c, CDRH1, CDRH2 and d, CDRH3 interactions with RBD residues. RBDs, (2) ACE2-blocking hNAbs that bind both up and down RBDs and can contact adjacent RBDs, (3) hNAbs that bind outside the ACE2 site and recognize up and down RBDs, and (4) Previously-described antibodies that do not block ACE2 and bind only up RBDs9. Class 2 comprised four hNAbs whose epitopes bridged RBDs, including a hNAb that used a long CDRH3 with a hydrophobic tip to bridge between adjacent down RBDs, thereby locking spike into a closed conformation. Epitope/paratope mapping revealed few interactions with host-derived or gene segment3,12,13,16,17,27C29, a majority of which are known or predicted15,26,28,30,31 to exhibit a common RBD binding mode resulting from the use of germline-encoded residues within the complementarity-determining regions 1 and 2 (CDRH1 and CDRH2) and a CDRH3 that is shorter than the average length (15 amino acids; IMGT32 CDR definition) in human antibodies33. Other SARS-CoV-2 RBD-binding antibodies are encoded by and hNAbs Pipequaline to elucidate rules for binding by four distinct anti-RBD antibody classes (Supplementary Table 2). The hNAbs chosen for structures are highly potent, achieving 90% neutralization in pseudotype virus assays at concentrations ranging from 22C140 ng/mL5, thus our structural analyses and classifications directly relate to understanding mechanisms of neutralization and potency differences between hNAbs. Class 1: hNAbs with short (9 and 12 residues) CDRH3s (Extended Data Fig. 1g) that were isolated from the same donor5. They share structural similarities with each other and with other hNAb binding mode.a, 3.2 ? Pipequaline cryo-EM density for C144-S trimer complex revealing C144 binding to a closed (3 RBDs down) spike conformation. b, Overlay of C102 Fab (from C102-RBD crystal structure; Extended Data Fig. 1) and C144 Fab (from C144-S structure) aligned on a RBD monomer. ACE2 (PDB 6M0J; Pipequaline light green surface) is usually aligned on the same RBD for reference. C144 adopts a distinct conformation relative to the C102-like hNAbs with short CDRH3s, a small subset of potently ERBB neutralizing encoded antibodies utilize longer CDRH3s ( 15 residues, IMGT definition32, Extended Data Fig. 1g)5,12. A recent structure of a RBD complexed with a hNAbs C144 and COVA2C39, the C121 CDRH2 SxxS motif forms a potential hydrogen bond network with residue E484RBD (Fig. 3b,?,jj). Overall, these results suggest a convergent mode of recognition by germline-encoded residues across diverse VH/VL gene segments for SARS-CoV-2, which may contribute to low levels of somatic hypermutation observed for these hNAbs (Extended Data Fig. 6, Extended Data Table 1). Class 3: hNAbs that bind outside the ACE2 binding site and recognize both up and down RBD conformations C135 is usually a potent hNAb that showed binding properties distinct from class 1, class 2, and the cross-reactive SARS-CoV antibody CR30225 (which we categorized as a class 4 antibody; Extended Data Table 1). To evaluate the mechanism Pipequaline of C135-mediated neutralization of SARS-CoV-2, we solved the cryo-EM structure of a C135-S complex to 3.5 ? (Fig. 4a, Extended Data Fig. 7), using an unbound C135 crystal structure for fitting (Supplementary Table 1). The structure revealed three C135 Fabs bound to an S trimer with 2 down and 1 up RBDs, although the C135-bound up RBD conformation was weakly resolved and therefore not modeled. C135 recognizes an glycopeptidic epitope similar to the cross-reactive SARS-CoV hNAb S30934, focusing on a region of the RBD near the N343RBD glycan and non-overlapping with the ACE2 binding site (Fig. 4b, Extended Data Fig. 7c,?,d).d). Despite differences in binding orientations between C135 and S309, targeting of the RBD epitope was mainly VH-mediated (the BSA of RBD around the C135 HC represented ~480?2 of ~700 ?2 total BSA) and included interactions with the core fucose moiety of the N343RBD glycan. The smaller C135 footprint relative to S309 (~700 ?2 versus ~1150 ?2 BSA, respectively; Extended Data Fig. 7c,?,d)d) focused on interactions with RBD residues R346RBD and N440RBD, which are engaged by residues from HC and LC CDR loops (Fig. 4c,?,d)d) and are not conserved between SARS-CoV-2 and SARS-CoV RBDs, rationalizing the lack of SARS-CoV cross-reactivity observed for C1355. Open in a separate window Physique 4. Cryo-EM structure of S complexed with the class 3 (non-ACE2.