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Structure-function relations of the SARS-CoV-2 spike protein and impact of mutations in the variants of concern*-3pt

This review covers the main features of the severe acquired respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, its interaction with the main entry receptor, the human angiotensin converting enzyme 2 (ACE2), and the subsequent membrane fusion step. The focus is on the structural organization of these proteins and mechanistic aspects of their interactions that lead to cytoplasmic release of the viral genome. The most potently neutralizing antibodies against SARSCoV-2 were shown to interfere with the spike/ACE2 interaction. I thus also review the location and the potential impact of mutations in the spike protein observed in the variants of concern that emerged concomitantly with acquired immunity in the population after one year of virus circulation. Understanding how these interactions affect the spike/ACE2 interactions and the subsequent spike-proteininduced membrane fusion reaction is important to stay one step ahead of the virus evolution and develop efficient countermeasures. Résumé. Cette revue couvre les principales caractéristiques de la protéine de spicule du coronavirus 2 du syndrome respiratoire acquis sévère (SRAS-CoV-2), son interaction avec le principal récepteur d’entrée, l’enzyme de conversion de l’angiotensine 2 (ACE2) humaine, et l’étape subséquente de fusion membranaire. L’accent est mis sur l’organisation structurale de ces protéines et les aspects mécanistiques de leurs interactions qui conduisent à la libération cytoplasmique du génome viral. Les anticorps neutralisants les plus puissants contre le SARS-CoV-2 bloquent l’interaction spike / ACE2. Je passe donc également en revue la localisation et l’impact potentiel des mutations sur le spicule observées dans les variants préoccupants qui sont apparus en même temps que l’immunité acquise dans la population après un an de circulation du virus. Comprendre comment ces mutations affectent les interactions spicule / ACE2 et la catalyse de la réaction de fusion membranaire est important pour garder une longueur d’avance sur l’évolution du virus et développer des contre-mesures efficaces.


Introduction
As of mid-May 2021, the SARS-CoV-2 pandemic has caused more than 160 million cases with more than 3.4 million deaths reported to the World Health Organization (https://covid19.who.int/). At the same time, over 1.4 million vaccine doses have been administered throughout the world, and a race with the virus has been engaged in which, as more people are immunized through natural infection or because of vaccination, new virus variants emerge and take over the previously dominating strains [1]. These variants are selected because they are more apt to circulate in the presence of herd immunity. It remains to understand how far the virus can evolve in the face of the human immune response while maintaining its fitness, and how efficient the current vaccines will be in the long term to stop its dissemination and the economic tall imposed on society.
The development of novel and efficient vaccines has been made at an unprecedented pace, in part because the timing was ripe to test novel strategies such as mRNA vaccines, which had only been tried as anti-cancer strategies [2,3]. Or such as the use of adenovirus vectors, which had been advanced during the 2014-2016 Ebola outbreak in West Africa, an epidemic that had receded before large clinical vaccine trials could be deployed [4]. What these successful anti-Covid-19 vaccines have in common is the immunogen that they carry, obtained by applying new concepts of structure-based vaccine design [5]. The virus spike protein was thus stabilized in an inactive state mimicking its otherwise metastable "prefusion" form present at the surface of infectious virus particles. Indeed, the spike protein is the sole target of virus neutralizing antibodies, but is intrinsically metastable as is must come apart to allow virus entry into cells by inducing the fusion of the viral envelope with the target cell membrane. Structural biology played a very important role in devising strategies to cope with its instability by engineering stable versions that preserve the relevant epitopes to be presented to the immune system to elicit strongly neutralizing antibodies. Previous studies on other enveloped viruses, such as HIV and the respiratory syncytial virus (RSV, responsible for severe bronchiolitis in infants and the elderly or immunocompromised individuals), which also display a highly unstable en-velope protein at their surface, had paved the way for the coronavirus work [6,7]. For both HIV and RSV, the situation is more complex as the epitopes targeted by the most potently and broadly neutralizing antibodies are only present in the intact ectodomain folded in its pre-fusion form. We now know, however, that the most potently neutralizing antibodies against the SARS-like viruses are directed to the receptor binding domain (RBD). This domain has the advantage that it can be produced independently of the rest of the spike, presenting by itself relevant epitopes to the immune system to elicit neutralizing antibodies [8]. Yet the formulation used in the current vaccines, using the full-length spike protein stabilized with two internal proline mutations, has proven remarkably efficient to stimulate the production of protecting antibodies, especially when administered in an mRNA form [9,10].
I provide below a description of the ACE2 receptor and the spike protein together with an analysis of the conservation of the surfaces of interaction in both proteins across mammals and across SARS-like coronaviruses. I also summarize the knowledge gathered over the years on the mechanism of membrane fusion driven by enveloped viruses and how they apply to SARS-CoV-2. I end by illustrating that the mutations observed in the variants of concern display a clear clustering on three regions of the spike. In particular, in the receptor binding domain, they map to a rim all around the surface of interaction with ACE2.

The sarbecoviruses
The Coronaviridae family is divided into two subfamilies. The classic coronaviruses are grouped within the sub-family Orthocoronavirinae, which are unrelated to those of the sarbecoviruses [13], suggesting a convergence in evolution to use the same receptor. Another finding worth mentioning is that, for most coronaviruses the receptor is a membrane bound peptidase. For instance, amino-peptidase N (APN) is used by the human seasonal α-coronavirus 229E [14] as well as the porcine α-coronaviruses [15].
APN is a metal-dependent aminopeptidase playing multiple physiological functions, pain regulation, blood pressure homeostasis, angiogenesis of tumor cells, among others [16]. Similarly, the β-coronavirus Middle-East respiratory syndrome virus (MERS-CoV) uses dipeptidyl peptidase 4 (DPP4) as receptor [17]. DPP4 is a serine protease cleaving at the N terminus of physiological peptides involved in controlling apoptosis, immune regulation and signal transduction [18][19][20]. The catalytic activity is not required for virus recognition of any of these peptidase receptors, and the binding site recognized by the spike protein is away from the catalytic site. Yet it is intriguing that various coronaviruses from different genera have converged to use cell-surface peptidases for entry, and the potential relations to their pathogenicity remain an open field of investigation [21].

The ACE2 receptor
In 2003 Farzan and colleagues identified the angiotensin-converting enzyme 2 (ACE2) as the main receptor for the severe acquired respiratory syndrome coronavirus (SARS-CoV) [22]. The new coronavirus, SARS-CoV-2, was shown to use the same receptor [23]. ACE2 is a type I single-pass transmembrane protein present at the cell surface of multiple tissues in various organisms. It is a component of the renin-angiotensin system (RAS) that controls blood volume and systemic vascular resistance, playing a crucial role in protection from high blood pressure and cardiovascular disease [24]. The other components of this hormone system are the soluble aspartic protease renin and the angiotensinogen (AGT) precursor, as well as the membrane-bound angiotensin converting enzyme (ACE) and different G-protein coupled receptors (GPRs) specific for the various angiotensin peptides derived from AGT cleavage. Cleavage of AGT by renin is the ratelimiting step [25] and is tightly regulated to maintain normal blood pressure, avoiding hypertension and other cardiovascular diseases. AGT is a 452 amino acid (aa) long circulating protein belonging to the serpin family of protease inhibitors [26,27], albeit with no inhibitor function. Renin cleaves AGT at its N-terminus to generate angiotensin I (Ang-I) [28,29], a decapeptide that is the substrate for ACE, which converts it to Ang-II ( Figure 1). The Ang II octapeptide hormone is important for blood pressure homeostasis through its interaction with the GPR AP1, leading to increased blood pressure. The role of the carboxypeptidase ACE2 is to remove the C-terminal phenylalanine residue of Ang II to make Ang (1-7) [30][31][32], which binds to the GPR Mas [33,34]. The Ang (1, 7)/GPR Mas interaction leads to vasodilation and protection from excessive blood pressure, contributing to vascular pressure homeostasis. Infection by a virus is known to cause downregulation of the expression if its entry receptor, as seen for instance with HIV and its CD4 receptor [35]. The reason is that low amounts of receptor in infected cells helps virus propagation away from the site in which the virus was replicated within an organism to more easily reach other tissues. SARS-CoV-2 downregulation of the ACE2 receptor may thus result in an imbalance in the Ang II/Ang(1/7) peptide ratio, potentially contributing to its pathogenicity.
Human ACE2 was first identified in 2000 [30,31] as a paralog of the angiotensin-converting enzyme (ACE), discovered in 1956 [38]. The corresponding gene, located at position p22.2 in the X chromosome, contains 18 exons, and gives rise to several ACE2 isoforms via alternative splicing. The longest isoform is 805 amino acids long and is expressed in a broad range of human tissues, including endothelial cells in blood vessels, enterocytes in the intestine, alveolar cells in the lung, nasal epithelial cells, all organs that are targeted by SARS-CoV-2. The organization of this ACE2 isoform is depicted in Figure 1B. Its ectodomain is composed of two structural domains [39]: an N-terminal catalytic domain with zinc metallopeptidase activity exhibiting 42% amino acid sequence identity with each the two catalytic domains of ACE [40] (Figure 2). The ACE2 protease domain is followed by a collectrin-like domain, which associates with the neutral amino acid transporter B 0 AT1 ( Figure 1C), at least in intestinal epithelial cells. It has been shown that this association is required for expression of the transporter on the luminal surface of intestinal epithelial cells [41,42]. The collectrin domain of human ACE2 shares 50% Figure 1. The renin-angiotensin system and the ACE2 structural organization. (A) ACE2 processing of the angiotensin peptides. Shown is a partial amino acid sequence (truncated to leave out residues 51 to 481, with the break marked by a wiggly symbol) of angiotensinogen (ATG). The amino-terminus of AGT is generated co-translationally by signalase cleavage in the ER lumen (grey scissors). Upon environmental clues, secreted ATG exposes its N-terminal segment for cleavage by renin to generate the decapeptide angiotensin I (Ang I), highlighted in a yellow background [28,29]. The renin cleavage site is labeled and marked by the black scissor to the right. The angiotensin converting enzyme (ACE) then cleaves Ang I at the site indicated by the left black scissor, to generate Ang II, a highly active peptide that stimulates vasoconstriction via interaction with its G-protein coupled receptor AP1 present at the plasma membrane of multiple cells. Unchecked Ang II leads to high blood pressure and vascular disease, and this effect is balanced by ACE2 (purple scissors below the sequence), which has a carboxypeptidase activity and cleaves the C-terminal residues of both, Ang I and Ang II, to generate Ang (1-9) and Ang (1-7), respectively.  -terminal residue of Ang II with about 400-fold the efficiency of Ang-1 cleavage to make Ang (1-9). Ang (1-7) induces vasodilation through its interaction with the GPR Mas. The activity of ACE-2 is therefore critical in the RAS system. Circled in green and red are, respectively, a glycosylated asparagine and a cysteine that forms a labile disulfide bond with another ATG cysteine located 120 aa downstream in the aa sequence (not shown). Both residues have been shown to play a role in recognition and cleavage by renin [29]. (B) Linear diagram of ACE, ACE2 and Collectrin, showing that ACE2 originated as a chimeric protein with its N-terminal domain derived from ACE and its Cterminus from collectrin. The domain boundaries are indicated, and vertical arrows point to different loops in the collectrin domain that are cleaved to release soluble ACE2 to the medium. ADAM17 cleaves in the loop 652-659, while TMPRSS2 cleaves in the loop 697-716 [36]. (C) Organization of ACE2 on membranes. The structure of ACE2 was determined by cryo-EM as a binary complex with the multispanning transmembrane amino acid transporter B 0 AT1 (left panel, PDB:6M18) and as a ternary complex with the SARS-CoV-2 spike's receptor-binding domain (RBD) (right panels, PDB:6M17) [37]. The plasma membrane is indicated in the left panel, as a guide. ACE2 is shown in surface representation whereas B 0 AT1 and the RBD are shown as ribbons in blue and purple, respectively. In the left panel, ACE2 is colored green and yellow according to domains as in the linear diagram, with the two protomers shown in different shades of green and yellow. Similarly, both protomers of the B 0 AT1 transporter are shown in different blue tints. Note that the transmembrane helices of ACE2 form integral part of the transporter and contribute to its dimer interface. In the right panel, ACE2 is colored according to its amino acid sequence conservation from an alignment of 24 ACE2 sequences from various mammals. This alignment shows that the surface conservation is high within the cleft that carries the active site. The RBD binds away from this cleft at an exposed surface which is variable across mammalian species.
sequence identity with human collectrin, a type I single-pass trans-membrane glycoprotein that binds amino acid transporters of the B 0 type, such as B 0 A1 and B 0 A3. These transporters, which belong to the solute carrier 6 (SLC6) family [43], are only active when collectrin is bound [44]. The activity of collectrin was also proposed to play a role in blood pressure homeostasis [45].
The spike protein binds at the membranedistalmost surface of ACE2, away from the active site cleft ( Figure 1C, right panels). Amino acid sequence comparisons show that the surface patch recognized by the RBD is variable across different mammalian species, contrary to the highly conserved cleft harboring the ACE2 catalytic site. The structure also shows that the two spike-interacting sites present in the (ACE2/B 0 A1) dimer are located such that two RBDs from the same spike cannot bind simultaneously, implying that two different spike trimers bind to one receptor dimer [37]. This situation has the potential of clustering the spikes on the virion side facing the cell membrane, where each spike trimer may bind up to three ACE2 dimers, each bound to a second spike and resulting in cluster formation.
No study has so far addressed the stoichiometry of binding and whether the proposed clustering has a functional effect for virus entry, either in triggering fusion directly at the plasma membrane or by stimulating endocytosis with subsequent fusion in late endosomes.

The coronavirus spike glycoprotein
The sarbecovirus S polypeptide chain is roughly 1300 amino acids long with multiple asparagine-linked glycans in its ectodomain. It forms a trimer anchored to the viral membrane via a C-terminal transmembrane helix ( Figure 2). S is activated by proteolytic cleavage to generate two subunits: S1 (roughly the Nterminal half of S), and S2, the membrane-anchored C-terminal half. SARS-CoV-2 features an insertion of a polybasic sequence at the S1/S2 cleavage site, which allows for S processing by furin, a trans-Golgi network resident protease in the virus producer cells.
Cleavage by furin appears not to take place extensively, and most S trimers were reported to be only partially cleaved (i.e., cleavage occurring in only one or two of the three protomers of the trimer) [47]. Such subunits S1 and S2, the transmembrane (TM) segments, the individual domains and the cleavage sites. The amino acid numbers correspond to the SARS-CoV-2 sequence, original Wuhan strain. The "head" and "stalk" regions indicated in B are also labeled. The fusion peptide is indicated by a white hashed region downstream the S2 cleavage site. A thin purple box encompasses a 140 residues long region of S2 (aa 772-912 in SARS-CoV-2 S), encompassing the FP and the S2 cleavage site, that was not resolved in the available cryo-EM structures of coronavirus S2 [46,47] (see also Figure 3). (B) Ribbon representation of the intact spike protein as modeled by molecular dynamics (MD) [48] on the viral membrane and based on intermediate resolution cryo-electron tomography structures of the spike on inactivated intact virus particles [49][50][51]. The polypeptide chain of the protomer in the foreground is colored according to domains as in A. The lipids on the MD modeled membrane and the complete sugar chains of the glycans attached to the protein are shown as sticks color-coded by atom type (carbon, nitrogen, oxygen and sulfur in light grey, blue, red and yellow, respectively). The approximate dimensions are provided on the side, with the flexible "stalk" and the more globular dynamic "head" portions of the trimeric spike labeled. The C-terminus of the TM segment in the intraviral side of the membrane is labeled in pink. (C, D) Cryo-EM structures of the spike ectodomain, showing only the head portion, with the front protomer colored according to domains as in A. The trimer is shown in the closed form with three RBDs in the "down" conformation (C) and with one RBD in the "up" conformation (D) in a partially open spike. On virions, the spike head displays a dynamic equilibrium between the closed conformation and partially open forms. Virions also display inactive post-fusion S2 spikes, presumably resulting from spontaneous S1 shedding and premature spike activation [49] (see Figure 3). The models displayed correspond to PDB:7bnm (C) and 7bnn (D), determined at a resolution of about 3.5 Å [52]. partial cleavage may have functional consequences, as proposed in Figure 3B.

The furin cleavage site insertion
This spike polybasic site has been shown to be involved in interactions with neuropilin 1 [56,57]. The neuropilins bind vascular endothelial growth factors (VEGFs) at the carboxyl-terminal sequence generated by furin cleavage of the VEGF precursor [58]. Neuropilins are present in the olfactory epithelium, and could play a role in the observed anosmia in Covid-19 patients [59] and potentially in the virus invasion of the central nervous system [60]. The same polybasic furin site has also been proposed to interact with the nicotinic acetylcholine receptor (nAChR) [61], by analogy to a similar motif present in the glycoprotein of rabies virus and in several snake neurotoxins [62]. No evidence has been provided so far, however, for a direct interaction between the nAChR and the polybasic site. Indirect evidence lies in the fact that ivermectin (IVM), an antiparasitic compound that is also a positive allosteric effector of the α7 neuronal nACHR [63] was shown to attenuate the neurological symptoms in the hamster model for Covid 19 [64]. IVM did not reduce the viral load in the infected hamsters, only the Covid 19 pathological symptoms, contradicting a previous report [57] suggesting that IVM had a direct antiviral effect. In any case, whatever the role of the polybasic sequence, independent studies have demonstrated that SARS-CoV-2 mutants in which it is deleted are less pathogenic than the parent virus [65,66].

The spike protein on the viral membrane
The S trimer projects about 25 nm from the viral membrane, displaying a prominent, globular yet dynamic "head" and a flexible "stalk" region (Figure 2A). The three-dimensional (3D) structure of the spike head has been determined by cryogenic electron microscopy (cryo-EM) and single-particle averaging for several coronaviruses, the first of which were reported back in 2016 [67,68] (reviewed in [69]) to resolutions of about 3 Å or better, including that of the SARS-CoV-2 spike [47,70,71]. The full spike has been visualized on SARS-CoV-2 virions by cryoelectron tomography analyses of inactivated virus particles using sub-tomogram averaging to reach a resolution beyond 1 nm [49][50][51]. S1, which remains non-covalently attached to S2 in the cleaved trimer, contains two structural domains, an N-terminal domain (NTD) and the RBD. The fold of both of these domains is independent of the rest of the chain (Figure 2). The structural studies have shown that the RBD displays a dynamic equilibrium between two conformations, termed "down" and "up". The spike head trimer has thus been captured in four different forms, a completely closed form with all three RBDs in the down conformation, an open conformation with all three RBDs up, and intermediately open forms with one or two RBDs up [52,54]. Only in the up conformation the RBD can engage ACE2, which shifts the equilibrium towards the open form of the spike.
5.1. The S2 subunit: a "class I" fusion protein S2 is responsible for catalyzing the membrane fusion reaction required for entry. This reaction follows S1 dissociation from the spike trimer upon ACE2 binding, which releases S2 from important constrains derived by folding within the context of the S precursor. Structural studies have characterized three structural classes of viral membrane fusion proteins (reviewed by [72]). S2 belongs to class I, together with the fusion proteins found in multiple enveloped viruses, such as paramyxoviruses, influenza viruses, retroviruses and other viral families. All of them undergo a functional conformational change from a metastable pre-fusion to a highly stable post-fusion form to drive fusion. The energy stored in the metastable form is coupled to target-membrane binding to catalyze lipid merger. In particular, the proteins of the three classes were shown to adopt a highly stable "hairpin" conformation in the post-fusion form. In the prototypic class I proteins, such as influenza virus HA2 or HIV-1 gp41, the N-terminal half forms a long "N-helix" that makes a central, parallel trimeric coiled coil, while the Cterminal half runs antiparallel to the N helix (in some cases as a "C-helix, like in gp41) along the grooves of the coiled coil. The resulting "hairpin" is such that its N-and C-terminal ends are at the same end of a long trimeric rod. A hydrophobic segment termed "fusion peptide" (FP) is located immediately upstream the N helix. This segment is responsible for insertion into the currently accepted main steps of the virus fusion process triggered by receptor binding. The fusion protein is anchored via its C-terminal end to the viral membrane, and is proteolytically activated by cleavage into an N-terminal subunit (NSU, in yellow), and a C-terminal subunit (CSU, in red and blue). The fusion peptide, indicated in green and labeled FP (i), is located immediately downstream the cleavage site. Upon proteolytic activation it becomes the N-terminus of the CSU. This activating cleavage renders the fusion protein metastable, with the CSU spring-loaded underneath an NSU crown (i). The CSU reacts to clues such as receptor binding or acidification of the milieu (i.e., in an endosome, depending on the virus) to trigger fusion via a major conformational rearrangement. shedding of the NSU (i.e., the S1 subunit) from the trimer (ii), which allows the CSU to spring out and project the FP into the cell membrane, where it inserts firmly in the first step of the fusogenic rearrangement (iii). This "elongated intermediate" form is only transient, and folds back into a "hairpin" conformation in which the Nterminal half (in red) remains bound to the target membrane in the form of a central, trimeric, α-helical coiled coil, whereas the C-terminal half of the CSU runs in the opposite way along the grooves of the central coiled coil. Hairpin formation thus entails bringing into juxtaposition the N-and C-terminal ends of the CSU, thereby forcing the two membranes against each other (iii, iv and v). Fusion occurs first by merging of the outer leaflets, a process called "hemi-fusion" (v) and is followed by formation of a fusion pore (vi), which then enlarges to give rise to a single fused membrane. (adapted from [53]). (B) Current understanding of the SARS-CoV-2 activation process to drive membrane fusion. The Spike trimer is shown with the S1 subunit in surface representation and S2 as ribbons. One protomer is in colors and the others in grey and black. The S1 subunit of the front protomer in the trimer is shown in yellow to match the NSU represented in A. The front S2 subunit is ramp colored blue-green-yellow-orange-red from its N-to C-terminus (see bar under panel C), highlighting that its N-terminal moiety is spring-loaded underneath the S1 crown, whereas its C-terminal portion enters the stalk, as indicated in Figures 2A and B. The ACE2 receptor in complex with B 0 AT1 in the cell membrane is shown in surface representation with the two protomers of the ACE2 dimer differentiated in dark and pale red, while B 0 AT1 is in light grey. The central step (labeled "(0)") shows the spike trimer with a closed head, which is in equilibrium with partially open forms (as the one shown in Figure 2C). ACE2 binding shifts the equilibrium towards open forms (step labeled (1)). Structural studies have shown that in the open head the S1/S2 interactions are weaker, especially when all three RBDs are in the up conformation [54]. It was proposed that fully furin processed spikes are unstable and spontaneously shed S1 after displaying the open-head conformation (step (−1), resulting in premature conformational change and insertion of the post-fusion S2 into the viral membrane (step (−2) [47]). In this panel, the post-fusion form is indicated with a hand-drawn connection between residues 772 and 912, which are disordered in the cryo-EM structures [46,47]. Note that the S2 conformation of the left panel corresponds to step (vi) in Figure 3A, and not to that of step (ii), which depicts the CSU still in the spring-loaded pre-fusion conformation.
Step (1) in B corresponds to a step in between (i) and (ii) in A, where the spike protein has already bound the ACE2 receptor. Pulling forces arising from the flow or other fluctuations in between the bound particle and the cell then result in S1 shedding, a step that requires completion of the cleavage between S1 and S2 on the spike. This cleavage is carried out by TMPRRSS2 at the surface of some cells, or by cathepsin upon endocytosis of the receptor-bound virions [55]. S1 shedding in this context leads to insertion of the fusion loop into the target cell membrane upon springing out of the HR1 helix to reach the target membrane, as indicated in step (iii) in A. The HR2 portion then completes the hairpin conformation by binding to the grooves of the central, trimeric coiled coil formed by the HR1 helix, which is the structure represented in step (3). (C) HR1 spring-out. The four panels represented illustrate the transition between steps (1) and (3) of Figure 2B, with only S2 represented in ribbons ramp-colored from N-to C-termini according to the color-coded bar underneath. For clarity, only one of the three protomers of the S2 trimer is shown. The left panel shows the HR1 moiety together with segments upstream the S2 cleavage site tucked underneath the S1 crown in the pre-fusion form. The S1 subunit and the ACE2 receptor are schematically diagrammed and colored according to Figure 3A in steps (i) and (ii). Dissociation of the receptor-bound S1 crown induces HR1 to spring out and reach the target membrane (second panel). Simultaneously, the S2 cleavage site, which is buried and inaccessible in the spring-loaded form of S2, becomes exposed, to be cleaved by either TMPRSS2 at the cell surface, or the cathepsins in endosomes. The curved black arrows indicate the subsequent refolding of S2 into the stable post-fusion hairpin, bringing the two membranes together as indicated in Figure 3A, steps (iv) to (vi). It is not known whether cleavage at the S2 site occurs before or after hairpin formation, and the two options are represented (third and fourth panel). The fusion peptide (FP) has been shown to be required at the S2 N-terminus for membrane fusion to take place, but the cleavage site becomes exposed only after the conformational change. Its location in the uncleaved form is too far to reach the target membrane (as labeled). We postulate that the region immediately upstream of HR1 is also involved in target membrane insertion. Amino acid sequence analyses indicate that this region, termed "internal fusion peptide" in the Figure, has a strong propensity to form an amphipathic helix. Its binding to the outer leaflet of the target membrane would allow the right leverage to bring the two membranes into apposition even if the S2 occurs after the conformational change. Otherwise the S2 trimer would to flip and end inserting into the viral membrane, as observed on particles and as indicated in Figure 3B, left panel. The effect of the IFP segment has not yet been experimentally tested, however. the target membrane during the first steps of the fusogenic conformational rearrangement, a step absolutely required to drive the fusion process, as outlined in Figure 3A. The prototypical class I fusion proteins, such as those in influenza virus or in retroviruses, have the fusion peptide at the N-terminal end, and the hairpin is formed exclusively by the region in between the FP and the C-terminal TM anchor. In the coronavirus spike protein, similar to the paramyxovirus counterpart, this is not the case as the fusion peptide is at the N-terminus of S2 , which is internal to S2 (see Figure 2A), and regions upstream the S2 cleavage site are also present as part of the hairpin. The structural similarities between coronavirus S2 and the paramyxovirus fusion (F) protein suggest that they had a common origin [46], forming a distinct subclass compared to the prototypical class I proteins. There are two heptad-repeat regions in the S2 moiety, termed HR1 and HR2 (labeled in Figure 2A), which have a strong potential to form alpha helices. In the prefusion S trimer, the HR1 region is spring-loaded in the form a bundle of short helical segments under a "crown" formed by the S1 moiety ( Figure 3), while HR2 is part of the flexible stalk ( Figure 2B). To stabilize the prefusion spike trimer used in the current vaccines was engineered to introduce proline residues at the connections between the short helices of the spring-loaded HR1 [73][74][75]. Because prolines are helix-breaking residues, the mutated HR1 cannot spring out to forming a long helix as shown in Figure 3C, resulting in an overall stabilized from that is non-fusogenic.

Membrane fusion mechanism
The S trimer becomes metastable upon proteolytic maturation into S1 and S2. Subsequent binding to ACE2 on cells shifts the equilibrium of the spike toward the open form, within which the S1/S2 interactions were shown to be weaker [54]. Since each spike is bound to more than one receptor, mechanical constraints have the potential of exerting forces pulling the S1 subunits in opposite directions, ripping off the spike head and releasing the receptorbound S1 moiety from the spike surface ( Figure 2B). Loss of the S1 crown on the activated spike in turn causes the HR1 moiety to spring out and form a long helix and projecting the FP a distance of about 15 nm to reach and stably insert into the target membrane ( Figure 3C). This inserted form of S2, bridging the two membranes at a distance of about 30 nm, is a transient long extended intermediate trimer. The HR2 moiety, which is initially distantly located, near the viral membrane, eventually binds to the grooves of the trimeric HR1 coiled coil to make the trimer of hairpins observed in the post-fusion form (shown in Figure 3C, right panel, where only one protomer of the trimer is shown for clarity). In its final conformation, S2 thus forces viral and target membrane against each other, overcoming the repulsion between the two outer leaflets, as in steps (iv) and (v) in Figure 3A. Repulsion arises when two membranes are brought under 1 nm distance due to the required dehydration of the lipid heads to allow direct contact [76,77]. The hairpin conformation of the fusion protein thus catalyzes the membrane fusion reaction by forcing the close apposition of the outer lipid bilayers, lowering the kinetic barrier for fusion, which is an overall exothermic reaction. Membrane fusion ensues via a hemifusion (step (v) in Figure 3A) in which only the outer leaflets merge, followed by formation of a fusion pore (step vi), which then broadens to make a single, fused membrane.

The spike protein in the SARS-CoV-2 variants of concern
New SARS-CoV-2 variants with mutations increasing inter-individual transmission or allowing immune evasion have emerged recently and are displacing the initial circulating viral strains [78][79][80][81]. Genomic changes in these VOCs have been associated with increased person-to-person transmission and/or escape from post-infection or post-vaccination immunity. Although the mutations in VOCs occur throughout the viral genome, there are key mutations in the spike protein that control virus transmission and/or are targeted by neutralizing antibodies. The first variant identified presented the D614G mutation in the spike [82], a mutation has now become fixed in the circulating virus and was shown to increase virus transmissibility [78,[83][84][85][86][87]. As demonstrated recently for the B.  (A) Linear diagram of the mature spike protein colored to match the diagram of Figure 3A. The grey bar underneath represents the complete polypeptide, including its signal sequence. On this bar are plotted the mutations appearing on the variants of concern annotated immediately below, as labeled.   Figure 4 lists the positions on the spike of the mutations that have appeared in the main VOCs, including the three new Indian sub-lineages. These mutations cluster in in several regions of the spike head, in particular the N-terminal domain. There is also clustering in the RBD and near the S1/S2 cleavage sites. Some mutations map to the HR1 helix and the chain reversal region in the post-fusion hairpin (between the red and blue halves of S2 colored as in the diagram of Figure 3A). Although the majority of the mutations map to the trimer surface, a number of them affect contact residues buried at the trimer interface (see Figure 4B, right panels). This is the case for the mutations in the S2 moiety and those that are in S1 near the S1/S2 site (from residue 570 on). An analysis of the amino acid conservation of the spike protein among sarbecoviruses predicted to use ACE2 and infecting different mammals ( Figure 5) showed that the location of the VOC mutations at the spike surface lies in variable regions, as expected. Importantly, although the most variable surface maps to the NTD, the ACE2 binding surface of the RBD is is also highly variable (compare the 4th and 5th columns in Figure 5A)-in line with the observation that the ACE2 patch that is recognized by the RBD is also variable across mammals ( Figure 1C). The open spike exposes a conserved region of S2 at the spike top that cor-responds to the spring-loaded S2 ( Figure 5A, right panels). This region does not appear to be immunogenic, most likely because the completely open spike is exposed only transiently, immediately before its irreversible fusogenic conformational change, as indicated by arrows in Figure 5B. The location and relative orientation of the ACE2 binding surface of the RBD (yellow patch in Figure 5B, especially in the 5th panel) is also compatible with a "ripping off" effect wen more than one ACE2 is bound, as they can pull the RBD in opposite directions, thereby disrupting the spike and inducing the conformational change.

VOC mutations in the RBD and escape to neutralizing antibodies
Although the immune response against the spike is directed to multiple sites [97], the RBD has been shown to be the target of the vast majority of the most potent human neutralizing antibodies against SARS-CoV-2. Multiple reports describing the cloning and isolation of monoclonal antibodies (mAbs) from convalescent individuals and vaccinees, together with structural studies by X-ray crystallography and/or cryo-EM have been reported [8,[98][99][100][101][102][103][104][105][106][107][108]. These studies attest to the potency of the RBD-specific mAbs. Overall, they have been categorized into four broad classes, depending on the location of their epitope at the RBD surface [98]. The most strongly neutralizing mAbs, acting with near picomolar efficiently, target the ACE2 binding patch (yellow surface in Figure 6) [103]. These antibodies are divided into two classes: those in class 1 require the RBD in the up conformation to bind, whereas those in class 2 bind The central two panels have the surface of ACE2 recognition in blue (labeled "ACE2 patch"), showing that this surface is not accessible in the closed spike. The conservation is plotted as indicated in the horizontal bar at the top. Comparison between the second third columns, and between fourth and fifth, shows that the ACE2 patch is part of a variable surface, in line with the observation that the ACE2 receptor surface that it recognizes also varies across species (see Figure 1C, right panels). The top views also show that in the open spike, a conserved surface of S2 (in green in the sixth column), becomes exposed. This surface, which corresponds to the spring-loaded HR1 helix, is non-immunogenic as is essentially not exposed on infectious particles. This To facilitate the description, the protomers have been labeled A, B and C. The four middle trimers display, in addition, the ACE2 patch in yellow, and the mutations in the variants listed in Figure 4 indicated in the same color code as in Figure 4. Note that these mutations map to regions that are variable across sarbecoviruses (compare with rows 2 and 5 above). The second row in B has all three RBDs down, the third panel has the RBD of protomer A up and the others down, , the fourth panel has RBDs A and C up, and the fourth panel all three RBDs up to make an open spike. Double arrows between panels indicate reversibility between the various conformations. Note that the three RBDs up conformation is unstable and appears to lead to irreversible S1 shedding, as indicated in Figure 3  irrespective of having the RBD up or down. The location of the corresponding epitopes is outlined in Figure 6D, which shows that the epitope lo class 1 mAbs includes a region that is buried in the closed conformation of the spike, whereas the class 2 does not (compare panel D, top with panel A, as well as the bottom panels of C and D). Two additional categories of antibodies have been identified in which the epitope is away from the ACE2 patch: class 3 mAbs target an exposed surface, whereas the epitope of class 4 is buried and require at least two RBDs in the up conformation for binding [109][110][111][112]. Class 4 mAbs are among those with the broadest reactivity across sarbecoviruses, [101], as can be seen by comparing panels D and B of Figure 6. The mutations in the VOCs all result in reduced binding of the mAbs of classes 1 and 2, suggesting that these are the dominant form controlling virus circulation in the population in general.
The RBD mutations that have appeared in the VOCs are not randomly distributed: they make a rim all around the periphery of the ACE2 binding surface ( Figure 6D, bottom panel). This is in contrast to the variability observed when comparing across sarbecoviruses, in which the whole surface is variable. The notion emerging from this analysis is that the virus samples all mutations that are still compatible with binding to the human ACE2 yet interfering with antibody binding. It remains to be seen how far the virus can continue this trend and escape the human immune response while still maintaining its fitness.

Concluding remarks
This review covers most of the structural information available on the coronavirus spike protein, its interaction with ACE2, and the mechanistic information that can be extracted from the structural studies. The location of the mutations in the new SARS-CoV-2 variants, distributed throughout the spike surface, further suggest that there is epistasis between them and also that a likely allosteric effect may expose differently the three cleavage sites to furin, a feature that appears to be functionally important. The mutations may also affect in multiple ways the conformational change triggered by receptor binding. The biology of the coronavirus spike is far from having revealed its secrets, and there remains a great deal to learn. Still, the amount of information gathered on this protein in 18 months of the pandemic has been unprecedented, as is the pandemic itself. Only by understanding the spike function in mechanistic detail-as well that of the other viral proteins involved in other steps of the viral cycle-can we expect to be better prepared to face future outbreaks of emerging zoonotic viruses such as novel coronaviruses.