key: cord-1015711-1dhtpjdb authors: Oliveira, A. Sofia F.; Ibarra, Amaurys Avila; Bermudez, Isabel; Casalino, Lorenzo; Gaieb, Zied; Shoemark, Deborah K.; Gallagher, Timothy; Sessions, Richard B.; Amaro, Rommie E.; Mulholland, Adrian J. title: A potential interaction between the SARS-CoV-2 spike protein and nicotinic acetylcholine receptors date: 2021-02-18 journal: Biophys J DOI: 10.1016/j.bpj.2021.01.037 sha: 38109cc6801bd14c3946a436264bf400e375a17a doc_id: 1015711 cord_uid: 1dhtpjdb Changeux et al. recently suggested that the SARS-CoV-2 spike protein may interact with nicotinic acetylcholine receptors (nAChRs), and that such interactions may be involved in pathology and infectivity. This hypothesis is based on the fact that the SARS-CoV-2 spike protein contains a sequence motif similar to known nAChR antagonists. Here, we use molecular simulations of validated atomically detailed structures of nAChRs, and of the spike, to investigate the possible binding of the Y674-R685 region of the spike to nAChRs. We examine the binding of the Y674-R685 loop to three nAChRs, namely the human α4β2 and α7 subtypes and the muscle-like αβγδ receptor from Tetronarce californica. Our results predict that Y674-R685 has affinity for nAChRs. The region of the spike responsible for binding contains a PRRA motif, a four-residue insertion not found in other SARS-like coronaviruses. The conformational behaviour of the bound Y674-R685 is highly dependent on the receptor subtype: it adopts extended conformations in the α4β2 and α7 complexes, but is more compact when bound to the muscle-like receptor. In the α4β2 and αβγδ complexes, the interaction of Y674-R685 with the receptors forces the loop C region to adopt an open conformation, similar to other known nAChR antagonists. In contrast, in the α7 complex, Y674-R685 penetrates deeply into the binding pocket where it forms interactions with the residues lining the aromatic box, namely with TrpB, TyrC1 and TyrC2. Estimates of binding energy suggest that Y674-R685 forms stable complexes with all three nAChR subtypes. Analyses of simulations of the glycosylated spike show that the Y674-R685 region is accessible for binding. We suggest a potential binding orientation of the spike protein with nAChRs, in which they are in a non-parallel arrangement to one another. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel strain of coronavirus that first appeared in China in late 2019 and causes the potentially fatal disease COVID-19. This virus initially infects respiratory epithelial cells by binding to the angiotensin-converting 2 enzyme (ACE2) receptor (1) . Although predominantly recognized as a respiratory disease (2, 3) , SARS-CoV-2 also causes severe inflammation and damage in other organs (4) (5) (6) (7) . Under certain conditions (and as with other coronaviruses (8) ), SARS-CoV-2 may enter the central nervous system (CNS) through the bloodstream by disrupting the blood-brain barrier or infecting the peripheral nerves (e.g. (7, (9) (10) (11) (12) ). Since it emerged as a human pathogen, SARS-CoV-2 has caused more than 80.8 million confirmed cases of COVID-19 and more than 1.7 million deaths worldwide, as of 28 th December, 2020 (13) . Several major risk factors for the development of COVID-19 have been identified, including age, J o u r n a l P r e -p r o o f heart disease, diabetes and hypertension (14) . Given the apparently low prevalence of smokers among hospitalised COVID-19 patients (15) (16) (17) , it was proposed that nicotine may offer some protective value to mitigate COVID-19 (the 'protection' hypothesis) (15) . It has been suggested that medicinal nicotine (either in patches, gum, or electronic delivery systems) should be investigated as a therapeutic option for this disease (e.g. (15, 18) ). Clinical trials for nicotine are underway (e.g. https://clinicaltrials.gov/ct2/show/NCT04429815). Alternative explanations to the protection hypothesis have been proposed (19) : the first relates to the failure in correctly identifying smokers upon hospital admission (19) , and the second is that hospitalised COVID-19 patients may be less likely to smoke as their comorbidities motivate them to quit ('smoking cessation' hypothesis) (19) . Based on the early observations of the lower than expected smoking prevalence in hospitalised COVID-19 patients, Changeux and colleagues suggested a role for nicotinic acetylcholine receptors (nAChRs) in the pathophysiology of COVID-19 via a direct interaction between these receptors and the viral spike glycoprotein (20) . This suggestion was based in the fact that the spike protein contains a sequence motif similar to known nAChR antagonists (20) (Figure S1 ), such as α-bungarotoxin from Bungarus multicinctus and glycoprotein from Rabies lyssavirus (formerly Rabies virus). Changeux et al. and others also proposed that COVID-19 might be controlled or mitigated by the use of nicotine, if the latter can compete with the virus for binding to these receptors (e.g. (9, 18, (20) (21) (22) (23) (24) ). If interactions with nAChRs are important, they may be relevant for some of the systemic effects observed in nAChRs are cation channels that belong to the pentameric ligand-gated ion channel family (25) . They are present in both the peripheral (at the skeletal neuromuscular junction and in the autonomic nervous system) and CNS (26) . The neuronal receptors have emerged as important targets for the treatment of Alzheimer's disease, schizophrenia, pain and nicotine addiction (26, 27) . Mutations of muscle nAChR can cause congenital myasthenia gravis (27) . A large repertoire of nAChR subtypes differ in the homo-or heteromeric assembly of five monomers arranged around a central channel axis (28) (29) (30) . Each nAChR subtype shows different selectivity for agonists and antagonists (28) (29) (30) . All nAChRs share the same basic architecture (Figure 1B) , formed of a large N-terminal extracellular domain (ECD), where the agonist binding site is located; a transmembrane domain (TMD) surrounding the ion channel; an intracellular domain (ICD); and a short extracellular C-terminal domain (CTD) (28) (29) (30) The ligand-binding pocket is located at the interface between two neighbouring subunits ( Figure 1B) and is formed by loops A, B and C from the principal subunit and D, E and F from the complementary subunit ( Figure S2) . The α4β2 nAChR is the most prevalent heteromeric subtype in the brain: it is implicated in diverse processes such as cognition, mood and reward, and is necessary for nicotine addiction (28) (29) (30) (31) . The homomeric α7 nAChR is also abundant and widely expressed in the CNS, where it contributes to cognition, sensory processing and attention (32) . The α7 subtype is also expressed on a variety of non-neuronal cells, such as immune cells, J o u r n a l P r e -p r o o f astrocytes, microglia, endothelial cells, where it contributes to anti-inflammatory pathways (33) (34) (35) . Due to its role in the downregulation of the production of pro-inflammatory cytokines (33) (34) (35) , it has been suggested that the α7 nAChR may be involved in the hyper-inflammation response that can be caused by SARS-CoV-2 (9, 18, 24, 36) . The muscle-type receptor derived from the electric organ of Tetronarce californica (formerly Torpedo californica) is one the most extensively studied nAChRs, and has provided significant structural insight into this receptor family. It is formed by two α and one each of β, δ and γ subunits, and has high sequence similarity (55%-80% identity) with its human counterpart (37) . For this reason, and because its structure is available (38) , we used it in this work as a proxy for the human muscle-type nAChRs. Muscle fatigue, myalgia and arthralgia are common symptoms in COVID-19 patients (e.g. (39) (40) (41) ). However, it is still unclear if these symptoms result from direct muscle damage from viral infection or from the body's inflammatory response (7, 39) . According to Changeux et al.'s 'nicotinic hypothesis', direct interaction between SARS-CoV-2 and nAChRs occurs via a small region in the viral spike protein (20) (Figures S3 and S4) . The spike is a fusion protein (42, 43) found on the surface of the virion that mediates entry into host cells. It is an extensively glycosylated homotrimer, with each monomer formed by three domains ( Figure 1A) : head, stalk and cytoplasmic tail (CT) (42) . The head comprises two subunits: S1, which binds the ACE2 receptor on host cells (42) , and S2, which facilitates membrane fusion (42) . The spike contains two proteolytic cleavage sites (42) : one ('furin cleavage' site) at the S1/S2 boundary thought to activate the protein (44) and a second in the S2 subunit that releases the fusion peptide (45) . The region suggested by Changeux et al. to be directly involved in the interaction with nAChRs spans from Y674 to R685 and is located in the head region of the protein, at the interface between the S1 and S2 domains, immediately preceding the S1/S2 cleavage point (42) (Figures 1A, S3 and S4 ). Furin cleaves the peptide bond after R685, thus separating it from its neighbour S686 (e.g. before viral exit from the host cell) (44) . Cleavage activation of viral glycoproteins is known to be important for infectivity and virulence (42, 44) . The Y674-R685 region contains a 4-residue, polybasic PRRA insertion not present in other SARS-CoV-related coronaviruses (46) that is homologous to several neurotoxins known to target nAChRs (20) . In SARS-CoV-2, abrogation of the PRRA motif moderately affects virus entry into cells (42, 44) . This motif has recently been shown experimentally to interact with neuropilin-1 receptors (47), T cell receptors (48) , and host cell glycans, such as heparin sulfate (49, 50) . The high sequence similarities between the Y674-R685 region and several known nAChR antagonists ( Figure S1) suggests that this region of the SARS-CoV-2 spike protein may bind to nAChRs, potentially acting as an antagonist (20) . Hence, it has been postulated that nicotine may have an effect in COVID-19 by competing and interfering with this binding. Note that an alternative region (G381 to K386 in the S1 subunit) in the spike protein has been hypothesized to interact with nAChRs (51), but glycosylation makes this unlikely. Here, we use molecular simulations to examine the 'nicotinic hypothesis' proposed by Changeux et al. (20) , in particular to test whether the SARS-CoV-2 spike protein can bind stably to nAChRs via the Y674-R685 region and identify interactions that may be involved in the stabilisation of the complexes. To test this, we have built structural models for the complexes formed by the 12-residue region from the spike (S-peptide) and the ECDs of three different nAChRs, namely the human α4β2, human α7 and muscle-like αβγδ receptor from Tetronarce californica (hereafter named αβγδ). These simulations build on our successful previous extensive simulations of nAChRs, which have e.g. identified a general mechanism for signal propagation in this receptor family (52) (53) (54) , and simulations of the spike (55-58) and its interactions (47, 59) . Structural models of the three SARS-CoV-2 S-peptide-nAChR complexes were built based on the cryoEM structure of the αβγδ receptor from Tetronarce californica with bungarotoxin (38) . αbungarotoxin is a neurotoxin that acts as a nAChR antagonist, directly competing with acetylcholine (60) , and has high sequence similarity with the Y674-R685 region of the spike protein of SARS-CoV-2 ( Figure S1 ). Twenty models were generated for each complex, and the one with the lowest Modeller objective function (61) (Figures 2 and S7 ) was used as the starting point for MD simulations (see the Supporting Information for more details). Three replicate simulations, each 300 ns long, were performed for each complex to investigate the peptide-receptor conformational behaviour and possible induced-fit effects. At the beginning of the simulations, the S-peptide was located in the binding pocket, bound by interactions with both the principal and complementary subunits (Figures 2 and S7) . A closeup view of the peptide-receptor interface reveals extensive contacts (Figures 2B and S7B) , mainly with the J o u r n a l P r e -p r o o f principal subunit. In all three complexes, the side-chain of R682 of the S-peptide binds as the recognised positively charged group, a strictly conserved pharmacophore of all nAChR ligands (62, 63) . As can be seen in Figure 2B , the guanidinium group of R682 is well-positioned inside the aromatic box, forming several cation-π interactions with TyrC1 (α4Y223, α7Y210, αY214 in the human α4β2, human α7 and muscle-like αβγδ receptor from Tetronarce californica, respectively), TyrC2 (α4Y230, α7Y217, αY222) and TyrA (α4Y126, α7Y115, αY117). Note that these cation-π interactions do not entirely mimic the binding of nicotine, as no interactions with TrpB are present (64) . R682 is part of the four-residue PRRA insertion not found in other SARS-like coronaviruses (46) , and it forms part of the furin cleavage site located the boundary between the S1 and S2 subunits (42) . Additional binding interactions with the peptide are also observed with different residues depending on the receptor subtype: in the α4β2 nAChR, hydrogen bonds involving the side-chains of α4Y223, α4E224, β2S192, β2D195 in the receptor and Q675, N679 and the main-chain nitrogen of A684 of the S-peptide are observed; in the α7 nAChR, two hydrogen bonds between α7D186 and α7Y210 in the receptor and S-peptide Q675 and Q677 are seen; in the αβγδ receptor from Tetronarce californica, hydrogen bonds involving αY214 and δD186 from the receptor and Q675, N679, R682 and R683 of the peptide are observed. The simulations show distinct patterns of dynamical behaviour for the S-peptide in the different receptor subtypes. In the α4β2 and α7 complexes, the peptide showed high positional and conformational variability, while in the αβγδ complex, it generally remained in the same pose throughout the simulation (Figures S8 and S10) . Similar behaviour is observed for the peptides in the two binding pockets in each complex. When bound to the α4β2 and α7 nAChR, the peptide adopted many different binding modes inside the pocket, ranging from highly compact to fully extended conformations ( Figure S10 ). In contrast, in the αβγδ receptor, the peptide was more compact ( Figure S10 ). The range of the radius of gyration values for the S-peptide in all three complexes is similar to that observed in the simulations of the full-length glycosylated SARS-CoV-2 spike protein embedded in a viral membrane (55) (Figure S6 ). Principal component analysis (PCA) of the peptide dynamics revealed different conformational behaviour of the peptide in the three complexes. When bound to the muscle-like receptor, the peptide shows limited dynamical freedom: it explores a restricted conformational space spanned by the first two principal components ( Figure S11 ). The number of hydrogen bonds between the peptide and the receptors over the simulations was determined ( Figure S12) . Two more H-bonds are observed in the αβγδ complex than in the α4β2 and α7 receptors ( Figure S12) . These additional interactions with the complementary subunit ( Figure S12 ) probably contribute to the increased stability of this complex and the more compact conformation of the peptide in the αβγδ receptor. Analysis of the distribution of the distance between the R682 of the peptide and the conserved aromatic residues forming the aromatic box shows the distinctive behaviour of the peptide when bound to different receptors ( Figure S13) . Interactions with R682, TyrC1 and TyrC2 are quite frequent in all three complexes, being present more than 60% of the time. To examine how deeply into the binding pocket the peptide inserts, we monitored the interactions of R682 with TrpB, a residue lining the back wall of the nAChR aromatic box. TrpB (α4W182, α7W171 and αW173) is highly conserved across the nAChR family, and it makes cation-π and H-bond interactions with the positively charged group on the ligands (62, 63) . In the α4β2 and αβγδ complexes, the S-peptide does not extend far into the pocket and interactions between R682 and TrpB are mostly absent ( Figure S13 ). In contrast, in the α7 complex, the peptide binds more deeply into the hydrophobic cavity, adopting conformations that allow not only for the direct contact between R682 and TrpB (Figures S14-S15) but also achieve optimal core-binding interactions (Figure 3) . In such configurations, other interactions are present in addition to those with TrpB, namely cation-π interactions with TyrC1 and TyrC2 ( Figure S15) . Although no direct contact between R682 and TyrA is observed, both residues are connected through a H-bond network mediated by Q675 from the S-peptide ( Figure S16 ). This is significant because interactions with TyrA, TrpB, TyrC1 and TyrC2 are known to be critical for ligand binding and to modulate gating in the α7 subtype (65-67). The binding of a ligand or a peptide can be expected to affect the conformational dynamics of the receptors (e.g. (52) (53) (54) (68) (69) (70) ). To investigate this, the Root Mean Square Fluctuation (RMSF) profiles of the C α atoms were determined for all three receptors. Distinct dynamic behaviours are observed for the binding site regions in the different subtypes (Figures S17-S19) . These differences are mostly in loops C and F, two structural motifs important for binding and selectivity (66, 71, 72) . Loop F shows decreased flexibility in the α4β2 complex, while loop C dynamics is more restricted in the muscle-like αβγδ receptor, compared to the other two subtypes. Table S1 for the ∆G bind for the individual replicates) and do not contain the entropic contribution to the binding energy. Average ∆G bind for the complexes (kJ/mol) α4β2 α7 α α α αβ β β βγδ (Tables 1 and S1 ). MM-PBSA calculations are an efficient and often useful method to estimate binding free energies (74, 75) , and are widely used to study protein-ligand interactions in medicinal chemistry (76) (77) (78) including in drug design for nAChRs (79, 80) . The favourable calculated binding energies suggest stable complex formation between the S-peptide and all three nAChRs (Tables 1 and S1), with different binding affinities depending on the subtype. In silico alanine-scanning mutagenesis was performed to identify important residues (referred to as 'hot-spots') in peptide−receptor association (Figures S20-S22) . Hotspots are residues with high energetic contributions to the thermodynamic stability of a given complex (81) . Alanine-scanning provides a detailed energy map of a protein-binding interface (81) . Here, we used the fast in silico method, BudeAlaScan (81), in which every residue, for both receptor and peptide, is mutated (singly, in turn) to alanine. Hotspots are determined by the difference between the binding free energies of the alanine mutant and wild-type complexes (∆∆G bind ) (81) . Hotspots were identified at the interface of the receptor, some of them common to all three subtypes ( Figure S23 and Tables 2 and S2). In particular, TyrC1 (α4Y223, α7Y210, αY214) and the negatively charged residues in the upper part of loop F (β2D195, α7D186, δD201, δE203) strongly stabilise the complex. In the human α7 nAChR, the substitution of several key agonist-binding residues in the aromatic box (namely TyrA (α7Y115), TyrC1 (α7Y210), TrpB (α7W171) and TrpD (α7W77)) by alanine is also predicted to destabilise the interface between the peptide and the receptor. Of residues in the peptide, Y674, R682 and R685 are the major contributors to stabilizing the interface (Figure S24 ). This analysis reinforces the critical role of R682 in binding to nAChRs. Since the beginning of the pandemic, the computational structural biology/biomolecular simulation community has investigated the SARS-CoV-2 spike protein in different states and conditions, and the complexes that it forms (e.g. (55) (56) (57) (58) (59) (82) (83) (84) (85) (86) ). Simulations have revealed the dynamics of the spike and its glycan shield (55, 57-59, 82, 83, 85, 86) and the effects of the binding of small molecules (56, 84) . Here, to further explore the 'nicotinic hypothesis', we show that the Y674-R685 region (corresponding to the S-peptide) is accessible for binding, using the available MD simulations of the fully glycosylated full-length SARS-CoV-2 spike protein in the open and closed states by Casalino et al. (55) We note that in these models, the Y674-R685 region was modelled de novo as it was entirely (open spike) or partially (closed spike) missing in the initial cryoEM structures (42, 43) (for more details see Supporting Information). In these simulations, the Y674-R685 region adopts conformations potentially compatible with binding to nAChRs ( Figure 4A ). Our analysis reveals that the Y674-R685 loop is only weakly shielded by the glycans, and is predominantly solvent-exposed (Figure 4 and Figure S5 ). Especially when the spike is in the closed state (Figure 4B) , the Y674-J o u r n a l P r e -p r o o f R685 loop appears highly accessible to a probe with a radius ranging from 1.4 Å to 15 Å. In contrast, in the open spike ( Figure 4C) , a larger variability of the accessible area is observed, preventing an unambiguous interpretation of the glycan shield effect on Y674-R685 for probes with a radius larger than 7 Å. The slightly different and less variable accessibility of the Y674-R585 loop observed in the closed spike when compared the open spike protein is in agreement with the sharper distribution of the radius of gyration calculated for this region in the closed spike ( Figure S6 ). This behaviour might indicate different binding propensity of the S-peptide in the open and closed spike states. We hypothesize that it might be linked to a different packing of the three spike monomers in the two states. We note that the accessibility of this region makes it available to bind other receptors that may also bind the PRRA motif, such as neuropilin-1. In summary, the findings reported here support the hypothesis that the SARS-CoV-2 spike protein can interact with nAChRs. Our calculations indicate stable binding of the spike protein to these receptors through a region adjacent to the furin cleavage site and corresponding to the Y674-R685 region. These calculations also show apparent subtype-specific interactions and dynamics for the Y674-R685 region. COVID-19 is known to cause a range of neurological (87, 88) , muscular (39) , and respiratory (89) symptoms and these predicted interactions may be relevant to understand the pathophysiology associated with this disease. Our results predict that the Y674-R685 region of the spike protein has affinity for nAChRs. The region in the spike responsible for binding to nAChRs harbours the PRRA motif and shares high sequence similarity with neurotoxins known to be nAChR antagonists. The guanidinium group of R682 is the key anchoring point to the binding pocket, where it forms several interactions with the residues that form the aromatic box. Analysis of the structure and dynamics of the full-length glycosylated spike shows that the Y674-R685 region protrudes outside the glycan shield, is solvent accessible (Figures 4 and S5) and is flexible (Figure S6) , showing that it is accessible to bind to nAChRs (and to other receptors such as neuropilins (47)). Modelling the interaction between the fulllength spike and nAChRs indicates that association is possible with the proteins in a non-parallel orientation to one another ( Figure S4) . Cryoelectron microscopy and tomography experiments, and coarse-grained simulations, show considerable bending and tilting of the spike. A tilt angle up to 60° relative to the normal axis of the membrane is observed (57, 58, 85, 90, 91) . This flexibility of the spike protein would facilitate binding to host nAChRs. Evidence that the interaction between the Y674-R685 region of the spike and nAChRs is possible comes from the recently characterized interaction of the spike protein with neuropilin-1 (47) , which was shown to occur via the same region as the one proposed here. Having explored various possible orientations, we find that only approximately non-parallel arrangements of the spike and receptor allow for their interaction. This non-parallel interaction may not be immediately obvious, but it is consistent with other observations and is possible for two principal reasons: first, membrane curvature and deformation, and second, bending of the stalk of the spike. Experiments (e.g. cryoEM and tomography) and coarse-grained simulations show a significant degree of stalk bending is possible, and that the spike can adopt a wide range of conformations with different degrees of bending (57, 85, 90, 91) given by the three flexible hinges in the spike protein (85) . In Intriguingly, in the α7 complexes, the peptide adopts binding modes that allow strong interactions within the aromatic box, raising the question of whether it promotes gating in this subtype. This is important because activation of α7 nAChR triggers anti-inflammatory signalling mechanisms in inflammatory cells, leading to a decrease in cytokine production, which may have relevance in understanding early COVID-19 pathology (9, 18, 24, 36) . If nicotine does indeed prove to have any clinical value, it is likely that it would be due to interfering with the association with nAChRs. If so, nicotine analogues (e.g. smoking cessation agents such as varenicline (92) , cytisine (93) and potentially cytisine variants (54)) could also find useful application for COVID-19. Given the promising results presented here, structural, mutational and single-channel studies will be of interest to test the importance of the interactions of the SARS-CoV-2 spike with nAChRs, and the potential relevance of these interactions to pathology and infectivity in COVID-19. To assist with further investigations, we make our simulation files and datasets available and openly accessible, in accordance with the sharing principles agreed to by our community for simulations relevant to COVID-19 (94) . References ( Conceptualization SARS-CoV-2 spike represents the closed state of the protein, after furin cleavage (55) . The spike protein is a homotrimer (42) : each monomer is shown in different colours, namely green, cyan and orange, with glycans depicted in pink. Each monomer is formed by three domains: head, stalk and cytoplasmic tail (CT) (42) . The Y674-R685 region is shown in red. (B) The cryoEM structure of the muscle-type receptor from Tetronarce californica (PDB code: 6UWZ) (38) . This receptor is a heteropentamer formed of two α (green), one β (blue), one δ (yellow), and one γ (orange) subunits. Each monomer is formed by four domains (28) (29) (30) : extracellular (ECD), transmembrane (TMD), intracellular (ICD) and C-terminal domain (CTD). The agonist binding site is located in the ECDs at the interface between two neighbouring subunits. Complexes formed by the S-peptide and three different nAChRs, namely the human α4β2, human α7 and the muscle-like αβγδ receptor from Tetronarce californica. The S-peptide (region Y674-R685) is highlighted in magenta, and the principal and complementary subunits of the receptors are coloured in green and cyan, respectively. These models show the conformation of the S-peptide bound to the first pocket at the beginning of the simulations. In the human α4β2 receptor, the binding pocket is formed by one α4 and one β2 subunit, whereas in the human α7 nAChR, the pocket is formed by two α7 subunits. In the αβγδ receptor, the two binding pockets are non-equivalent: one is formed by an α and a δ and the second by an α and a γ subunits. (B) Closeup view of the peptide-receptor interaction region. Residues involved in binding of the S-peptide are shown with sticks. Note that the sidechain of R682 in the S-peptide is located inside the aromatic box establishing cation-π interactions with some of the highly conserved aromatic residues lining the pocket. 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See also Figures S13-S16 for more details about the behaviour of the S-peptide when bound to α7 nAChR 55) of the glycosylated full-length SARS-CoV-2 spike in the closed state showing Y674-R685 loop protruding into the solvent. The protein is depicted with a grey surface, whereas 674-685 loop is shown as a cyan ribbon. The glycans are illustrated with blue sticks. (B-C) The ASA of the of residues 674-685 (corresponding to the S-peptide), and the area shielded by glycans, at multiple probe radii from 1.4 Å (water molecule) to 15 Å are calculated using the available MD trajectories of the full-length models of the glycosylated SARS-CoV-2 spike protein in the closed (B) and open states (C) from Casalino et al. (55). The area of 674-685 shielded by the glycans is presented in blue, whereas the grey line represents the accessible area of 674-685 in the absence of glycans J o u r n a l P r e -p r o o f The authors declare no competing interests.