Structural and Computational Studies of the SARS-CoV-2 Spike Protein Binding Mechanisms with Nanobodies: From Structure and Dynamics to Avidity-Driven Nanobody Engineering

Abstract

Nanobodies provide important advantages over traditional antibodies, including their smaller size and robust biochemical properties such as high thermal stability, high solubility, and the ability to be bioengineered into novel multivalent, multi-specific, and high-affinity molecules, making them a class of emerging powerful therapies against SARS-CoV-2. Recent research efforts on the design, protein engineering, and structure-functional characterization of nanobodies and their binding with SARS-CoV-2 S proteins reflected a growing realization that nanobody combinations can exploit distinct binding epitopes and leverage the intrinsic plasticity of the conformational landscape for the SARS-CoV-2 S protein to produce efficient neutralizing and mutation resistant characteristics. Structural and computational studies have also been instrumental in quantifying the structure, dynamics, and energetics of the SARS-CoV-2 spike protein binding with nanobodies. In this review, a comprehensive analysis of the current structural, biophysical, and computational biology investigations of SARS-CoV-2 S proteins and their complexes with distinct classes of nanobodies targeting different binding sites is presented. The analysis of computational studies is supplemented by an in-depth examination of mutational scanning simulations and identification of binding energy hotspots for distinct nanobody classes. The review is focused on the analysis of mechanisms underlying synergistic binding of multivalent nanobodies that can be superior to single nanobodies and conventional nanobody cocktails in combating escape mutations by effectively leveraging binding avidity and allosteric cooperativity. We discuss how structural insights and protein engineering approaches together with computational biology tools can aid in the rational design of synergistic combinations that exhibit superior binding and neutralization characteristics owing to avidity-mediated mechanisms.

Keywords: ACE2 host receptor; allosteric interactions; binding energy hotspots; biophysical methods; molecular dynamics; mutational scanning; signal transmission.

Figure 1

A schematic representation and comparison of conventional antibodies, heavy chain-only antibodies, and VHHs nanobodies (A,B). Adapted from https://app.biorender.com/biorender-templates (accessed on 3 February 2022). The antigen recognition site of a conventional antibody is formed jointly by the variable regions of the heavy (VH) and light (VL) chains. A nanobody corresponds to the variable region of a heavy chain only antibody. Immunization of camel, llama, alpaca as repertoire source to develop nanobodies (C) against interested targets allowed for a growing number of high-affinity nanobodies that effectively target the SARS-CoV-2 S proteins and induce effective neutralization. Cryo-EM structures of the SARS-CoV-2 S trimer in the complex with Nb6 nanobody, pdb id 7KKK. Nb6 nanobodies are shown in yellow (D). The SARS-CoV-2 S trimer in the complex with VHH E nanobody, pdb id 7KSG. VHH E is in yellow spheres (E). The SARS-CoV-2 S trimer in the complex with VHH V/VHH E nanobody, pdb id 7B18. VHH VE is in yellow/orange spheres. VHH E part in yellow and VHH V is in orange (F). The structures are shown in full spheres with protomers A,B,C colored in green, red, and blue. The rendering of SARS-CoV-2 S structures was done using the interactive visualization program UCSF ChimeraX.

Figure 2

Structural classification and superposition of the SARS-CoV-2 S-RBD complexes with high-affinity nanobodies. (A) Structural superposition of the S-RBD complexes with class I nanobodies targeting the ACE2-binding region. The representative class I nanobodies shown are Nb6 (pdb id 7KKK), Nb20 (pdb id 7JVB), Sb45 (pdb id 7KLW), MR17 (pdb id 7C8W), SR4 (pdb id 7C8V), Sb14 (pdb id 7MFU), Sb16 (pdb id 7KGK), WNb2 (pdb id 7LX5), VHH-E (pdb id 7B14), C2 (pdb id 7OAO), H3 (pdb id 7OAP), NM1230 (pdb id 727), and Nanosota-1 (pdb id 7KM5). The S-RBD is shown in green surface. The superimposed nanobodies are shown in ribbons. A typical binding epitope for this class of nanobodies is shown in red-colored surface. The positions of K417, E484, and N501 are shown in cyan-colored surface on the RBD. For clarity, only E484 can be seen. (B) Structural superposition of the S-RBD complexes with class II nanobodies targeting the cryptic binding site in the core RBD region. The representative class II nanobodies shown included: C1 (pdb id 7OAP), F2 (pdb id 7OAY), Sb68 (pdb id 7KLW), VHH-U (pdb id 7KN5), VHH-V (pdb id 7KN6), VHH-W (pdb id 7KN7), NM1226 (pdb id 7NKT), WNb10 (pdb id 7LX5), and SR31 (pdb is 7D2Z). The S-RBD is shown in green surface. The superimposed nanobodies are shown in ribbons. Both ACE2-binding epitope and class II binding epitopes are shown in red-colored surface. The positions of K417, E484, and N501 are shown in cyan-colored surface on the RBD. The class II nanobodies target the S-RBD core region that is distinct from the ACE2- binding site.

Figure 3

Structural details and binding modes for the class I nanobodies in complexes with S-RBD. The representative class I nanobodies shown are Nb6 (pdb id 7KKK), Nb20 (pdb id 7JVB), Sb45 (pdb id 7KLW), MR17 (pdb id 7C8W), SR4 (pdb id 7C8V) (top panel) and Sb16 (pdb id 7KGK), WNb2 (pdb id 7LX5), VHH-E (pdb id 7B14), Sb14 (pdb id 7MFU), and H3 (pdb id 7OAP) (bottom panel). The S-RBD is shown in green surface. The nanobodies are shown in ribbons. A typical binding epitope for this class is shown in red-colored surface. A high degree of similarity in binding modes can be seen for Nb6, Nb20, Sb45, MR17, WNb2, and VHH-E. A highly unusual binding mode was observed for SR4 and Sb16. The unique binding modes are seen for Sb14 and H3.

Figure 4

Structural details and binding modes for the class II nanobodies in complexes with S-RBD. The structures are shown for VHH-U (pdb id 7KN5), VHH-V (pdb id 7KN6), VHH-W (pdb id 7KN7), NM1226 (pdb id 7NKT) (top panel) and Sb68 (pdb id 7KLW), WNb10 (pdb id 7LX5), C1 (pdb id 7OAP), and SR31 (pdb id 7D2Z) (bottom panel). The S-RBD is shown in green surface. The nanobodies are shown in ribbons. Both ACE2- binding epitope and class II binding epitopes are shown in red-colored surface. A similarity of binding modes is seen for VHH U, VHH W, and NM1226. The unique binding modes in the cryptic binding site are seen for Sb68, WNb10, and especially SR31 nanobody.

Figure 5

Mutational profiling of the SARS-CoV-2 S binding with class I nanobodies. (A) The mutational scanning heatmap for the SARS-CoV-2 S complex with Nb6 nanobody and structural map of the S-RBD binding epitope in the complex with Nb6. The S-RBD is shown in green surface. The epitope residues are shown in red and the binding energy hotspots from are shown in blue surface. (B) The mutational scanning heatmap and structural map for the SARS-CoV-2 S complex with Nb20 nanobody. The annotations for the epitope residues and the binding energy hotspots are as in panel A. (C) The mutational scanning heatmap and structural map for the SARS-CoV-2 S complex with VHH E nanobody. The annotations for the epitope residues and the binding energy hotspots are as in panel A. (D) The mutational scanning heatmap and structural map for the SARS-CoV-2 S complex with Sb45 nanobody. The annotations for the epitope residues and the binding energy hotspots are as in panel A. The heatmaps show the computed binding free energy changes for 19 single mutations on the binding epitope sites. The squares on the heatmap are colored using a 3-colored scale from blue to yellow with yellow indicating the largest unfavorable effect on binding.

Figure 6

Mutational profiling of the SARS-CoV-2 S binding with class I nanobodies. (A) The mutational scanning heatmap and structural map for the SARS-CoV-2 S complex with Sb68 nanobody. The S-RBD is shown in green surface. The epitope residues are shown in red and the binding energy hotspots from are shown in blue surface. (B) The mutational scanning heatmap and structural map for the SARS-CoV-2 S complex with VHH U nanobody. The annotations for the epitope residues and the binding energy hotspots are as in panels A,B. (C) The mutational scanning heatmap and structural map for the SARS-CoV-2 S complex with VHH V nanobody. (D) The mutational scanning heatmap and structural map for the SARS-CoV-2 S complex with VHH W nanobody. The heatmaps show the computed binding free energy changes for 19 single mutations on the binding epitope sites. The squares on the heatmap are colored using a 3-colored scale from blue to yellow with yellow indicating the largest unfavorable effect on binding.