key: cord-330473-f03ka7bd
authors: Yuan, Meng; Wu, Nicholas C.; Zhu, Xueyong; Lee, Chang-Chun D.; So, Ray T. Y.; Lv, Huibin; Mok, Chris K. P.; Wilson, Ian A.
title: A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV
date: 2020-03-14
journal: bioRxiv
DOI: 10.1101/2020.03.13.991570
sha: 
doc_id: 330473
cord_uid: f03ka7bd

The outbreak of COVID-19, which is caused by SARS-CoV-2 virus, continues to spread globally, but there is currently very little understanding of the epitopes on the virus. In this study, we have determined the crystal structure of the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein in complex with CR3022, a neutralizing antibody previously isolated from a convalescent SARS patient. CR3022 targets a highly conserved epitope that enables cross-reactive binding between SARS-CoV-2 and SARS-CoV. Structural modeling further demonstrates that the binding site can only be accessed when at least two RBDs on the trimeric S protein are in the “up” conformation. Overall, this study provides structural and molecular insight into the antigenicity of SARS-CoV-2. ONE SENTENCE SUMMARY Structural study of a cross-reactive SARS antibody reveals a conserved epitope on the SARS-CoV-2 receptor-binding domain.

(3). Such a high degree of sequence similarity raises the possibility that cross-reactive 48 epitopes may exist. A recent study has shown that CR3022, which is a human 49 neutralizing antibody that targets the receptor-binding domain (RBD) of SARS-CoV (4),

can bind to the RBD of SARS-CoV-2 (5). This finding provides an opportunity to uncover 51 a cross-reactive epitope. 52 53 CR3022 was previously isolated from a convalescent SARS patient and is encoded by 54 germline genes IGHV5-51, IGHD3-10, IGHJ6 (heavy chain), and IGKV4-1, IGKJ2 (light 55 chain) (4). Based on IgBlast analysis (6), the IGHV of CR3022 is 3.1% somatically 56 mutated at the nucleotide sequence level, which results in eight amino-acid changes 57 from the germline sequence, whereas IGKV of CR3022 is 1.3% somatically mutated 58 resulting in three amino-acid changes from the germline sequence (fig. S1). We 59 therefore determined the crystal structure of CR3022 with the SARS-CoV-2 RBD at 3.1 60 Å resolution (table S1 and 

implying their likely importance in the affinity maturation process.

Out of 28 residues in the epitope (defined as residues buried by CR3022), 24 (86%) are 68 conserved between SARS-CoV-2 and SARS-CoV (Fig. 1D ). This high sequence 69 conservation explains the cross-reactivity of CR3022. Nonetheless, despite having a 70 high conservation in the epitope residues, CR3022 Fab binds to SARS-CoV RBD (K d = 1 71 nM) with a much higher affinity than to SARS-CoV-2 RBD (K d = 115 nM) (Table 1 

Mass spectrometry analysis has shown that a complex glycan is indeed present at this 79 N-glycosylation site in . An N-glycan at N370 would fit into a groove 80 formed between heavy and light chains ( fig. S4C ), which could increase contact and, 81 hence, binding affinity to CR3022. We then tested whether CR3022 was able to 82 neutralize SARS-CoV-2 and SARS-CoV in an in vitro microneutralization assay (7).

While CR3022 could neutralize SARS-CoV, it did not neutralize SARS-CoV-2 at the 

as [9] [10] [11] . Interestingly, the epitope of CR3022 does not overlap with the 90 ACE2-binding site ( Fig. 2A) . Structural alignment of CR3022-SARS-CoV-2 RBD complex 91 with the ACE2-SARS-CoV-2 RBD complex (11) further indicates that binding of CR3022 92 would not clash with ACE2 (12). This analysis implies that the neutralization mechanism 93 of CR3022 does not depend on direct blocking of receptor binding, which is consistent 94 with the observation that CR3022 does not compete with ACE2 for binding to the RBD 95 (5). Unlike CR3022, most known SARS RBD-targeted antibodies compete with ACE2 for 96 binding to RBD (4, [13] [14] [15] [16] . The epitopes of these antibodies are very different from that 97 of CR3022 (Fig. 2B ). In fact, it has been shown that CR3022 can synergize with other 98 RBD-targeted antibodies to neutralize SARS-CoV (4). Although CR3022 itself cannot 99 neutralize SARS-CoV-2 in this in vitro assay, whether CR3022 can synergize with other 100 SARS-CoV-2 RBD-targeted monoclonal antibodies for neutralization remains to be 101 determined.

Recently, the cryo-EM structure of homotrimeric SARS-CoV-2 S protein was determined 104 (17, 18) and demonstrated that the RBD, as in other coronaviruses (19, 20) adopts two 105 different dispositions in the trimer. The RBD can then undergo a hinge-like movement to 106 transition between "up" or "down" conformations ( Fig. 3A ). ACE2 host receptor can only 107 interact with the RBD when it is in the "up" conformation, whereas the "down" 108 conformation is inaccessible to ACE2. Interestingly, the epitope of CR3022 is also only 109 accessible when the RBD is in the "up" conformation ( Fig. 3 , B and C). Furthermore, the 110 ability for CR3022 to access the RBD also depends on the relative disposition of the 111 RBD on the adjacent protomer. CR3022 can only access RBD when the targeted RBD 112 on one protomer of the trimer and the RBD on the adjacent protomer are both in the "up" 113 conformation. The variable region of CR3022 would clash with the RBD on the adjacent 6 protomer if the latter adopts a "down" conformation ( Fig. 3D) . As a homotrimer, the S 115 protein could potentially adopt four possible RBD configurations, namely none-"up", 116 single-"up", double-"up", and triple-"up". It appears that CR3022 can only bind to the S 117 protein when it is in double-"up" or triple-"up" configuration. Specifically, one molecule of 118 CR3022 can be accommodated in the double-"up" configuration ( Fig. 3E) , whereas three 119 molecules of CR3022 could potentially be accommodated in the triple-"up" configuration 120 (Fig. 3F ). Previous cryo-EM studies have also shown that the recombinant SARS-CoV S 121 protein is mostly found in the none-"up", single-"up", or double-"up" conformations (19, 122 21), but rarely in the triple-"up" conformation, even with ACE2 receptor bound (21, 22) .

Together with the fact that CR3022 was isolated from a convalescent SARS patient (4) 

Therefore, although CR3022 does not neutralize SARS-CoV-2 in vitro despite its 146 reasonable binding affinity, it is possible that this epitope can confer in vivo protection.

The potential existence of non-neutralizing protective antibodies to SARS-CoV-2 148 highlights the need for an effective SARS-CoV-2 infection mouse model, which has yet 149 to be established.

Since there is currently great urgency in the efforts to develop a vaccine against SARS-

CoV-2, characterizing the epitopes on SARS-CoV-2 S protein is extremely valuable.

Much work is now ongoing in isolating human monoclonal antibodies from SARS-CoV-2 154 patients. We anticipate that these investigations will decipher the antigenic properties 

Potent neutralization of severe acute respiratory syndrome (SARS)

coronavirus by a human mAb to S1 protein that blocks receptor association

Molecular and biological characterization of human 200 monoclonal antibodies binding to the spike and nucleocapsid proteins of severe 201 acute respiratory syndrome coronavirus

Development and characterisation of neutralising monoclonal 203 antibody to the SARS-coronavirus

Structure of severe acute respiratory syndrome coronavirus 205 receptor-binding domain complexed with neutralizing antibody

Cryo-EM structure of the 2019-nCoV spike in the prefusion 208 conformation

Structure, function, and antigenicity of the SARS-CoV-2 spike 210 glycoprotein

Cryo-EM structures of MERS-CoV and SARS-CoV spike

Robyn Stanfield for assistance in data collection, and Andrew Ward for discussion

This work was supported by NIH K99 AI139445

Yersin scholarship (to H.L.), Bill and Melinda Gates Foundation OPP1170236 (to I

National Natural

Science Foundation of China (NSFC)/Research Grants Council (RGC)

CDR loops are labeled. Cyan: epitope residues that are conserved between 300

Green: epitope residues that are not conserved between 301

D) Epitope residues that are important for binding to 302 CR3022 are labeled. Epitope residues are defined here as residues in SARS

RBD with buried surface area > 0 Å 2 after Fab CR3022 binding as calculated with PISA 304 (35). (E) Several key interactions between CR3022 and SARS-CoV-2 RBD are 305 highlighted. CR3022 heavy chain is colored in orange, CR3022 light chain in yellow, and 306 SARS-CoV-2 RBD in cyan

The relative binding position of CR3022 with respect to receptor ACE2 and 309 other SARS-CoV RBD monoclonal antibodies. (A) Structures of CR3022-SARS-CoV-310 2 RBD complex and ACE2-SARS-CoV-2 RBD complex

SARS-CoV-2 RBD. ACE2 is colored in green, RBD in light grey, and CR3022 in yellow

Structural superposition of CR3022-SARS-CoV-2 RBD complex

80R-SARS-CoV RBD complex (PDB 2GHW) (37), 314 and m396-SARS-CoV RBD complex

Binding of CR3022 depends on the RBD configurations on the S protein

RBD in the S proteins of SARS-CoV-2 and SARS-CoV can adopt either an "up" 318 conformation (blue) or a "down" conformation (red). PDB 6VSB (cryo-EM structure

17) is shown. (B-C) CR3022 epitope (cyan) on the RBD is 320 exposed in (B) the "up

E-F) The double-"up" and triple-"up

One CR3022 molecule can be accommodated per S protein in the double-"up" 325 configuration, and (F) three CR3022 molecules could potentially be accommodated per 326 S

We thank Henry Tien for technical support with the crystallization robot, Jeanne

Matteson for contribution to mammalian cell culture, Wenli Yu to insect cell culture,