SARS-CoV-2 S protein ACE2 interaction reveals novel allosteric targets

The Spike (S) protein is the main handle for SARS-CoV-2 to enter host cells through surface ACE2 receptors. How ACE2 binding activates proteolysis of S protein is unknown. Here, we have mapped the S:ACE2 interface and uncovered long-range allosteric propagation of ACE2 binding to sites critical for viral host entry. Unexpectedly, ACE2 binding enhances dynamics at a distal S1/S2 cleavage site and flanking protease docking site ~27 Å away while dampening dynamics of the stalk hinge (central helix and heptad repeat) regions ~ 130 Å away. This highlights that the stalk and proteolysis sites of the S protein are dynamic hotspots in the pre-fusion state. Our findings provide a mechanistic basis for S:ACE2 complex formation, critical for proteolytic processing and viral-host membrane fusion and highlight protease docking sites flanking the S1/S2 cleavage site, fusion peptide and heptad repeat 1 (HR1) as allosterically exposed cryptic hotspots for potential therapeutic development. One Sentence Summary SARS-CoV-2 spike protein binding to receptor ACE2 allosterically enhances furin proteolysis at distal S1/S2 cleavage sites

The COVID-19 pandemic caused by the SARS-CoV-2 virus has sparked extensive efforts 44 to map molecular details of its life cycle to drive vaccine and therapeutic discovery.(1) SARS- class I viral fusion protein that has distinctive 'head' and 'stalk' regions ( Fig. 1A). 54 A characteristic feature of SARS-CoV-2 is proteolysis of Pre-fusion S protein by host 55 proteases into S1 and S2 subunits. The S1 subunit comprises an N-terminal domain (NTD) and a 56 receptor binding domain (RBD) that interacts with the host receptor Angiotensin converting 57 enzyme-2 (ACE2)(10, 11) to initiate viral entry into the host.(12) Cryo-electron tomography (cryo-58 ET) has been used to capture the distribution and organization of trimeric S protein on the intact 59 virion,(9) revealing that 25 ± 9 S protein trimers decorate a single virion with a small percentage 60 (3%) of embedded S proteins in a post-fusion state adopting an extended helical conformation. 61 The first virus-host interaction is mediated by the viral S protein with the host ACE2 receptor.(10) 62 Binding to ACE2 primes the S protein for proteolysis at S1/S2 cleavage site into individual S1 and

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Localizing subunit specific dynamics and domain motions of S protein trimer 81 Structural snapshots of the ACE2 binding to the SARS-CoV-2 S protein interface have 82 been obtained with the RBD alone.(10, 16, 21-23) In this study, we have mapped this interface for 83 the S protein construct (1-1208) with mutations at the S1/S2 cleavage site (PRRAS to PGSAS) 84 and proline substitution at 986-987,(16) to block proteolysis during expression and purification 85 (Fig. S1A). The S protein and isolated RBD constructs showed high affinity binding to ACE2 (Fig.   86 S1B). We measured dynamics of a trimer of this near-full length S protein by amide hydrogen-87 deuterium exchange mass spectrometry (HDXMS). Pepsin proteolysis generated 321 peptides 88 with high signal to noise ratio, accounting for ~87% of the entire S protein (Fig. S2). Glycosylation of at least 22 sites have been predicted on S protein.(24) Average deuterium exchange at these 90 reporter peptides was monitored for comparative deuterium exchange analysis of S protein, ACE2 91 receptor and S:ACE2 complex, along with a specific ACE2 complex with the isolated RBD. While 92 glycosylation is an important posttranslational modification, our HDXMS study has measured 93 deuterium exchange of non-glycosylated segments of S protein alone. Deuterium exchange (t = 1 94 and 10 min) across all peptides of the free S protein trimer are shown in (Fig. 2). We built an around the HR2 domain, compared to the rest of the protein (Fig. S3, Fig. S4).

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The deuterium exchange heat map showed the highest relative exchange in the S2 subunit 105 (Fig. S3) and helical segments, while peptides spanning the fusion peptide showed relatively lower 106 deuterium exchange. Individually, S1 and S2 subunits showed different intrinsic deuterium 107 exchange kinetics, where the average relative fractional deuterium uptake (RFU) of S1 subunit 108 (~0.25) was lower than the average RFU (~0.35) of S2 subunit (Fig. S3, Table S1). Moreover, 109 peptides connecting the RBD to the remainder of the S protein showed greater deuterium the 'down'-conformation relative to the hinge region (Fig. 2D, Fig. S4A). Interestingly, a part of 114 the receptor binding motif, specifically residues 476-486, exhibited a higher degree of flexibility 115 based on its average atomic fluctuations ( Fig. 2A, 4C), suggesting that binding to ACE2 receptor 116 would be required to stabilize its motion.

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The NTD of the S protein showed low overall RFU (~0.2), consistent with its well-118 structured arrangement of β-sheets connected by loops (Fig. 1B). Importantly, certain regions

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Overall, the S2 subunit showed relatively higher RFU than the S1 subunit, with each 130 domain exhibiting specific conformational changes (Fig. 1E, Fig. S4). Peptides spanning the 131 region immediately downstream of the S1/S2 cleavage site showed the highest deuterium uptake 132 (0.6), reflecting the rapid dynamics it undergoes for facilitating cleavage of S protein into two 133 subunits. Congruently, our MD simulations revealed the unstructured loop housing the S1/S2 134 cleavage site (residues 677-689) to be highly dynamic ( Figure S4C), with RMSFs reaching >1.0 135 nm. It is important to note that the S1/S2 cleavage site has been abrogated in the construct of the 136 S protein used in this study to block proteolytic processing into S1 and S2 subunits during 137 expression in host cells. We thus observed lower deuterium uptake (and lower RMSF values) at 138 peptides in the central helix and connector domain, suggesting that these act as the central core of   critically at the S1/S2 cleavage sites (Fig. 4D). Even though the construct used in this study has 187 the proteolysis site mutated, it still resulted in increased dynamics at this S1/S2 locus. Furthermore, 188 this region exhibited high RMSF values during simulations. (Fig. S4B). These results clearly 189 indicate that ACE2 binding induces allosteric enhancement of dynamics at this locus, providing 190 mechanistic insights into the conformational switch from the pre-fusion to fusogenic intermediate.

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Differences in deuterium exchange between free S protein and S:ACE2 complex shows 192 stabilization at ACE2 interacting site and local destabilization at peptides juxtaposed to S1/S2 193 cleavage site (residues 931-938). This suggests that ACE2 binding potentiates peptide of residues 194 931-938 and other high exchanging regions flanking the S1/S2 cleavage site for enhanced furin 195 protease binding and cleavage. Importantly, these results suggest that the S1/S2 cleavage site is a 196 critical hotspot for S protein dynamic transitions for viral entry into the host, and therefore 197 represents a new target for inhibitory therapeutics against the virus. 199 Considering the indispensable role of ACE2 binding in SARS-CoV-2 infection, it is crucial to 200 assess the effects of S protein and RBD binding on ACE2 dynamics. We therefore mapped the 201 corresponding binding sites of RBD, both isolated and within the Spike, on ACE2. The S:ACE2 202 complex represents the prefusion pre-cleavage state wherein full-length S protein is bound to the 203 ACE2 receptor (Fig. 1B ii), while the RBDisolated:ACE2 complex represents the post-furin cleavage 204 product formed by the S1 subunit and ACE2 (Fig. 1B iii). Previous studies have shown that 14 key 205 amino acids of RBD interact with ACE2, wherein mutations at 6 amino acids resulted in higher  (Fig. S7, Fig. S8). We observed a reduction in  conformational changes promoting Furin (red) proteolysis at the S1/S2 cleavage site (red arrows, 337 leading to dissociation of S1 and S2 subunits, mechanism of which is unknown. (iii) The residual 338 ACE2-bound S1 subunit stably bound to ACE2 and S2 subunits dissociate (iv) Conformational (RBDS) mapped on to the structure of RBD extracted from S protein model (see Table S2). High