In a recent study published in bioRxiv*, researchers determined cryo-electron microscopic (cryo-EM) structures of angiotensin-converting enzyme 2 (ACE2) receptor traps.
Study: Computational pipeline provides mechanistic understanding of Omicron variant of concern that neutralizes engineered ACE2 receptor traps. Image credit: Kateryna Kon/Shutterstock
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has acquired multiple mutations throughout the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV-2 Omicron has 37 mutations in the spike protein. The N-terminal (NTD) and receptor-binding domains (RBD) of the spike protein contain 11 and 15 mutations, respectively, resulting in lower neutralization by plasma from convalescent or fully vaccinated subjects.
Previously, the authors designed ACE2 receptor traps for SARS-CoV-2 neutralization. They were computationally designed and optimized for affinity using yeast surface visualization. The ACE2 domains were fused to the fragment crystallizable (Fc) domain of human IgG1 for additional binding avidity and to a neonatal Fc receptor to increase half-life. The structures of computationally designed (CVD293) and affinity-matured (CVD313) ACE2-Fc fusion constructs bound to RBD have not been determined.
The study and conclusions
In the present study, the researchers shortened the bond length between ACE2 and Fc to generate a new construct (CVD432) and solved cryo-EM structures of the engineered traps (CVD293 and CVD432) bound to the spike protein complete. First, the authors verified that CVD293 and CVD432 neutralize wild-type (WT) pseudotyped viruses.
The WT spike-CVD293 complex had a 1-RBD-up state with full ACE2 occupancy and an appreciable percentage of 1-RBD-up state with partial ACE2 occupancy, a 2-RBD-up state with 1-ACE2 occupancy, and 1- RBD-up state without ACE2 occupancy, per trimer. In contrast, the WT spike-CVD432 complex showed a 1-RBD up state with full ACE2 occupancy, a 2-RBD up state with 2-ACE2 occupancy, an all-RBD down state, and another partial state or not. Occupation ACE2 states 1 or 2 RBD-up.
The team then developed a multi-model workflow that combined cryo-EM and Rosetta protein modeling to calculate the average predicted interface energy. Overlapping 10-residue stretches of the ACE2 interface from each cryo-EM model (spike-CVD239 and spike-CVD432) were subjected to a CartesianSampler engine in Rosetta to generate 2000 models for each 10-residue stretch. These models were all-atom minimized in the cryo-EM map using the FastRelax engine. The refinement protocol was repeated to generate nearly 8000 models.
The atomic models were ranked according to the Rosetta scores and 80 models were selected. The interface helix residues of the 80 models were superimposed to examine the convergence of side chain conformations and intermolecular interactions. They noted that residue Q35 in CVD293 and CVD432, a high side chain root mean square deviation (RMSD) residue, formed a hydrogen bond with residue Q493 of the WT spike in more than 90% of the models atomic
Furthermore, they noted that the low average side chain RMSD residues in CVD293 and CVD432 formed hydrophobic interactions with the corresponding spike residues. The authors reported that low average side chain RMSD hydrophobic residues designed into the receptor traps improved the binding affinity.
The predicted interface energy for CVD293 was lower (-58 Rosetta energy units, REU) than the average interface energy of the 80 models (-45 REU) due to differences in the interactions mediated by the side chain Residue Q35 designed in CVD293 had the largest average side chain RMSD per residue.
Multiple SARS-CoV-2 Omicron spike mutations have been identified in other variants, although the variant has 14 unique modifications that enhance its binding affinity. The researchers then assessed the binding of Omicron RBD to engineered ACE2 traps. To this end, they generated models of the Omicron RBD-CVD293 and Omicron RBD-CVD432 complex by overlapping and replacing WT RBD in local cryo-EM refinement with Omicron RBD and minimized the complexes.
The interface energy for Omicron RBD-CVD293 complex residues was 10.77 REU and -8.5 REU for Omicron RBD-CVD432 complex, the interface energy for Omicron RBD-WT ACE2 was be -4.99 REU. The authors performed biolayer interferometry (BLI) of CVD293 or CVD432 with Omicron RBD to test whether the predicted interface energies corresponded to apparent binding affinities.
The dissociation constants (KD) for Omicron RBD-CVD293 (4.2 nM) and Omicron RBD-CVD432 (0.53 nM) were 10- and 100-fold lower than those of Omicron RBD-WT RBD, respectively. In addition, neutralization assays were performed with pseudotyped recombinant vesicular stomatitis virus with Delta or Omicron spike. CVD293 and CVD432 neutralized Delta and Omicron pseudoviruses, with 2- to 20-fold improvements in half-maximal inhibitory concentrations (IC50) over WT spike.
Conclusions
In summary, the authors determined cryo-EM structures of engineered ACE2 traps complexed with WT spike. Although informative, cryo-EM provided limited resolution at the ACE2-RBD interface, prompting the development of a cryo-EM pipeline: the multimodel Rosetta.
This pipeline revealed that distributed binding interactions at the interface between the two proteins were more effective than one or two interactions at the interface and that the stability of the individual proteins was as important as the stability of the complex.
Furthermore, Omicron RBD binding to receptor traps was experimentally validated by BLI and pseudovirus neutralization, demonstrating that ACE2 traps designed for WT spike were robust against mutant spike (of Omicron). Overall, the study exemplified how cryo-EM and computational modeling could be combined to improve the design-build-test cycle for designing biotherapeutics.
*Important news
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice/health-related behavior, or be treated as established information.