key: cord-0878755-h64p2vao authors: Anand, Sai Priya; Chen, Yaozong; Prévost, Jérémie; Gasser, Romain; Beaudoin-Bussières, Guillaume; Abrams, Cameron F.; Pazgier, Marzena; Finzi, Andrés title: Interaction of human ACE2 to membrane-bound SARS-CoV-1 and SARS-CoV-2 S glycoproteins date: 2020-09-08 journal: bioRxiv DOI: 10.1101/2020.09.07.286567 sha: 02f999e6e7e815d286a7c89e71767e766acf4563 doc_id: 878755 cord_uid: h64p2vao A novel severe acute respiratory (SARS)-like coronavirus (SARS-CoV-2) is responsible for the current global coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The viral entry of SARS-CoV-2 depends on an interaction between the receptor binding domain of its trimeric Spike glycoprotein and the human angiotensin converting enzyme 2 (ACE2) receptor. A better understanding of the Spike/ACE2 interaction is still required to design anti-SARS-CoV-2 therapeutics. Here, we investigated the degree of cooperativity of ACE2 within both the SARS-CoV-2 and the closely related SARS-CoV-1 membrane-bound S glycoproteins. We show that there exist differential inter-protomer conformational transitions between both Spike trimers. Interestingly, the SARS-CoV-2 spike exhibits a positive cooperativity for monomeric soluble ACE2 binding when compared to the SARS-CoV-1 spike, which might have more structural restrains. Our findings can be of importance in the development of therapeutics that block the Spike/ACE2 interaction. CoV-1 membrane-bound S glycoproteins. We show that there exist differential inter-protomer 29 conformational transitions between both Spike trimers. Interestingly, the SARS-CoV-2 spike 30 exhibits a positive cooperativity for monomeric soluble ACE2 binding when compared to the 31 SARS-CoV-1 spike, which might have more structural restrains. Our findings can be of importance 32 in the development of therapeutics that block the Spike/ACE2 interaction. CoV-2 viruses [7, 8] , is a type I membrane protein [9] and the soluble version of ACE2 has been 48 shown to bind both S glycoproteins and block viral entry [10] . Currently, recombinant human 49 ACE2 is being tested as a treatment option for patients with COVID-19 to decrease viral 50 replication (NCT04335136). Phylogenetic analyses have demonstrated that SARS-CoV-2 and SARS-CoV-1 are closely related, 53 with an ~80% genomic sequence identity [11, 12] . Moreover, recent studies have also compared 54 the spike glycoproteins of SARS-CoV-1 and SARS-CoV-2, with a 76% amino acid sequence 55 identity between the two and a 74% amino acid sequence identity between their RBDs [13] which 56 directly contribute to the engagement of ACE2. Available structural and functional data reveal 57 several similarities in how both interact with ACE2: first, the contact interface of ACE2 and the 58 RBDs of the two spikes are largely similar [6] , second, ACE2 binding epitopes on both RBDs are 59 4 of 20 inaccessible in the fully closed spike conformation [6, 14] , third, effective receptor engagement 60 requires both the "up" orientation and a slight rotation of the RBD [15] , and fourth, in the ACE2-61 free condition, spike trimers on the virion surface can equilibrate between closed (3-RBD-down) 62 and open (one or more RBDs up) states [1, 2, 15, 16] . On the other hand, the existing structural 63 data for SARS-CoV-1 and the recent information that has become available in last few months 64 regarding the architecture and conformational status of the soluble or virion bound SARS-CoV-2 65 spike point toward important differences in their thermodynamics. The affinity of SARS-CoV-1 S 66 and SARS-CoV-2 S glycoproteins for soluble monomeric ACE2 (sACE2) has been determined, 67 with the latter having a 10 to 20-fold higher binding affinity. It has been suggested that this could 68 be a critical factor explaining the higher transmissibility of SARS-CoV-2 [1, 6, 17]. Additionally, 69 recent studies have characterized the nature of the interaction between an engineered dimeric 70 ACE2-Fc fusion protein and the trimeric SARS-CoV-2 S proteins, showing a high affinity 71 interaction superior to that seen with sACE2 [18, 19] . ACE2-Fc is also able to neutralize SARS- CoV-2 S more efficiently than SARS-CoV-1 S [20] . In this study, we attempted to better 73 understand the interaction between sACE2 or ACE2-Fc and the trimeric membrane-bound SARS-74 CoV-1 S or SARS-CoV-2 S glycoproteins by evaluating the cooperative binding of each of the 75 ligands and receptors. Our results further highlight conformational differences between the SARS- CoV-1 and SARS-CoV-2 spike glycoproteins. of 1 x 10 6 cells/mL at 37°C with 8 % CO2 with regular agitation (150 rpm). Cells were transfected 98 with a plasmid coding for soluble ACE2 or ACE2-Fc using ExpiFectamine 293 transfection 99 reagent, as directed by the manufacturer (Invitrogen). One week later, cells were pelleted and 100 6 of 20 discarded. Supernatants were filtered using a 0,22 µm filter (Thermo Fisher Scientific). The 101 recombinant sACE2 protein was purified by nickel affinity columns (Invitrogen) and ACE2-Fc 102 was purified using Protein A affinity column (Cytiva), as directed by the manufacturers. The 103 protein preparations were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots 104 at -80°C until further use. To assess purity, recombinant proteins were loaded on SDS-PAGE gels 105 and stained with Coomassie Blue. 148 sACE2, To better understand the interaction between membrane-bound SARS-CoV-1 and SARS-CoV-2 S 150 glycoproteins with their receptor, human ACE2, we sought to determine the cooperativity of ACE2 151 within the respective trimers. To asses this, we calculated the Hill coefficient, which is the 152 steepness of a concentration-response curve and reflects the degree of cooperativity between a 153 ligand and its receptor [23, 24] . Briefly, HEK293T cells were transfected with plasmids expressing 154 the full-length SARS-CoV-1 S and SARS-CoV-2 S glycoproteins. We also tested the SARS-CoV-155 2 S D614G mutant that is associated with higher infectivity and is now the strain circulating can engage adjacent S protomers. To evaluate if the differential Hill coefficients observed between 165 SARS-CoV-2 and SARS-CoV-1 was conserved among different RBD ligands, we tested two 166 additional RBD-binding ligands: ACE2-Fc, a molecule presenting two ACE2 domains (residues 167 1-615) fused to a Fc fragment and the CR3022 monoclonal antibody, which is specific to the 168 SARS-CoV-1 RBD and has been shown to cross-react strongly with the RBD of SARS-CoV-2 169 9 of 20 and does not compete with the binding of ACE2 [28, 29] . Interestingly, no differential 170 cooperativity between SARS-CoV-2 and SARS-CoV-1 was observed for ACE2-Fc, suggesting 171 that the enhanced avidity provided by ACE2-Fc which allows for multiple spike protomers to bind 172 is able to overcome potential structural restraints present in the SARS-CoV-1 S ( Figure 1B) . Of 173 note, we observed negative cooperativity of CR3022 for all tested S glycoproteins ( Figure 1C Next, we tested the ability of sACE2 and ACE2-Fc to neutralize SARS-CoV-1 and SARS-CoV-2 181 spike bearing pseudovirions. We observed a higher neutralization potency of sACE2 against 182 SARS-CoV-2 S (wt or D614G) when compared to SARS-CoV-1 (Table 1) and SARS-CoV-2 S with a higher efficiency compared to monomeric sACE2 ( Figure 2B ; Table 188 1), further supporting previous observations that ligand multimerization enhances potency by 189 providing higher avidity [18, 20, 31] . Interestingly, we observed that sACE2 IC50 was only reached In contrast, available data reveal differences in the conformational dynamics of the SARS-CoV-1 209 spike which seems to have less propensity to engage multiple ACE2 monomers. With a ratio of Based on the structural dynamic data described above we propose a model as shown in Figure 3 . SARS-CoV-2: red) that permit equilibration among four distinct states: all-RBD-down ("0"), one-414 RBD-up ("1"), two-RBD-up ("2"), and three-RBD-up ("3"). We hypothesize that the SARS-CoV- Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis Structural basis of receptor recognition by SARS-CoV-2 A pH-318 dependent switch mediates conformational masking of SARS-CoV-2 spike Structures and distributions of SARS-CoV-322 2 spike proteins on intact virions Characterization 324 of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for 325 development of RBD protein as a viral attachment inhibitor and vaccine Trimeric SARS-CoV-2 Spike interacts with 331 dimeric ACE2 with limited intra-Spike avidity SARS-CoV-2 and three related 334 coronaviruses utilize multiple ACE2 orthologs and are potently blocked by an improved 335 ACE2-Ig CoV-2 spike pseudotyped virus by recombinant ACE2-Ig The membrane-proximal 340 intracytoplasmic tyrosine residue of HIV-1 envelope glycoprotein is critical for basolateral 341 targeting of viral budding in MDCK cells Cross-sectional evaluation of 347 humoral responses against SARS-CoV-2 Spike The Hill equation revisited: uses and misuses Cooperativity in Binding 351 Processes: New Insights from Phenomenological Modeling Tracking Changes in SARS-CoV-2 Spike: Evidence that 358 D614G Increases Infectivity of the COVID-19 Virus Decline of humoral responses against SARS-CoV-2 Spike in convalescent individuals The D614G mutation in the SARS-CoV-2 spike protein reduces S1 shedding and 366 increases infectivity Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-369 specific human monoclonal antibody Human monoclonal antibody 373 combination against SARS coronavirus: synergy and coverage of escape mutants Neutralization of 379 SARS-CoV-2 by Destruction of the Prefusion Spike The sequence of human ACE2 is suboptimal for binding the S spike protein of 381 SARS coronavirus 2 Controlling the SARS-CoV-2 spike glycoprotein conformation Cryo-EM structure of the SARS coronavirus 386 spike glycoprotein in complex with its host cell receptor ACE2