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Engineered disulfide reveals structural dynamics of locked SARS-CoV-2 spike [1]

['Kun Qu', 'Structural Studies Division', 'Medical Research Council Laboratory Of Molecular Biology', 'Cambridge', 'United Kingdom', 'Infectious Diseases Translational Research Programme', 'Department Of Biochemistry', 'Yong Loo Lin School Of Medicine', 'National University Of Singapore', 'Qiuluan Chen']

Date: 2022-10 

The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct “S-R/x3” to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.

Spike (S) proteins on the surface of SARS-CoV-2 initiate viral infection by binding to cell receptors and mediating the fusion of virus and cell membranes. Several conformations of S have been identified that are thought to exist at different steps of the virus life cycle, for example assembly, receptor-binding and entry. The function of a conformation termed “locked” has not been clearly understood, due to its transience. Here, we engineered a disulfide bond in SARS-CoV-2 S to stabilise the locked conformation for structural and biochemical studies. This allowed us to distinguish two distinct locked-1 and locked-2 conformations in S from the initial SARS-CoV-2 strain, only the locked-2 conformation still exists after introduction of the D614G mutation. Based on structural and biochemical characterizations, we propose that the locked conformations of S prevent the premature opening of the receptor binding domain during virus assembly and egress through intracellular compartments. Our engineered S provides a useful tool to facilitate structural research, serological research, and design of immunogens.

Funding: This study was supported by funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (ERC-CoG-648432 MEMBRANEFUSION to JAGB), the Medical Research Council as part of United Kingdom Research and Innovation (MC_UP_A025_1011 to APC; MC_UP_1201/16 to JAGB), Wellcome (210711/Z/18/Z to APC), the Max-Planck Society (to JAGB), and the Natural Science Fund of Guangdong Province (2021A1515011289 to XX). XX acknowledges start-up grants from the Chinese Academy of Sciences and Bioland Laboratory (GRMH-GL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Considering the above observations, the role of the locked conformation in the SARS-CoV-2 life cycle is unclear. Here, based on our previous locked spike structure [ 4 ], we engineered a disulfide bond which stabilizes the SARS-CoV-2 spike in the locked conformation. This construct provides a tool to characterize the structure and dynamics of the locked form of S and to assess how mutations and other environmental factors could affect its structure and function. Our data reveal multiple distinct locked conformations and suggest that structural dynamics of domain D modulates the conformational state of the SARS-CoV-2 spike. Newly obtained information regarding the “locked” spike allowed us to consider its functional role in the context of SARS-CoV-2 life cycle.

Among the observed conformational states of SARS-CoV-2 spike, the locked conformation is unique in that it has not been observed for the highly related SARS-CoV-1 spike. Other coronavirus spikes do have locked-like conformations featuring FPPR motifs that preventing RBD opening, but for many of them, stochastic formation of an open conformation has not been observed [ 16 ]. The early cryo-EM studies of purified SARS-CoV-2 spike ectodomain did not identify the locked conformation, and we previously found that only a small fraction of trimeric ectodomains adopt the locked conformation [ 4 ], suggesting this conformation is transient. However, the locked conformation continued to be identified in other cryo-EM studies: spikes in locked conformation have been identified in purified full-length spike proteins including the transmembrane region [ 8 , 9 ]; insect-cell-expressed spike ectodomains can also adopt the locked conformation, perhaps due to stabilizing lipid in the insect cell media [ 10 ]. However, curiously, the locked conformation was not observed in the high-resolution structures of spike proteins on virions, where only closed and open conformation spikes were observed [ 17 ].

In the locked and closed conformations, the three copies of RBD in each spike trimer lie down on the top of the spike such that each RBD interacts in trans with the NTD of the neighbouring S protomer, hiding the receptor binding site. In the locked conformation, the three RBDs are in very close proximity at the 3-fold axis, and a disulfide-bond stabilized helix-turn-helix motif (FPPR motif) is formed below the RBD that can sterically hinder RBD opening [ 4 , 8 – 10 ]. Additionally, a linoleic acid is bound within the RBD and this ligand has been proposed to stabilize the locked conformation [ 10 ]. The locked conformation is adopted in solution by the Novavax NVX-CoV2373 vaccine candidate [ 8 ]. In structures of the locked conformation, both NTD and RBD are well resolved, suggesting that the locked spike has a rigid overall structure. In the closed conformation, the RBDs are slightly further apart than in the locked conformation and the FPPR motif is not folded [ 4 , 8 – 10 ]. As a result, the NTD and RBD exhibit considerable dynamics and are less well resolved than the core part of the protein in cryo-EM structures. Closed SARS-CoV-2 spike usually coexist with spikes exhibiting an open conformation [ 5 , 6 ]. In the open conformation, one or more RBDs are raised up to expose the receptor binding loops. ACE2 or antibody binding to these loops appears to maintain the spike in an open conformation [ 13 , 14 ]. It is believed that opening of the cleaved spike ultimately leads to structural transition into the postfusion conformation. The virus uses the dramatic structural refolding from the prefusion conformation to the lowest-energy postfusion conformation to drive membrane fusion [ 15 ].

SARS-CoV-2 spike protein has been captured in four distinct conformations by cryo-electron microscopy (cryo-EM): three prefusion conformations–locked [ 4 , 8 – 10 ], closed [ 5 , 6 ], open [ 5 , 6 ]; and one post-fusion conformation [ 9 ]. The different conformations of S expose different epitopes [ 11 ] and therefore induce different immune responses [ 12 ]. The different conformations must therefore play distinct roles in the infection cycle.

(A) Structure of the S-R/PP spike in the locked state (PDB: 6ZP2). Structural domains are coloured and labelled. The box indicates the location of the zoomed-in view in the right panel. Residues 427 and 987 that were mutated to cysteine to form the x3 disulfide bond are indicated. (B) Coomassie-stained SDS-PAGE gels to assess expression of the S-R/x3 and S-R/x3/D614G S proteins and confirm formation of a disulphide bond. NR or R indicates protein samples were prepared in non-reducing or reducing conditions. (C) Negative stain EM images of the purified S-R/x3 and S-R/x3/D614G spikes.

The S protein of SARS-CoV-2 is a large, 1273 residue, type I fusion protein, that can be processed by host proteases into two parts, S1 and S2. In the prefusion, trimeric form, S1 forms multiple folded domains. From N-terminus to C-terminus, they are the N-terminal domain (NTD, also called domain A, residues 13–307), receptor binding domain (RBD, also called domain B, residues 330–528), Domain C (residues 320–330 and 529–592), and Domain D (residues 308–319 and 593–699) [ 4 – 6 ]. Neutralising antibodies have been identified that target NTD and RBD [ 7 ]. S2 contains the fusion peptide (817–832), S2’ cleavage site (811–815), and a fusion peptide proximal region (FPPR) C-terminal to the fusion peptide (previously referred to by us as the 833–855 motif [ 4 ]) that we and others have previously suggested may take part in regulating RBD opening [ 4 , 8 , 9 ] ( Fig 1A ).

The spike (S) protein of coronaviruses is responsible for interaction with cellular receptors and fusion with the target cell membrane [ 1 ]. It is the main target of the immune system, therefore the focus for vaccine and therapeutics development. Most candidate Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines utilise S protein or its derivatives as the immunogen [ 2 ] and a number of antibodies targeting the SARS-CoV-2 S protein are under development for COVID-19 treatment [ 3 ].

Results and discussion

Design of a spike protein ectodomain stabilized in the locked conformation We did not previously observe a locked conformation for S-R (S with a deletion at the furin cleavage site leaving only a single arginine residue), while for S-R/PP (S-R additionally containing the widely-used, stabilizing double-proline mutation) the locked conformation was rare, <10% of S trimers when imaged by cryo-EM [4]. The scarcity of the locked conformation for the expressed ectodomain makes it difficult to study. To overcome this challenge, based on the locked SARS-CoV-2 S-R/PP structure [4], we engineered cysteines residues replacing positions D427 and V987 to generate a new disulfide-stabilized S protein S-R/x3. We predicted that S-R/x3 should be predominantly in the locked conformation (Fig 1A). Under non-reducing conditions, S-R/x3 ran exclusively as trimer in SDS-PAGE gel, and the trimer is converted to monomer under reducing conditions (Fig 1B). This behaviour is similar to our previously engineered S-R/x2, suggesting efficient formation of disulfide bond between the 2 engineered cysteines during protein expression. Negative stain electron microscopy (EM) images of purified S-R/x3 show well-formed S trimers (Fig 1C). We additionally introduced a D614G substitution into the S-R/x3 spike to obtain S-R/x3/D614G. Similar to S-R/x3, purified S-R/x3/D614G ran as disulfide-linked trimers in SDS-PAGE gel under non-reducing conditions, and negative stain EM images show well-formed trimers (Fig 1B and 1C).

Biochemical properties of the x3 spike protein We tested the sensitivity of the x3 disulfide bond to reduction by dithiothreitol (DTT), and compared this to the sensitivity of the previous described x2 disulfide bond, which stabilizes the spike in the closed state [4]. After 5 min of incubation with 2.5 mM DTT, the x3 disulfide in both S-R/x3 and S-R/x3/D614G spikes was substantially reduced, and 5 min of incubation with 20 mM DTT was sufficient for almost complete reduction (Fig 2A). In contrast, a 60 min incubation with 20 mM DTT was needed to achieve near-complete reduction of the x2 disulfide bond (Figs 2A and S1A). The x3 and x2 disulfides both include C987 in S2, therefore, different local chemical environments of the other engineered cysteines at position 427 and 413 respectively, are likely to be responsible for the difference in susceptibility to reduction by DTT. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Biochemical properties of purified x3 spikes comparing to x2 spikes. (A) Reduction of x3 and x2 disulfide bonds under native conditions by 5 min incubation with indicated concentrations of DTT, reactions were stopped by excess amount of iodoacetamide before SDS-PAGE. (B) binding of ACE2-Fc to different spike proteins in the absence and presence of 20 mM DTT. Spike proteins were serial diluted to 1500, 500, 166.7, 55.6, 18.5, 6.17, 2.06 and 0 nM and used as analytes in the assays. Kinetic parameters (k on , k off ) and dissociation constants derived from kinetic analyses (K Dkin ) are summarised in the table. https://doi.org/10.1371/journal.ppat.1010583.g002 We tested the receptor binding properties of S-R/x3 and S-R/x3/D614G in the absence and presence of DTT. In the absence of DTT, S-R/x3 and S-R/x3/D614G bind ACE2-Fc with weak responses (<0.4) and low affinities (>100 nM) (Fig 2B), while under similar conditions, S-R and S-R/PP bind ACE2-Fc with maximum responses between 0.9–1.0 and affinities of ~1 nM [12]. In the presence of 20 mM DTT, S-R/x3 and S-R/x3/D614G bound ACE2-Fc with maximum responses of 0.8–1.0 and affinities in the low nano-molar range (Fig 2B). In contrast, likely due to the inefficiency of x2 disulfide bond reduction, S-R/x2 and S-R/x2/D614G only moderately increased responses and affinities under reducing conditions (Fig 2B). These observations are consistent with our prediction that the x2 and x3 disulphide bonds prevent RBD opening and thereby prevent strong receptor binding, and that reduction of the x3 disulphide by DTT permits RBD opening and receptor binding. Entirely in line with the receptor binding data, we found that the antibody CR3022, which binds a cryptic epitope exposed only in the open RBD, is only able to bind S-R/x3 and S-R/x3/D614G spikes in the presence of DTT, and does not bind S-R/x2 and S-R/x2/D614G spikes (S1B Fig).

Cryo-EM Structures of S-R/x3/D614G Introduction of the D614G substitution into SARS-CoV-2 S spike ectodomain constructs with the double proline stabilizing modification has been reported to lead to a higher fraction of purified S in the open conformation [19,20]. Consistent with this, we found that 63% of purified S-R/D614G spikes adopted an open conformation (the remainder are in a closed conformation) (S2 Fig), compared to ~18% of S-R spikes in the open conformation [4]. We previously speculated that the D614G substitution alters the structure of the locked conformation, modulating spike structural dynamics [4]. Therefore, we determined the structure of the S-R/x3/D614G spike. The proportion of locked conformations is greatly reduced compared to S-R/x3, constituting only ~19% of total particles (S2 Fig). Classification did not identify two different locked conformations of the S-R/x3/D614G –all locked spikes were in the locked-2 conformation. As in the S-R/x3 locked-2 structure, residues 617–641 are ordered, and linoleic acid is bound (Fig 3D) confirming that the D614G spike retains the ability to bind linoleic acid, a molecule that has been suggested to be important for regulating conformational state of the spike [10]. Despite the presence of the x3 disulfide bond, 81% of the particles are in a closed conformation. The closed conformation accommodates the disulfide bond by motion of the RBD towards S2 (S5G Fig).

Structural transition of locked spike to closed conformation and the effect of low pH The S-R/x2 spike can be stored at 4°C for at least 30 days without significant loss of particles by negative stain EM [4]. We observed that the S-R/x3 spike can also be stored at 4°C for 40 days retaining well-formed trimers in negative stain EM. We performed cryo-EM imaging of the remaining trimers, finding that they had transitioned into the “closed” conformation (S2 Fig), and that linoleic acid was no longer bound in the RBD. This suggests that both the locked-1 and locked-2 conformations are metastable at pH 7.4, and spontaneously transition into the closed conformation. It is consistent with the general observation that most published ectodomain structures (not stabilized by the x3 crosslink) adopt the closed/open conformations rather than the locked conformation. We adjusted the buffer pH of the now “closed” spike to pH 5.0 and incubated overnight before determining its structure by cryo-EM. We found that low pH treatment had converted most of the closed spike to a conformation similar to the locked-2 conformation (Figs 3F and S2), however, in the acid reverted locked-2 structure the linoleic acid binding pocket is unoccupied (Fig 3F).

The structure of domain D differs in locked-1 and locked-2 conformations The locked-1 and locked-2 conformations differ primarily at residues 617–641 within domain D, and this structural perturbation is associated with differences in the FPPR motif and in residues around 318–319 which are located in the junction region connecting domain C and D (Figs 3A, 3B, 4A and 4B). In the S-R/x3 locked-1 structure, the long sidechains of R634 and Y636 interdigitate with the sidechains of F318 and Y837 through cation-π and π-π stacking interactions (dashed red lines in Fig 4A. In this interaction, R634 in domain D is sandwiched between F318 of the domain C/D junction region, and Y837 of the FPPR motif, bridging these three structural features. These interactions appear to be further stabilized by hydrophobic contacts between the aromatic sidechains of F318 and Y636 and the domain D hydrophobic core. This arrangement positions loop residue R634 approximately 9 Å above D614 (Fig 4A). It appears likely that this zip-locking interaction not only maintains residues 617–641 in a loop structure but also restrains motion of domain C relative to domain D. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Structural changes in domain D between spikes in locked-1, locked-2 and closed conformations. (A)—(F) structures of domain D in S-R/x3 and S-R/x3/D614G spikes of different conformations. Domain C is in light blue, Domain D green, FPPR yellow. Selected amino acid sidechains are shown and key amino acid sidechains are marked. The disordered loops are represented by dashed lines. C617 and N641 are marked with stars to highlight the dynamic region in between. π-π and cation-π interactions stabilising locked conformations are highlighted with dashed lines. Positions of hydrophobic residues with omitted side chains interacting with the domain D hydrophobic core are marked by black dots. The hydrophobic core formed by the domain D beta sheet is shown as a transparent molecular surface. (G) overlay of the S1 backbone from different conformations of the S-R/x3 spike. (H) the region within the box in panel (G) is zoomed to highlight the movements of the domain C/D junction and domain C between locked and closed conformations. https://doi.org/10.1371/journal.ppat.1010583.g004 In the locked-1 structure, we and others observed a salt bridge between D614 and K854 within the FPPR motif [4,9,10] (Fig 4A). We suggest that substitution of negatively charged D614 to neutral G alters the local electrostatic interactions, preventing R634 binding between F318 and Y837, and triggering the 617–641 loop refolding. This would provide an explanation for the absence of the locked-1 conformation in S-R/x3/D614G. In the locked-2 structure, 617–641 loop refolds into two short alpha helices, and the sidechains of F318 and R319 at the domain C/D junction reorient (Fig 4B). These structural changes allow formation of new electrostatic cation-π interactions between R319 and aromatic residues W633 and F592 (dashed red lines in Fig 4B) and formation of hydrophobic interactions between residues in the 617–641 loop and the hydrophobic core of domain D formed by the beta-sheet structure (Fig 4B). In the acid-reverted locked-2 structure of S-R/x3, F592 is no longer within cation-π interaction distance of R319 and instead forms cation-π interactions with H625 which is positively charged under low pH (Fig 4D).

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010583

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