hACE2-Induced Allosteric Activation in SARS-CoV versus SARS-CoV-2 Spike Assemblies Revealed by Structural Dynamics

Abstract

SARS-CoV and SARS-CoV-2 cell entry begins when spike glycoprotein (S) docks with the human ACE2 (hACE2) receptor. While the two coronaviruses share a common receptor and architecture of S, they exhibit differences in interactions with hACE2 as well as differences in proteolytic processing of S that trigger the fusion machine. Understanding how those differences impact S activation is key to understand its function and viral pathogenesis. Here, we investigate hACE2-induced activation in SARS-CoV and SARS-CoV-2 S using hydrogen/​deuterium-exchange mass spectrometry (HDX-MS). HDX-MS revealed differences in dynamics in unbound S, including open/​​closed conformational switching and D614G-induced S stability. Upon hACE2 binding, notable differences in transduction of allosteric changes were observed extending from the receptor binding domain to regions proximal to proteolytic cleavage sites and the fusion peptide. Furthermore, we report that dimeric hACE2, the native oligomeric form of the receptor, does not lead to any more pronounced structural effect in S compared to saturated monomeric hACE2 binding. These experiments provide mechanistic insights into receptor-induced activation of Sarbecovirus spike proteins.

Examining Functional Linkages Between Conformational Dynamics, Protein Stability and Evolution of Cryptic Binding Pockets in the SARS-CoV-2 Omicron Spike Complexes with the ACE2 Host Receptor: Recombinant Omicron Variants Mediate Variability of Conserved Allosteric Sites and Binding Epitopes

“…The recent HDX-MS studies of ACE2-induced allosteric activation of the S protein demonstrated allosteric changes upon ACE2 binding that extend to the hinge region and the top of the central helical bundle of the S2 subunit [38]. The experiments showed that ACE2 binding allosterically perturbs and primes HR1 and regions flanking the S1/S2 cleavage site for cleavage [36–38].…”

“…The recent HDX-MS studies of ACE2-induced allosteric activation of the S protein demonstrated allosteric changes upon ACE2 binding that extend to the hinge region and the top of the central helical bundle of the S2 subunit [38].…”

“…The experiments showed that ACE2 binding allosterically perturbs and primes HR1 and regions flanking the S1/S2 cleavage site for cleavage [36][37][38]. Targeting the predicted conserved cryptic pocket in the hinge apex region may potentially interfere with ACE2-induced destabilization of the HR1 helical structure (residues 930-960) that facilitates the pre-to-post-fusion spike conformational change.…”

“…HDX-MS studies of the unbound S protein trimer also showed the highest relative exchange in the S2 subunit and helical segments of the stalk regions (central helix CH and heptad repeats HR1 and HR2), while a comparative analysis of the S protein and S-ACE2 complex suggested that ACE2 binding allosterically enhances dynamics at a distal S1/S2 cleavage site and can modulate dynamics of the stalk hinge region, thereby highlighting the role of stalk and proteolysis sites as dynamic allosteric centers in the prefusion state [37]. Another HDX-MS study of S-ACE2 binding confirmed that the ACE2 can induce the enhanced dynamics throughout the entire S protein, with allosteric changes being propagated to the S2 hinge region and to the central helical bundle of the S2 subunit [38]. Overall, these studies suggested that a considerable conformational plasticity can be preserved in both open and stable closed S trimers including more rigid S2 subunit, while Omicron mutations and ACE2 binding can allosterically modulate the S dynamics at distal functional regions.…”

“…Strikingly, mutational scanning of the inter-protomer contacts in the XBB.1 trimer showed a distinct pattern, revealing fewer inter-protomer sites and visibly sparse distribution of these sites in the NTD and RBD (Figure 7C). Nonetheless, the distribution displayed several stability hotspots across all S regions including NTD (residues 38,43,47,135,168,199,200,225,232,235), RBD (residues 396,413), CTD1 (residues 560-563), (residues 1047,1079). These results suggested that XBB.1 trimer may be less densely packed and has smaller inter-protomer interface which may contribute to the reduced stability and greater plasticity of XBB.1 variant.…”