key: cord-332185-a96r1k7a authors: Zhang, Shuyuan; Qiao, Shuyuan; Yu, Jinfang; Zeng, Jianwei; Shan, Sisi; Lan, Jun; Tian, Long; Zhang, Linqi; Wang, Xinquan title: Bat and pangolin coronavirus spike glycoprotein structures provide insights into SARS-CoV-2 evolution date: 2020-09-22 journal: bioRxiv DOI: 10.1101/2020.09.21.307439 sha: doc_id: 332185 cord_uid: a96r1k7a In recognizing the host cellular receptor and mediating fusion of virus and cell membranes, the spike (S) glycoprotein of coronaviruses is the most critical viral protein for cross-species transmission and infection. Here we determined the cryo-EM structures of the spikes from bat (RaTG13) and pangolin (PCoV_GX) coronaviruses, which are closely related to SARS-CoV-2. All three receptor-binding domains (RBDs) of these two spike trimers are in the “down” conformation, indicating they are more prone to adopt this receptor-binding inactive state. However, we found that the PCoV_GX, but not the RaTG13, spike is comparable to the SARS-CoV-2 spike in binding the human ACE2 receptor and supporting pseudovirus cell entry. Through structure and sequence comparisons, we identified critical residues in the RBD that underlie the different activities of the RaTG13 and PCoV_GX/SARS-CoV-2 spikes and propose that N-linked glycans serve as conformational control elements of the RBD. These results collectively indicate that strong RBD-ACE2 binding and efficient RBD conformational sampling are required for the evolution of SARS-CoV-2 to gain highly efficient infection. studies revealed that the SARS-CoV-2 S trimer, similar to that of SARS-CoV, needs 58 to have at least one RBD in an "up" conformation to bind hACE2 17-23 . Therefore, a 59 spike trimer with all three RBDs "down" is in a receptor-binding inactive state, and 60 the conformational change of at least one RBD from "down" to "up" switches the 61 4 spike trimer to a receptor-binding active state 18 Overall structures of RaTG13 and PCoV_GX spikes 106 The overall structures of homotrimeric RaTG13 and PCoV_GX spikes resemble the 107 previously reported pre-fusion structures of coronavirus spikes (Fig. 1A ). Both spikes 108 have a mushroom-like shape (~150 Å in height and~120 Å in width), consisting of a 109 cap mainly formed by β-strands and a stalk mainly formed by α-helices (Fig. 1A) . 110 Like other coronaviruses, the RaTG13 and PCoV_GX spike monomers are composed 111 of the S1 and S2 subunits with a protease cleavage site between them (Fig. 1B,1C) . 112 The structural components of the spike include the N-terminal domain (NTD), RBD 113 (also called the C-terminal domain, CTD), subdomain 1 (SD1) and subdomain 2 (SD2) 114 in the S1 subunit; and the upstream helix (UH), fusion peptide (FP), connecting 115 region (CR), heptad repeat 1 (HR1), central helix (CH), β-hairpin (BH), subdomain 3 116 (SD3) and heptad repeat 2 (HR2) in the S2 subunit (Fig. 1D, Fig. S5 ). Table S1 . 419 A novel coronavirus associated with severe acute 421 respiratory syndrome Isolation of a novel coronavirus from a man with pneumonia 425 in Saudi Arabia A new coronavirus associated with human respiratory disease in 428 A Novel Coronavirus from Patients with Pneumonia in China A pneumonia outbreak associated with a new coronavirus of 432 probable bat origin Origin and evolution of pathogenic coronaviruses Identifying SARS-CoV-2-related coronaviruses in Malayan 437 pangolins Isolation of SARS-CoV-2-related coronavirus from Malayan 439 pangolins Are pangolins the intermediate host of the 2019 novel 441 coronavirus (SARS-CoV-2)? Associated with the COVID-19 Outbreak Recombination, reservoirs, and the modular 447 spike: mechanisms of coronavirus cross-species transmission SARS-CoV-2 Cell Entry Depends on ACE2 and 450 TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor Structure of the SARS-CoV-2 spike receptor-binding domain 453 bound to the ACE2 receptor Structural basis for the recognition of SARS-CoV-2 by 456 full-length human ACE2 Structural and Functional Basis of SARS-CoV-2 Entry by 459 18 Using Human ACE2 Structural basis of receptor recognition by SARS-CoV-2 Cryo-electron microscopy structures of the SARS-CoV spike 464 glycoprotein reveal a prerequisite conformational state for receptor binding Cryo-EM structure of the SARS 467 coronavirus spike glycoprotein in complex with its host cell receptor ACE2 Cryo-EM structure of the 2019-nCoV spike in the prefusion 473 conformation SARS-CoV-2 and bat RaTG13 spike glycoprotein 475 structures inform on virus evolution and furin-cleavage effects A pH-dependent switch mediates conformational masking of 478 SARS-CoV-2 spike. bioRxiv Receptor binding and priming of the spike protein of 480 SARS-CoV-2 for membrane fusion SARS-CoV-2 and three related coronaviruses utilize multiple 483 ACE2 orthologs and are potently blocked by an improved ACE2-Ig Structural insights into coronavirus entry Adaptation of SARS-CoV-2 in BALB/c mice for testing vaccine 488 efficacy A mouse-adapted model of SARS-CoV-2 to test COVID-19 countermeasures Functional and Genetic Analysis of Viral Receptor ACE2 Cryo-EM structures of MERS-CoV and SARS-CoV spike 495 glycoproteins reveal the dynamic receptor binding domains Glycans on the SARS-CoV-2 Spike Control the Receptor 498 Immunogenicity and structures of a rationally designed 501 prefusion MERS-CoV spike antigen EMAN2: an extensible image processing suite for electron 504 microscopy New tools for automated high-resolution cryo-EM structure 506 determination in RELION-3 MotionCor2: anisotropic correction of beam-induced 508 motion for improved cryo-electron microscopy Real-time CTF determination and correction Quantifying the local 513 resolution of cryo-EM density maps SWISS-MODEL: homology modelling of protein 516 structures and complexes UCSF Chimera--a visualization system for exploratory 519 research and analysis Coot: model-building tools for molecular graphics PHENIX: a comprehensive Python-based system for 525 macromolecular structure solution MolProbity: all-atom structure validation for 528 macromolecular crystallography EMRinger: side chain-directed model and map validation 531 for 3D cryo-electron microscopy 6VXX) RBD in wheat，and SARS-CoV-2 (PDB ID: 6ZGE) RBD in marine; 602 remaining regions shown in gray. (B) Detailed structures of the RBD-glycans 603 interface are shown 6ZGE/6VXX) RBDs are colored the same as in A. Glycans are shown as red sticks 605 and Asn-linked glycans are labeled. Sequence alignment of the SARS-CoV-2 RaTG13 and PCoV_GX RBD-interacting glycosylation sites is shown in the bottom 607 panel. Some sequences between the three sites are omitted and indicated by black dots Amino acid positions of asparagines are indicated above the sequences according to 609 Asparagines (N) are colored red and threonines (T) are colored blue Binding affinities and cell entr y of the differ ent spikes. (A) Binding curves 612 of immobilized hACE2 with the SARS-CoV-2, PCoV_GX or RaTG13 spike. Data are 613 shown as different colored lines and the best fit of the data to a 1:1 binding model is 614 shown in black. (B) The cell entry efficiencies of pseudotyped viruses as measured by 615 luciferase activity. SARS-CoV-2 C) The representative micrographs 617 and 2D classification results of negative-staining EM. Both spikes were incubated 618 with 4-fold molar ratio of hACE2. The red box shows the complex of the PCoV_GX 619 spike with hACE2 534 We thank the Tsinghua University Branch of China National Center for Protein Fig.4 The r esidues and glycans inter acting with one RBD of the differ ent spikes. 599 (A) The residues and glycans interacting with one RBD are shown as salmon spheres. 600 The RaTG13 RBD is colored in magenta, PCoV_GX RBD in green, SARS-CoV-2 601