key: cord-1040075-yf83p34u
authors: Zhou, Tongqing; Tsybovsky, Yaroslav; Gorman, Jason; Rapp, Micah; Cerutti, Gabriele; Chuang, Gwo-Yu; Katsamba, Phinikoula S.; Sampson, Jared M.; Schön, Arne; Bimela, Jude; Boyington, Jeffrey C.; Nazzari, Alexandra; Olia, Adam S.; Shi, Wei; Sastry, Mallika; Stephens, Tyler; Stuckey, Jonathan; Teng, I-Ting; Wang, Pengfei; Wang, Shuishu; Zhang, Baoshan; Friesner, Richard A.; Ho, David D.; Mascola, John R.; Shapiro, Lawrence; Kwong, Peter D.
title: Cryo-EM Structures of SARS-CoV-2 Spike without and with ACE2 Reveal a pH-Dependent Switch to Mediate Endosomal Positioning of Receptor-Binding Domains
date: 2020-11-17
journal: Cell Host Microbe
DOI: 10.1016/j.chom.2020.11.004
sha: a076606a6d77396558a91c6b62de7dc78628df84
doc_id: 1040075
cord_uid: yf83p34u

The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the human ACE2 receptor and to facilitate virus entry, which can occur through low pH-endosomal pathways. To understand how ACE2 binding and low pH impact spike conformation, we determined cryo-EM structures –at serological and endosomal pH– delineating spike recognition of up to three ACE2 molecules. RBDs freely adopted ‘up’ conformations required for ACE2 interaction, primarily through RBD movement combined with smaller alterations in neighboring domains. In the absence of ACE2, cryo-EM structures revealed single-RBD-up conformations to dominate at pH 5.5, resolving into a solitary all-down conformation at lower pH. Notably, a pH-dependent refolding region (residues 824-858) at the spike-interdomain interface displayed dramatic structural rearrangements and mediated RBD positioning through coordinated movements of the entire trimer apex. These findings provide insight into how receptor interactions and endosomal pH alter RBD positioning and potentially facilitate immune evasion from RBD-up binding antibody.

Like other beta-coronaviruses, entry by SARS-CoV-2 involves its trimeric spike 55 glycoprotein, a type 1 fusion machine that undergoes large scale conformational changes between 56 prefusion and postfusion conformations to facilitate fusion of viral and host cell membranes. The 57 exact entry process for SARS-CoV-2 is still being defined, but is known to involve spike with spike trimer at a 6:1 molar ratio at pH 7.4 and collected single-particle cryo-EM data on a 100 Titan Krios. We obtained structures at 3.6-3.9 Å resolution and observed spike to bind ACE2 at 101 stoichiometries of 1:1, 1:2, and 1:3, with prevalences of 16%, 44%, and 40%, respectively 102 ( Figures 1A and S1 ; Table S1 ). ACE2 binding introduced trimer asymmetry. First, comparison of 103 single-RBD-up conformations of the spike, for ACE2-bound and ligand-free structures, revealed 104 recognition of ACE2 to induce a small (~2 Å) movement of RBD ( Figure S2) . Second, while the 105 membrane-proximal region of the spike in these complexes remained 3-fold symmetric, the ACE2- 106 binding regions showed asymmetry with, for example, superposition of the double-ACE2-bound 107 complex onto itself based on membrane-proximal regions leading to displacement of ACE2 108 molecules by almost 11 Å ( Figure 1B) . The full complement of trimer superpositions ( Figure   109 S3A) revealed preferential ways to align trimers moving from single-to double-to triple-ACE2 110 bound conformations. Analysis of domain movements indicated the large movement of RBD (from 111 down to up conformation) required to accommodate ACE2 binding to be accompanied by more 112 subtle movements of neighboring domains (Figure 2A) , and we delineated the coordinated 113 interprotomer domain movements that were involved in raising RBD (Figures 2B,C and S3 ). 114 However, the largest movement in S2 between single-and triple-ACE2-bound spikes occurred at 115 the flexible C-terminus of S2, with an overall rmsd for S2 subunit of <1 Å between single-, 116 double-, and triple-ACE2 bound trimers (Figure S3A,B) . Thus, ACE2-receptor engagement 117 required RBD to be in the 'up' position, but we could see no clear evidence for the binding of 118 ACE2 priming S2 for substantial structural rearrangement, beyond the raising of RBD and a 119 reduction of RBD interactions with S2. 7 particles had all RBDs down. For all three of these prevalent classes, unlike the consensus 146 structure, density for all RBD domains was well resolved (Figure S4C, panel C) , indicating 147 multiple different orientations of RBD in the spike at pH 5.5. In the remaining classes, the RBD 148 did not assume a defined position, suggesting RBD mobility at pH 5.5. 149 To determine how even lower pH impacted spike conformational heterogeneity, we sought 150 to obtain a cryo-EM structure of the ligand-free spike at even lower pH. We collected cryo-EM 151 datasets at both pH 4.5 and 4.0. Single particle analysis of the pH 4.5 dataset comprising 603,476 152 particles resolved into an all-RBD-down conformation, and we refined this map to 2.5 Å resolution 153 ( Figures 3B and S4 , Table S3); single particle analysis of the pH 4.0 dataset comprising 911,839 154 particles resolved into a virtually identical all-RBD-down conformation (root-mean square 155 deviation (rmsd) between the two structures of 0.9 Å) (Figures 3C and S4 , Table S3 ). The 156 similarity of the pH 4.5 and pH 4.0 structures indicated spike conformational heterogeneity to be 157 reduced between pH 5.5 and 4.5, and then to remain unchanged as pH was reduced further. The pH 158 4.0 map was especially well-defined at 2.4-Å resolution (Table S3) Refolding at spike domain interfaces underlies conformational rearrangement 163 To identify critical components responsible for the reduction of conformational heterogeneity 164 between pH 5.5 and lower pH and to shed light on the spike mechanism controlling the positioning 165 of RBDs, we analyzed rmsds between the pH 5.5 structures and the all-down pH 4.0 conformation 166 with an 11-residue sliding window to identify regions that refolded (Figures 4A, top, and S5) . The switch domain, which included aspartic acid residues at 830, 839, 843 and 848 and a 207 disulfide linkage between Cys840 and Cys851, was located at the nexus of SD1 and SD2 from one 208 protomer, and HR1 (in the S2 subunit) and NTD from the neighboring protomer. This region showed Unprotonated-switches were exemplified by switches B and C at pH 5.5 and perhaps best by 213 switch B in the pH 5.5 single-RBD up structure ( Figure 5A, C, (Figure 5C, left) . Notably, all four of the unprotonated-switch aspartic acids faced solvent 219 and appeared to be negatively charged. Figure S6B ). 269 With CR3022 IgG, apparent affinities to spike and RBD were sub-nanomolar at serological pH, 270 though with a 10-fold difference (0.49 and 0.052 nM to spike and RBD, respectively) ( Figure 7C ). 271 At pH 5.5, this 10-fold difference was retained (1.7 and 0.23 nM, respectively). However, at pH 272 4.5, CR3022 still bound to RBD (1.1 nM), but its apparent affinity to spike was dramatically 273 reduced with a K D >1000 nM -an apparent affinity difference we estimated to be >1000-fold 274 ( Figures 7C and S6C) . Because CR3022 still bound strongly to the isolated RBD, we attributed 275 the dramatically reduced apparent affinity of CR3022 for spike at low pH to conformational 276 constraints of the spike ( Figure 7D ). 277 Overall, the pH-induced retraction of RBDs through the spike adopting an all-down 278 conformation can be described as a "conformational masking"-energy barrier, which CoV-2 spike evasion from CR3022 neutralization to depend on the reduced affinity of al., 2020) provide additional contexts by which to interpret the structural results described here. 315 Benton and colleagues suggest three-ACE2 to destabilize the prefusion spike, but in the context of 316 ACE2-bound to '2P'-stabilized spikes, no substantial changes in S2 conformation were induced by 317 ACE2 binding. Meanwhile, the fascinating motions described by Ke and colleagues and by 318 Turoňová and colleagues involve regions of spike that are below the ordered regions of S2 that we 319 described here. Lastly, smFRET analysis suggest an on-path intermediate as the basis for the 320 observed ACE2-induced trimer asymmetry; it will be fascinating to see if smFRET analysis of 321 soluble trimers and at endosomal pH can provide insight into the pH-induced alterations in spike 322 conformation that we observe here. 323 We note that the critical switch region (residues 824-858) displays remarkable structural 324 diversity within coronaviruses, segregating into three structural clusters ( Figure S7) . Each of the 325 structures within these clusters generally comprises two helices, linked by a disulfide, in distinct (Punjani et al., 2017) . We note that some classes of unbound spike were also observed in both 562 datasets; however, particle picking was optimized for complexes so the fraction was low. The 3D 563 reconstructions were performed using C1 symmetry for all complexes as the ACE2-RBD region 564 showed flexibility that prohibited typical symmetry operations in the triple-bound complexes. 565 However, the RBD-ACE2 region was assessed in greater detail through focused refinement A829  D830  A831  G832  F833  I834  K835  Q836  Y837  G838  D839  C840  L841  G842  D843  I844  A845  A846  R847  D848  L849  I850  C851  A852  Q853  K854  F855 Late endosomeearly lysosome pH 5.5-4.5

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