Free energy perturbation calculations of mutation effects on SARS-CoV-2 RBD::ACE2 binding affinity (preprint)

The strength of binding between human angiotensin converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of viral spike protein plays a role in the transmissibility of the SARS-CoV-2 virus. In this study we focus on a subset of RBD mutations that have been frequently observed in sequenced samples from infected individuals and probe binding affinity changes to ACE2 using surface plasmon resonance (SPR) measurements and free energy perturbation (FEP) calculations. We find that FEP performance is significantly better than that of other computational approaches examined here, in part due to its ability to account for protein structure relaxation resulting from the mutation of interfacial residues. Moreover, analysis of FEP trajectories offers physical insights not available from other methods. Notably, FEP calculations successfully predict the observed cooperative stabilization of binding by the Q498R N501Y double mutant present in the Omicron variant and offer a physical explanation for the underlying mechanism. Our results furthermore suggest a strategy as to how to effectively deploy FEP methods in the optimization of neutralizing antibodies.

Neutralizing antibody 5-7 defines a distinct site of vulnerability in SARS-CoV-2 spike N-terminal domain (preprint)

Antibodies that potently neutralize SARS-CoV-2 target mainly the receptor-binding domain or the N-terminal domain (NTD). Over a dozen potently neutralizing NTD- directed antibodies have been studied structurally, and all target a single antigenic supersite in NTD (site 1). Here we report the 3.7 [A] resolution cryo-EM structure of a potent NTD-directed neutralizing antibody 5-7, which recognizes a site distinct from other potently neutralizing antibodies, inserting a binding loop into an exposed hydrophobic pocket between the two sheets of the NTD {beta}-sandwich. Interestingly, this pocket has been previously identified as the binding site for hydrophobic molecules including heme metabolites, but we observe their presence to not substantially impede 5-7 recognition. Mirroring its distinctive binding, antibody 5-7 retains a distinctive neutralization potency with variants of concern (VOC). Overall, we reveal a hydrophobic pocket in NTD proposed for immune evasion can actually be used by the immune system for recognition.

Potent SARS-CoV-2 Neutralizing Antibodies Directed Against Spike N-Terminal Domain Target a Single Supersite (preprint)

Numerous antibodies that neutralize SARS-CoV-2 have been identified, and these generally target either the receptor-binding domain (RBD) or the N-terminal domain (NTD) of the viral spike. While RBD-directed antibodies have been extensively studied, far less is known about NTD-directed antibodies. Here we report cryo-EM and crystal structures for seven potent NTD-directed neutralizing antibodies in complex with spike or isolated NTD. These structures defined several antibody classes, with at least one observed in multiple convalescent donors. The structures revealed all seven antibodies to target a common surface, bordered by glycans N17, N74, N122, and N149. This site - formed primarily by a mobile {beta}-hairpin and several flexible loops - was highly electropositive, located at the periphery of the spike, and the largest glycan-free surface of NTD facing away from the viral membrane. Thus, in contrast to neutralizing RBD-directed antibodies that recognize multiple non-overlapping epitopes, potent NTD-directed neutralizing antibodies target a single supersite.

Molecular Dynamics Analysis of a Flexible Loop at the Binding Interface of the SARS-CoV-2 Spike Protein Receptor-Binding Domain (preprint)

Since the identification of the SARS-CoV-2 virus as the causative agent of the current COVID-19 pandemic, considerable effort has been spent characterizing the interaction between the Spike protein receptor-binding domain (RBD) and the human angiotensin converting enzyme 2 (ACE2) receptor. This has provided a detailed picture of the end point structure of the RBD-ACE2 binding event, but what remains to be elucidated is the conformation and dynamics of the RBD prior to its interaction with ACE2. In this work we utilize molecular dynamics simulations to probe the flexibility and conformational ensemble of the unbound state of the receptor-binding domain from SARS-CoV-2 and SARS-CoV. We have found that the unbound RBD has a localized region of dynamic flexibility in Loop 3 and that mutations identified during the COVID-19 pandemic in Loop 3 do not affect this flexibility. We use a loop-modeling protocol to generate and simulate novel conformations of the CoV2-RBD Loop 3 region that sample conformational space beyond the ACE2 bound crystal structure. This has allowed for the identification of interesting substates of the unbound RBD that are lower energy than the ACE2-bound conformation, and that block key residues along the ACE2 binding interface. These novel unbound substates may represent new targets for therapeutic design.

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

Understanding protective mechanisms of antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We discovered a new antibody, 910-30, that targets the SARS-CoV-2 ACE2 receptor binding site as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. We performed sequence and structural analyses to explore how antibody features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer revealed its binding interactions and ability to disassemble spike. Despite heavy chain sequence similarity, biophysical analyses of IGHV3-53/3-66 antibodies highlighted the importance of native heavy:light pairings for ACE2 binding competition and for SARS-CoV-2 neutralization. We defined paired heavy:light sequence signatures and determined antibody precursor prevalence to be ~1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These data reveal key structural and functional neutralization features in the IGHV3-53/3-66 public antibody class to accelerate antibody-based medical interventions against SARS-CoV-2.

A pH-dependent switch mediates conformational masking of SARS-CoV-2 spike (preprint)

The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the ACE2 receptor and to facilitate virus entry. Antibodies can engage RBD but some, such as CR3022, fail to inhibit entry despite nanomolar spike affinity. Here we show the SARS-CoV-2 spike to have low unfolding enthalpy at serological pH and up to 10-times more unfolding enthalpy at endosomal pH, where we observe significantly reduced CR3022 affinity. Cryo-EM structures -at serological and endosomal pH- delineated spike recognition of up to three ACE2 molecules, revealing RBD to freely adopt the up conformation. In the absence of ACE2, single-RBD-up conformations dominated at pH 5.5, resolving into a locked 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 and spike shedding of antibodies like CR3022. An endosomal mechanism involving spike-conformational change can thus facilitate immune evasion from RBD- up-recognizing antibody. HighlightsO_LIReveal spike at serological pH to have only ~10% the unfolding enthalpy of a typical globular protein, explaining how antibodies like CR3022 can bind with avidity C_LIO_LIDefine an endosomal mechanism whereby spike binds ACE2, but sheds CR3022, enabling immune evasion from potentially neutralizing antibody C_LIO_LIDetermine cryo-EM structures of the SARS-CoV-2 spike along its endosomal entry pathway-at pH 5.5, 4.5, and 4.0, and in complexes with ACE2 receptor at pH 7.4 and 5.5 C_LIO_LIShow spike to exclusively adopt an all RBD-down conformation at the low pH of the late endosome-early lysosome C_LIO_LIReveal structural basis by which a switch domain mediates RBD position in response to pH C_LI

Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes (preprint)

Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from [~]0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes.