key: cord-0869986-pm26g722
authors: Han, Pengcheng; Li, Linjie; Liu, Sheng; Wang, Qisheng; Zhang, Di; Xu, Zepeng; Han, Pu; Li, Xiaomei; Peng, Qi; Su, Chao; Huang, Baihan; Li, Dedong; Zhang, Rong; Tian, Mingxiong; Fu, Lutang; Gao, Yuanzhu; Zhao, Xin; Liu, Kefang; Qi, Jianxun; Gao, George F.; Wang, Peiyi
title: Receptor binding and complex structures of human ACE2 to spike RBD from Omicron and Delta SARS-CoV-2
date: 2022-01-06
journal: Cell
DOI: 10.1016/j.cell.2022.01.001
sha: 4cad9d52b2448a6020e4605e667489a69f86090a
doc_id: 869986
cord_uid: pm26g722

COVID-19 pandemic continues worldwide with many variants arising, especially those of variants of concern (VOCs). A recent VOC, Omicron (B.1.1.529), which obtains a large number of mutations in the receptor-binding domain (RBD) of the spike protein, has risen to intense scientific and public attention. Here we studied the binding properties between the human receptor ACE2 (hACE2) and the VOC RBDs and resolved the crystal and cryo- EM structures of the Omicron RBD-hACE2 complex, as well as the crystal structure of Delta RBD-hACE2 complex. We found that, unlike Alpha, Beta and Gamma, Omicron RBD binds to hACE2 at a similar affinity compared to that of the prototype RBD, which might be due to compensation of multiple mutations for both immune escape and transmissibility. The complex structures of Omicron-hACE2 and Delta-hACE2 reveal the structural basis of how RBD-specific mutations bind to hACE2.

, which may be explained by its higher 63 replication rates and S-mediated entry (Mlcochova et al., 2021) . 64 In contrast to other VOCs that emerged when natural immunity is predominant, In this study, we examined several VOCs, including Alpha, Beta, Gamma, Delta 77 and Omicron, and demonstrated that Omicron variant RBD binds to hACE2 with 78 comparable affinity to that of the prototype. We then determined both the crystal and Omicron is close related to Beta and Gamma ( Figure S1B ). 99 We then explored the binding capacity of Omicron RBD to hACE2 in comparison 100 J o u r n a l P r e -p r o o f to the prototype and other variants, and pangolin-origin GD/1/2019. We purified RBDs 101 from the prototype SARS-CoV-2 (the first strain of SARS-CoV-2 which was isolated 102 from a clinical patient on Jan 6, 2020, GISAID: EPI_ISL_402119, Tan et al., 2020) and 103 the variants, as well as pangolin-origin GD/1/2019 and tested their binding to hACE2-104 expressing BHK-21 cells using flow cytometry assay (Figure 2A) . RBDs from VOCs 105 and GD/1/2019 bound to hACE2-expressing cells to comparable levels with prototype 106 RBD, with 73.3%-86.3% positive cells in staining experiments. We further measured 107 the binding affinities of the RBDs to hACE2 with surface plasmon resonance (SPR) 108 assay ( Figure 2C , Table S1 ). The results showed that the prototype SARS-CoV-2 binds Omicron RBD-hACE2 crystal complex structure is larger than its ASA in cryo-EM, 144 with 1727.2 Å 2 and 1709.5 Å 2 , respectively. Similar result is also observed in the Delta 145 RBD-hACE2 complexes, with an ASA of 1704.0 Å 2 for the crystal structure and 1656.6 146 Å 2 for the cryo-EM structure, respectively.

As previously reported , the binding interface of hACE2 for 148 SARS-CoV-2 RBD is distributed over two patches. In Patch 1 of Omicron RBD-hACE2 149 complex, S19 of hACE2 forms H-bonds with both A475 and N477 of RBD. Q24 of 150 hACE2 interacts with N487 of RBD through an H-bond. Y83 of hACE2 contacts Y489 151 and N487 of RBD by H-bonds, and it also forms π-π stacking interaction with F486 of 152 RBD. H34 from hACE2 contacts Y453 of RBD through an H-bond ( Figure 3D ). E35 153 of hACE2 forms salt bridge with R493 from RBD ( Figure 3D ). Residue F486 from 154 RBD packs against a small hydrophobic pocket in the interface formed by F28, L79, 155 M82, and Y83 of hACE2 ( Figure 3D ). In Patch 2 of Omicron RBD-hACE2 complex, 156 residue D38 from hACE2 forms an H-bond with Y449 and a salt bridge with R498 of with G502 of RBD.

In Patch 1 of Delta RBD-hACE2 complex, S19, Q24, K31, H34 and Y83 from 162 hACE2 contact residues of A475, N487, Q493, Y453 and Y489 from RBD through H-163 bonds, respectively. Y83 of hACE2 forms an H-bond with Y498 from RBD and π-π 164 stacking interaction with F486 of RBD. D30 from hACE2 binds to K417 of RBD with 165 a salt bridge. F486 of RBD can also constitute to strong hydrophobic interactions with 166 F28, L79, M82, and Y83 of hACE2 ( Figure 3E ). In Patch 2, E37, D38, Y41 and K353 167 from hACE2 form H-bonds with Y505, Y449, T500 and G496 on the RBD, respectively.

Q42 of hACE2 contacts both Y449 and Q498 from RBD through H-bonds ( Figure 3E ). 169 It should be noted that both of the two substitutions, specifically the L452R and T478K, 170 locate outside the RBD-hACE2 binding interface, which is consistent with the 171 observation that similar binding affinities with ACE2 for both Delta and prototype 172 RBDs were obtained ( Figure 3A, Table1) . 173 Next, we focused on the substitutions of RBD on the hACE2 binding interface. We respectively. Similar results have also been observed in the previous study (Niu et al., 194 2021), while Q498 was mutated to H498 enhancing its binding affinity to human, 195 mouse or rat ACE2. On the other hand, K478 is located away from hACE2 binding 196 surface and may only influence hACE2 binding allosterically. In this study, we determined both X-ray crystallography and cryo-EM structures of 229 Omicron RBD-hACE2 complex, and X-ray crystallography structure of Delta RBD- This study did not generate custom computer code. The sitting-drop method was used to obtain the Omicron RBD-hACE2 and Delta 584 RBD-hACE2 complex crystals. In detail, purified complex proteins were concentrated 585 to 5 and 10 mg/mL. Then, 0.8 μL protein was mixed with 0.8 μL reservoir solution. 

Data collection, processing, and refinement statistics were summarized in Table S2 . All 603 structural figures were generated using Pymol software (https://pymol.org/2/). Table 3 . 

New SARS-CoV-2 variants-clinical, public health, and 666 vaccine implications

PHENIX: a comprehensive Python-based system for 669 macromolecular structure solution

Reduced neutralization of SARS-CoV-2 omicron variant by vaccine sera and monoclonal 673 antibodies. medRxiv

SARS-CoV-2: Virology, epidemiology, immunology and vaccine development

Humoral immune response to circulating SARS-CoV-2 variants elicited by inactivated and RBD-subunit 679 vaccines

529 escapes the majority of SARS-CoV-2 neutralizing antibodies of diverse epitopes

MolProbity: all-atom structure validation for 685 macromolecular crystallography

Persistence and evolution of SARS-CoV-2 in an immunocompromised host

SARS-CoV-2 evolution in an immunocompromised host reveals shared 691 neutralization escape mechanisms

Coot: model-building tools for molecular graphics

Features and development of Coot

SARS-CoV-2 B. 1.617 emergence and sensitivity to vaccine-elicited 698 antibodies

Emergence of SARS-CoV-2 spike protein escape mutation Q493R after treatment for COVID-19

COVID-19 expands its territories from humans to animals

Multiple SARS-CoV-2 variants escape neutralization by 706 vaccine-induced humoral immunity

Investigating the mutational 708 landscape of the SARS-CoV-2 Omicron variant via ab initio quantum mechanical modeling

Changes in symptomatology, reinfection, and transmissibility associated 712 with the SARS-CoV-2 variant B.1.1.7: an ecological study

Emergence of Q493R mutation in SARS-CoV-2 spike protein during 715 bamlanivimab/etesevimab treatment and resistance to viral clearance

Molecular insights into receptor binding of recent emerging SARS-CoV-2 variants

SARS-CoV-2 D614G variant exhibits efficient replication ex vivo and 721 transmission in vivo

Serum sample neutralisation 723 of BBIBP-CorV and ZF2001 vaccines to SARS-CoV-2 501Y. V2

Quantifying the local resolution of cryo-EM density 728 maps

The emergence, genomic diversity and global spread of

CoV-2 501Y. V2 variants lack higher infectivity but do have immune escape

Binding 734 and molecular basis of the bat coronavirus RaTG13 virus to ACE2 in humans and other species

Cross-737 species recognition of SARS-CoV-2 to bat ACE2

Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike

Functional 742 and genetic analysis of viral receptor ACE2 orthologs reveals a broad potential host range of SARS-CoV

The reproductive number of the Delta variant of SARS-CoV-2 is far higher 745 compared to the ancestral SARS-CoV-2 virus

Bat-to-human: spike features determining 'host jump'of 747 coronaviruses SARS-CoV, MERS-CoV, and beyond

SARS-CoV-2 B. 1.617. 2 Delta variant replication and immune evasion

Neutralization of SARS-CoV-2 lineage B. 1.1. 7 pseudovirus by BNT162b2 vaccine-753 elicited human sera

Structural basis for continued antibody evasion by the SARS-CoV-2 receptor 756 binding domain

Molecular basis of cross-species ACE2 interactions with SARS-CoV-2-like viruses of pangolin origin

Processing of X-ray diffraction data collected in oscillation mode

UCSF chimera -A visualization system for exploratory research and analysis

Mapping neutralizing and immunodominant sites on the 767

SARS-CoV-2 spike receptor-binding domain by structure-guided high-resolution serology

Genome characterization and potential 770 risk assessment of the novel SARS-CoV-2 variant Omicron (B. 1.1. 529). Zoonoses

CTFFIND4: Fast and accurate defocus estimation from electron 773 micrographs

Omicron has extensive but incomplete escape of Pfizer BNT162b2 elicited neutralization and requires 777 ACE2 for infection

SARS-CoV-2 variant B.1.1.7 is susceptible to neutralizing antibodies elicited by 780 ancestral spike vaccines

Serine 477 plays a crucial role in 782 the interaction of the SARS-CoV-2 spike protein with the human receptor ACE2

Prospective mapping of viral mutations that escape antibodies used to treat COVID-19

Epidemiology, 787 J o u r n a l P r e -p r o o f genetic recombination, and pathogenesis of coronaviruses

A novel coronavirus 789 genome identified in a cluster of pneumonia cases -Wuhan

CoV-2 variants of concern (vocs): an impending global crisis

The global epidemic of the SARS-CoV-2 Delta variant, key 794 spike mutations and immune escape

Structural and functional basis of SARS-CoV-2 entry by using human ACE2

MolProbity: More and better reference data for improved all-atom 799 structure validation

Broad 801 host range of SARS-CoV-2 and the molecular basis for SARS-CoV-2 binding to cat ACE2

SARS-CoV-2 variant prediction and antiviral drug design are enabled by RBD in 804 vitro evolution

The 806 significant immune escape of pseudotyped SARS-CoV-2 Variant Omicron

The molecular basis for SARS-CoV-2 binding to dog ACE2

MotionCor2: 811 anisotropic correction of beam-induced motion for improved cryo-electron microscopy

Evidence of escape of SARS-CoV-2 variant B. 1.351 from natural 815 and vaccine-induced sera

Cryo-electron microscopy structures of the N501Y SARS-CoV-2 spike 818 protein in complex with ACE2 and 2 potent neutralizing antibodies

New tools for automated high-resolution cryo-EM structure determination in RELION-3

 338 We are grateful to Drs G. Wong and G. Shang for revising the manuscript. We are 339 grateful to the PMI-IMCAS Public Technology Service Center for its support on FACS