key: cord-0961966-eskhjtfe authors: Wu, Nicholas C.; Yuan, Meng; Liu, Hejun; Lee, Chang-Chun D.; Zhu, Xueyong; Bangaru, Sandhya; Torres, Jonathan L.; Caniels, Tom G.; Brouwer, Philip J.M.; van Gils, Marit J.; Sanders, Rogier W.; Ward, Andrew B.; Wilson, Ian A. title: An alternative binding mode of IGHV3-53 antibodies to the SARS-CoV-2 receptor binding domain date: 2020-07-27 journal: bioRxiv DOI: 10.1101/2020.07.26.222232 sha: 2b0d299de7958b57c2e49ba3e8a6fb3ab7add369 doc_id: 961966 cord_uid: eskhjtfe IGHV3-53-encoded neutralizing antibodies are commonly elicited during SARS-CoV-2 infection and target the receptor-binding domain (RBD) of the spike (S) protein. Such IGHV3-53 antibodies generally have a short CDR H3 due to structural constraints in binding the RBD (mode A). However, a small subset of IGHV3-53 antibodies to the RBD contain a longer CDR H3. Crystal structures of two IGHV3-53 neutralizing antibodies here demonstrate that a longer CDR H3 can be accommodated in a different binding mode (mode B). These two classes of IGHV3-53 antibodies both target the ACE2 receptor binding site, but with very different angles of approach and molecular interactions. Overall, these findings emphasize the versatility of IGHV3-53 in this common antibody response to SARS-CoV-2, where conserved IGHV3-53 germline-encoded features can be combined with very different CDR H3 lengths and light chains for SARS-CoV-2 RBD recognition and virus neutralization. IGHV3-53 antibodies generally have a short CDR H3 due to structural constraints in CoV-2 RBD to 2.35 and 1.72 Å resolutions, respectively ( Figure 1A and Table S1 ). Both Table S2 ). In addition, COVA2-04 binds to the side of the ridge, whereas COVA2-39 binds Interestingly, some of these germline-encoded residues are also involved in binding of 129 COVA2-39 (mode B), but to a different location and, hence, to different residues on the 130 RBD. VH Y33 of the 32NY33 motif is retained and forms a π−π stacking interaction between 131 its aromatic ring with the aromatic side chain of Y489 ( Figure 3C ). Although VH N32 in RBD by at least 50-fold ( Figure 1D and Figure S4A ). The increased binding for 53TGGT56 147 is likely due to the methyl groups in VH T53 and T56, which make additional van der Waals 148 interactions ( Figure S4B ). Similar to the 53SGGS56 motif, 53TGGT56 in COVA2-39 takes part in a type I beta turn along 151 with VH Y52, which is the first residue (i) in the turn (YTGG). The partial positive dipole 152 from the aligned amides at the amino-end of the turn forms a charged interaction with the 153 RBD E484 carboxyl group. Moreover, VH Y52 is an important residue for binding SARS- CoV-2 RBD in binding mode B, but not in binding mode A ( Figure 3E ). VH Y52 of COVA2-155 39 forms a π−π interaction with backbone peptide bond between RBD G485 and F486. CDR H3 of COVA2-04 is highly buried by the RBD and the light chain ( Figure 4A ). This 8 observation substantiates the notion that IGHV3-53 in binding mode A has strong 168 structural constraints on CDR H3 length. In contrast, in binding mode B, the longer CDR 169 H3 of COVA2-39 is largely solvent exposed ( Figure 4B ). This observation explains why Final 2Fo-Fc electron density map for the ridge-anchoring pocket around RBD F486 is 50 contoured at 2.0 σ. PHENIX: a 388 comprehensive Python-based system for macromolecular structure solution Identification of neutralizing human 392 monoclonal antibodies from Italian Covid-19 convalescent patients Structures of human antibodies bound to SARS-CoV-2 spike reveal common epitopes 397 and recurrent features of antibodies Potent 400 neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability Potent neutralizing antibodies against SARS-CoV-2 identified by 404 high-throughput single-cell sequencing of convalescent patients' B cells Modelling the binding of COVA2-39 and COVA2-04 on the 13 homotrimeric SARS CoV-2 spike (S) protein. (A-B) The SARS-CoV-2 S trimer is shown 14 with one RBD in the up conformation (cyan) and two RBDs in the down conformation 15 (orange) (PDB: 6VSB (pink) to the RBDs in the up conformations is modelled (cyan) and (D) COVA2-39 (pink) cannot bind to their epitopes in the 19 RBD in the down conformation as the epitopes are partially buried. Epitopes are colored 20 in yellow. (E) COVA2-04/RBD crystal structure was fitted here to the negative-stain 21 electron microscopy (nsEM) reconstruction of the Fab COVA-2-04/SARS CoV-2 spike 22 protein complex that was previously generated