Cryo-EM reveals conformational variability in the SARS-CoV-2 spike protein RBD induced by two broadly neutralizing monoclonal antibodies

Abstract

SARS-CoV-2 spike proteins play a critical role in infection by interacting with the ACE2 receptors. Their receptor-binding domains and N-terminal domains exhibit remarkable flexibility and can adopt various conformations that facilitate receptor engagement. Previous structural studies have reported the RBD of the spike protein in "up", "down", and various intermediate states, as well as its different conformational changes during ACE2 binding. This flexibility also influences its interactions with the neutralizing antibodies, yet its role in the antibody complexes remains understudied. In this study, we used cryo-electron microscopy to investigate the structural properties of two broadly neutralizing monoclonal antibodies, THSC20.HVTR04 and THSC20.HVTR26. These antibodies were isolated from an unvaccinated individual and demonstrated potent neutralization of multiple SARS-CoV-2 variants. Our analysis revealed distinct binding characteristics and conformational changes in the spike RBD upon binding with the monoclonal antibodies. The structural characterization of the spike protein-monoclonal antibody complexes provided valuable insights into the structural variability of the spike protein and the possible mechanisms for antibody-mediated neutralization.

Fig. 1. Cryo-EM maps of the spike (S) protein-Fab complex: (A) high-resolution EM map showing the two RBDs (in the up conformation) of the S trimer bound to two independent Fab4 units. Top and side views of the complexes are shown. (B) High-resolution EM map of one-up RBD of the S trimer bound to one Fab26; the top and side views of the complexes are shown. S protein protomers (Pro 1, 2, 3) are colored gold, magenta, and cyan, respectively, while the Fab is colored orange-red.

Fig. 2. Outline of different conformational states captured from the Fab4- and Fab26-bound S trimer complexes: (A) cryo-EM map represents state I where two Fab4 units are bound to two “up” RBD conformation protomers. The colors of the first, second, and third protomers are gold, magenta, and cyan, respectively. Fab is colored orange-red. (B) Cryo-EM map of state II; two Fab4 units bound to “up” RBD and one Fab4 bound to partially open RBD. (C) State III represents an atomic model of three Fab4 units bound to the open form of the protomers. Shifting of all the RBDs from one state to another upon binding to Fab4 is shown by arrows. (D–F) Atomic models of the different conformations of the captured Fab4-S complexes (states I, II and III, respectively). (G) Cryo-EM map of state I represents two Fab26 units bound to two “up” RBD conformation protomers. The shift of one RBD while binding to Fab26 is shown by the arrow. (H) Cryo-EM map of state II represents three Fab26 units bound to the partial open form of the protomers. To accommodate the binding of Fabs to the protomers, all RBDs undergo conformational changes upon binding to Fab26 and adopt a partially open form, which is shown by the arrows. (I and J) Atomic models of the two different captured conformations of the Fab26-S complexes (states I and II respectively). (K and L) Bar graphs represent the number of particles present in the 2-Fab and 3-Fab binding conformations of Fab4 and Fab26 in the S protein complexes, respectively.

Fig. 3. Structural basis for the accommodation of Fab4 and Fab26 in the SARS-CoV2 S protein: cryo-EM maps fitted to the atomic model, generated for the highest-resolution EM maps obtained from the (A) S protein-Fab4 complex and (B) S protein-Fab26 complex. The colors of the first, second, and third protomers are gold, magenta, and cyan, respectively, and Fab4 and 26 are colored orange-red. (C) Heat map represents epitope residue selectivity and their instances in the different conformations captured. Accordingly, the frequency of epitope involvement in antibody interaction is colored linearly in green, whereas zero involvement is shown in yellow. It demonstrates the potent epitope residues in all the binding modes/conformational states. (D–G) Details of the RBD interactions with Fab4. (D and F) Interfacial area shows the CDR regions of Fab4 and 26 with RBD, respectively. CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are highlighted in light salmon, Indian red, maroon, cornflower blue, medium blue, and dark blue respectively, and the RBD region is colored hot pink. (E) Important interacting residue V445 of the S protein makes contact with a hydrophobic pocket of Fab4 comprising the residues P57, V105, and P106. (G) F486 is shielded in a hydrophobic pocket of Fab26 comprising L34 and G112 residues.

Fig. 4. Distinct binding features of Fab4 and Fab26 in the RBD region of the S protein with respect to ACE2: (A) Licorice diagram of the S protein bound to Fab4 and ACE2, and the magnified view of the conformational variable region of RBD bound to Fab4 and ACE2. ACE2 is shown in lime green, Fab4 is shown in hot pink, Fab4-bound S protein is shown as a black ribbon, and the ACE2-bound S protein is displayed in gold. (B) Licorice diagram of the conformationally variable region and the clashing region of Fab26- and ACE2-bound RBD. (C and D) Overlapping region of Fab4- and Fab26-bound RBD with ACE2-bound RBD is shown in the silhouette form. (E) High-resolution 3D structure obtained for the S protein binding loop along with Fab4 and Fab26 in the RBD region highlights the distinct targeting ability.

Fig. 5. Comparison of the conformational changes in the S protein during the formation of Fab4- and Fab26-bound complexes: (A–C) superimposition of individual protomer 1 (F), protomer 2 (G), and protomer 3 (H) from three different states of the Fab4-S protein complex provides insights into the dynamics of each protomer across the states. A magnified view of the RBD region is shown in the right corner of each image. (D–E) Similarly, a comparison of protomer 1 (I), and protomer 2 (J) in states I and II of the Fab26-S protein complexes is shown. Apart from minor angle shifts, significant angles are highlighted in the figures. (F–J) Protomer conformations captured within each state are compared, showing the structural changes in the RBD domain of each complex state. Protomer superimposition shows the protomers for (F–H) states I, II and III of the Fab4 complexes and (I and J) states I and II of the Fab26 complexes. Protomers 1, 2 and 3 are coloured magenta, gold and cyan, respectively.