at pH 5.5, frequently adopting single-RBD-up states needed for ACE2 interaction, reducing the pH to 4.5 or 4.0 — mimicking the late endosome/early lysosome environment—causes the spike to resolve into an almost exclusive "all-RBD-down" conformation. This dramatic structural rearrangement is driven by the protonation of aspartic acid residues within the switch domain, which breaks the necessary interprotomer interactions, locking the RBDs in a masked, downward position. This low-pH, all-RBD-down state suggests a powerful means of immune evasion by conformationally masking the spike, severely reducing the apparent affinity of RBD-up-recognizing antibodies (like CR3022) as the virus traffics through the low-pH compartment.

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