key: cord-0756404-n9epgdph
authors: Khelashvili, George; Plante, Ambrose; Doktorova, Milka; Weinstein, Harel
title: Ca2+-dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide
date: 2021-02-23
journal: Biophys J
DOI: 10.1016/j.bpj.2021.02.023
sha: 8c44cbb7e71c7bb7a6b87e96450c19d0af0e5448
doc_id: 756404
cord_uid: n9epgdph

Cell penetration after recognition of the SARS-CoV-2 virus by the ACE2 receptor, and the fusion of its viral envelope membrane with cellular membranes, are the early steps of infectivity. A region of the Spike protein (S) of the virus, identified as the “fusion peptide” (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca2+ ions to the FPs of corona viruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca2+ binding to the SARS-CoV-2-FP, the role of Ca2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca2+-bound SARS-CoV-2-FP and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics (MD) simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca2+-bound peptide models with lipid membranes SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca2+ ions. These findings provide novel mechanistic insights regarding the role of Ca2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.

The corona viruses (CoV) are envelope viruses that infect human and animal cells via fusion of the viral envelope membrane with cellular membranes 1 . Members of the CoV family include the well-known agents of recent pandemics such as severe acute respiratory syndrome CoV (SARS-CoV), the Middle East respiratory syndrome CoV (MERS-CoV) 2 , and the severe acute respiratory syndrome CoV-2 (SARS-CoV-2) virus responsible for the currently ongoing CoVID-19 pandemic 3, 4 .

The first point of the connection between the SARS-CoV-2 virus and the human cell is at the ACE2 receptor 1, [5] [6] [7] , and is now quite well characterized structurally [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] . It is mediated by the Spike protein which is a multi-domain homo-trimer glycoprotein anchored in the viral envelope membrane 5, 18 . Each monomer of the spike protein consists of two subunits, S1 which includes the receptor binding domain (RBD) that attaches to the ACE2 receptor 6, 7 , and S2 which includes a segment termed "fusion peptide" that is central to the fusion process leading to cell infection by CoVs 5, 19 . A catalytic cleavage site (the S1/S2 site) is recognized by a furin-like protease that can separate the two component domains of the Spike monomer 20 . Host cell recognition of both SARS-CoV and SARS-CoV-2 involves the angiotensin converting enzyme-2 (ACE2) which binds the RBD on S1. The binding of the S1 domain to the host receptor exposes another cleavage site, the S2' site on the S2 domain, to processing by cell surface enzymes of the host (primarily TMPRSS2) 7 . Cleavage of the S2' site occurs at the N-terminus of the fusion peptide (FP) segment, and a "tectonic" change in the conformation of the S2 domain repositions the FP regions of each of the three monomers close to one another to form a "spearhead" of the Spike, ready to penetrate the cell membrane while still attached to the S1 trimeric bundle 12, 21 .

Fusion Peptide insertion is thought to result in destabilization of the target cell membrane 22 as an essential step in allowing the entire virus to penetrate into the cell. In the cell, the FP spearheads the fusion of the viral capsule membrane with cell membranes to release the encapsulated genetic material for replication. Thus far, even the rapid pace of discovery of structural information about the SARS-CoV-2 virus 8-13,23 , has not yielded molecular level data about the vital step of engagement of the viral Spike protein with the membrane, much less the structure of its spearhead. Even less is known at the needed molecular level about the J o u r n a l P r e -p r o o f mechanism by which the FPs in the spearhead are involved in the fusion of the cell and virus membranes, despite the wealth of information that has recently accumulated from molecular dynamics (MD) simulations on SARS-CoV-2 models (e.g., see Refs. [24] [25] [26] [27] [28] [29] ).

Here we present results from an atomistic-level MD investigation of the interactions of SARS-CoV-2 FPs with membranes, and the consequences of these interactions on membrane properties. Biophysical and structure/function studies of the SARS-CoV-2 FP 30 as well as of the FPs from SARS-CoV 31 and MERS-CoV 32 , which are close homologs of SARS-CoV-2, had produced important insights about the functional significance of specific regions of the FP such as the large number of conserved acidic residues in the two consecutive fragments of the FP termed FP1 and FP2 (residues 816-835 and 835-854, respectively, in SARS-CoV-2), and the LLF hydrophobic motif 19, 31, 33 (L821/L822/F823 in SARS-CoV-2, see Figure 1 ). Studies on SARS-CoV-2 FP 30 and SARS-CoV 31 have suggested that together, the F1 and F2 fragments form a bipartite membrane interaction platform, and that acidic residues in both FP1 and FP2 promote membrane binding through their interactions with Ca 2+ ions 30, 31 . These findings about the functional role of Ca 2+ are echoed by results obtained for FPs from other related viruses, such as MERS-CoV 32 and Ebola virus 34 . Indeed, in the MERS-CoV FP one of these acidic residues, E891 in the N-terminal (FP1) part (corresponding to E819 in SARS-CoV-2 numbering, Figure 1A ) emerged as crucial for Ca 2+ interactions and fusion-related membrane perturbation effects 32 . In contrast to the SARS-CoV FP and SARS-CoV-2 FP, however, isothermal titration calorimetry (ITC) experiments with the MERS-CoV FP showed that the latter binds only a single Ca 2+ ion 32 .

Notably, much of the structural and functional inferences obtained from these studies appears to be in excellent agreement with, and recapitulated, by the recently published results obtained from equivalent experiments with the SARS-CoV-2 FP 30 .

The involvement of the conserved LLF motif in the FP1 fragment of the SARS-CoV-FP as a critical structural element for membrane insertion and fusogenicity of the peptide was identified from mutagenesis studies 19, 31, 33 . Also, perturbation of the lipid bilayer by Ca 2+dependent FP-membrane interactions was shown with Electron Spin Resonance (ESR) experiments to affect the organization of the lipid head group and backbone atoms of the lipids at the interaction site, but not the central region of the membrane hydrophobic core 30,31, 32,34 . J o u r n a l P r e -p r o o f Still, the loci of Ca 2+ binding to CoV FPs and their role in any specific (but unknown) modes of FP interaction with the membrane, had remained undetermined. This has hindered the interpretation of any measurable effects of viral interactions with the membrane, and thus any approaches to mitigate infectivity by targeting this essential region.

We addressed these fundamental unknowns about the SARS-CoV-2-FPs and their structurebased dynamic mechanisms with extensive atomistic ensemble molecular dynamics (MD) simulations. From analyses of the various trajectories, we predicted multiple ways in which Ca 2+ ions engage the FP's acidic residues singly and in pairs. From massive simulations of spontaneous membrane binding of all feasible variants of Ca 2+ -bound SARS-CoV-2-FP we found two preferred modes of membrane penetration. In one of these modes the inserted hydrophobic residues are F833/I834 from the FP2 region of the peptide which, in the full spike context are closely tethered to the S2 domain. The second more prevalent mode of penetration involves the N-terminal portion of the F1 segment, which is free after the S2' cleavage. In this mode the hydrophobic insertion consists of the highly conserved residues L822/F823 (LLF motif) and is achieved by the binding of Ca 2+ ions to the D830/D839 pair and the E819/D820 pair of acidic residues. Notably, our extensive control simulations to evaluate the role of Ca 2+ binding in the process showed that membrane binding/insertion of the SARS-CoV-2 FP is greatly diminished overall in the absence of Ca 2+ ions. Moreover, when bound the peptide exerted structural changes in the lipid bilayer whose nature and magnitude showed dependence on the local peptide concentration. Together, these findings provide new mechanistic insights and important molecular-level details about the SARS-CoV-2-FP penetration into the membrane and the structural dynamics underlying the key role of Ca 2+ binding in this process.

All computations were based on the atomistic structure of the FP (residues 816-854) from the full-length SARS-CoV-2 spike protein model described in Ref. 27 . In this structure, residues J o u r n a l P r e -p r o o f Cys840 and Cys851 form a disulfide bond (see Figure 1 ). For these studies, the FP segment from the full-length model was isolated and capped with neutral N-and C-termini (ACE and CT3, respectively, in the CHARMM force-field nomenclature). Protonation states of all the titratable residues were predicted at pH 7 using Propka 3.1 software 35 .

For the atomistic MD simulations of the SARS-CoV-2-FP in water, the peptide was embedded in a rectangular solution box and ionized using VMD tools ("Add Solvation Box" and "Add Ions", respectively) 36 . The box of dimensions ~90 Å x 80 Å x 82 Å included an ionic solution mixture of 49 Na + , 51 Cl -, 2 Ca 2+ ions, and ~18000 water molecules. The total number of atoms in the system was 54,510.

The system was equilibrated with NAMD version 2.12 37 following a multi-step protocol during which the backbone atoms of the SARS-CoV-2-FP as well as Ca 2+ ions in the solution were first harmonically constrained and subsequently gradually released in four steps (totaling ~3ns), changing the restrain force constants k F from 1, to 0.5, to 0.1 kcal/ (mol Å 2 ), and 0 kcal/ (mol Å 2 ). These simulations implemented all option for rigidbonds, 1fs (for k F 1, 0.5, and 0.1 kcal/ (mol Å 2 )) or 2fs (for k F of 0) integration time-step, PME for electrostatics interactions 38 , and were carried out in NPT ensemble under isotropic pressure coupling conditions, at a temperature of 310 K. The Nose-Hoover Langevin piston algorithm 39 was used to control the target P = 1 atm pressure with the "LangevinPistonPeriod" set to 200 fs and "LangevinPistonDecay" set to 50 fs. The van der Waals interactions were calculated applying a cutoff distance of 12 Å and switching the potential from 10 Å.

After this initial equilibration phase, the velocities of all atoms in the system were reset and ensemble MD runs were initiated with OpenMM version 7.4 40 during which the system was simulated in 18 independent replicates, each for 640ns (i.e., cumulative time of ~11.5 µs).

These runs implemented PME for electrostatic interactions and were performed at 310K temperature under NVT ensemble. In addition, 4fs time-step was used, with hydrogen mass repartitioning and with "friction" parameter set to 1.0/picosecond. Additional parameters for these runs included: "EwaldErrorTolerance" 0.0005, "rigidwater" True, and "ConstraintTolerance" 0.000001. The van der Waals interactions were calculated applying a cutoff distance of 12 Å and switching the potential from 10 Å.

Using the CHARMM-GUI web server 41 , we designed a symmetric lipid membrane composed of a 3:1:1 mixture of POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), and cholesterol. The total number of lipids in the bilayer was 600. The membrane was solvated and ionized (with 0.15 mM salt concentration), for a total of ~163,000 atoms.

This system was equilibrated with NAMD version 2.12 following the standard multi-step protocol provided by CHARMM-GUI, and then simulated with unbiased MD for 25ns. After this step, three independent replicates were run, each 75ns long. These runs implemented all option for rigidbonds, 2fs integration time-step, PME for electrostatics interactions, and were carried out in NPT ensemble under semi-isotropic pressure coupling conditions, at a temperature of 298 K. The Nose-Hoover Langevin piston algorithm was used to control the target P = 1 atm pressure with the "LangevinPistonPeriod" set to 50 fs and "LangevinPistonDecay" set to 25 fs. The van der Waals interactions were calculated with a cutoff distance of 12 Å and switching the potential from 10 Å. In addition, "vdwForceSwitching" was set to yes.

Interactions of a single, or multiple, FPs with lipid membranes were started by placing 1, 3, 6, 9, or 12 replicas of the Ca 2+ -bound SARS-CoV-2-FPs in the proximity of a bilayer composed of 3:1:1 POPC/POPG/Cholesterol that had been pre-equilibrated for 25ns as described above.

Parallel simulations of a single SARS-CoV-2-FP in the absence of Ca 2+ ions were carried out with the same pre-equilibrated 3:1:1 POPC/POPG/Cholesterol bilayer. The starting structure for the FP for the latter runs was the FP model from Ref. 27 J o u r n a l P r e -p r o o f Each system was equilibrated with NAMD version 2.12 following the same multi-step protocol described above during which the backbone atoms of the FP as well as the Ca 2+ ions were first harmonically constrained and subsequently gradually released in four steps. After this phase, the velocities of all atoms of the system were reset, and ensemble MD runs were initiated with OpenMM version 7.4. Table 1 lists the number of replicates and simulation times for each construct. These runs implemented PME for electrostatic interactions and were performed at 298K temperature under NPT ensemble using semi-isotropic pressure coupling, with 4fs time-steps, using hydrogen mass repartitioning and with "friction" parameter set to 1.0/picosecond. Additional parameters for these runs included: "EwaldErrorTolerance" 0.0005, "rigidwater" True, and "ConstraintTolerance" 0.000001. The van der Waals interactions were calculated applying a cutoff distance of 12 Å and switching the potential from 10 Å.

For all simulations we used the latest CHARMM36 force-field for proteins and lipids 42 , as well as the recently revised CHARMM36 force-field for ions which includes non-bonded fix (NBFIX) parameters for Na + and Ca 2+ ions 43 .

Lateral pressure profiles were calculated for selected FP-membrane systems (see Results) as well as for the pure lipid bilayer system described above. For the latter, the last halves of the trajectories of the three 75ns-long independent replicates were combined into a single trajectory for the pressure analysis. Similarly, for the FP-membrane complexes three 75ns long MD trajectory replicas were accumulated in NAMD and the last halves of these trajectories were concatenated and used for analysis. These simulations used the CHARMM36 force field, were initiated from the corresponding last frames of the OpenMM trajectories and were performed with the parameters used in the last step of the multi-stage equilibration protocol described above.

The pressure profiles were calculated with NAMD following the protocol described in Ref. 44 . The concatenated trajectory for analysis for each system comprised ~26,000 frames with output frequency of 2 ps. The trajectory was first centered on the average z position of the terminal methyl carbons of the lipid chains set at (x,y,z) = (0,0,0). The bilayer was then divided J o u r n a l P r e -p r o o f into a discrete number of equal-thickness (~0.8 A) slabs in the direction normal to the bilayer plane. The kinetic, bonded and non-bonded contributions to the pressure were obtained separately from the Ewald contributions, and the two were added to produce the lateral components of the pressure tensor ( , ) in each slab. The normal component was calculated as described earlier (see SI in Ref. 44 ) with the formula:

where L is the length of the simulation box in the z direction, and ( ) represents the tangential component of the pressure tensor. From here, the pressure in each slab was ( ) = ( ) − .

The FP of the SARS-CoV-2 spike protein (SARS-CoV-2-FP) harbors 6 acidic residues (E819, D820, D830, D839, D843, and D848) that are conserved in many coronaviruses, such as SARS-CoV and MERS-CoV (see Figure 1 ). As the binding of Ca 2+ ions was found experimentally to modulate the ability of the FPs from these CoVs to insert into lipid membranes, all these residues had to be considered putative Ca 2+ -binding sites 31,32,34 30 . To identify the modes of Ca 2+ interaction with the SARS-CoV-2-FP and to elucidate the function-related consequences of these interactions, we first used atomistic MD simulations of SARS-CoV-2-FP in aqueous solution to explore the spontaneous binding of Ca 2+ and its effect on the conformation of the peptide (see Methods).

Analysis of 18 independent 640ns MD trajectories amounting to >11.5µs sampling (stable binding of Ca 2+ is expected to bring the side chains of the coordinating residues close to each other).

As shown in Figure 2A , we observed simultaneous association of 2 Ca 2+ ions with different pairs of residues in the FP (see red and blue rectangles) in 4/18 trajectories (Replica IDs 6, 9, 14, and 16). In 3/18 simulations (Replica IDs 1, 10, and 17 in Figure 2A ), binding of only one Ca 2+ ion per FP was observed, with various pairs of residues involved. In the remaining trajectories we found instances in which a Ca 2+ ion was associated with a single acidic residue in the FP (the events marked by grey-striped rectangles in Figure 2A ). Since in such cases the coordination of the bound Ca 2+ is not optimal and leads to low affinity/stability, these systems were not considered for further analysis.

Overall, the binding stoichiometry in the simulations was ~1.8, in agreement with the experimentally measured value of ~1.9 for SARS-CoV-2 30 Figure S1B showing the fraction of frames in which a particular FP J o u r n a l P r e -p r o o f residue is part of a helix. We note that these findings agree well with recently published results from CD experiments on SARS-CoV-2-FP showing that the FP is mostly unfolded in solution 30 .

We investigated with MD simulations the spontaneous binding to lipid bilayers of each of the seven structural models of the Ca 2+ -bound SARS-CoV-2-FP in Figure 2B . Each of these structures was placed initially next to a membrane bilayer model composed of 600 lipids in a 3:1:1 mixture of POPC/POPG/Cholesterol that mimics the lipid composition used in recent experimental Ca 2+ binding assays in cognate systems 30, 34 . For each of these seven starting points the MD simulations were run in 6 independent replicates of 1 µs length, amounting to 42µs total run time (see Table 1 ) Figure 2B ) and illustrates the preferred modes of peptide-membrane association. A residue was considered to be in contact with the membrane if the z-directional distance between its C α atom and the neighboring lipid phosphorus atoms (P-atoms) was < 4Å (see Figure S2 captions for more details).

The contact frequency results show that the modes of association with the membrane involve different structural segments in the 7 replicas that represent the different modes of Ca 2+ binding shown in Figure 2B . Thus, Rep6 and Rep9 engage with the bilayer mostly via two segments: the middle region of the peptide (residues 830-840) and the C-terminal part (see also Interestingly, in all cases, the peptide segments interacting with the membrane contained residues that were coordinating Ca 2+ ions ( Figure S3 ) suggesting that the Ca 2+ ions enhance the interactions. In turn, MD simulations of the SARS-CoV-2-FP-membrane system without bound Ca 2+ ions registered only sporadic and much weaker binding of SARS-CoV-2-FP to the membrane ( Figure S4 ).

To obtain a measure of the timescales of the interactions characterized in Figure S2 , we first converted all trajectories into time-series of bound and unbound states ( Figure S5 , panels A-G) and then calculated lifetimes for each bound state. The distribution of the lifetimes ( Figure S5 , panel H) identified relatively long-lived complexes, ~850ns and ~770ns, respectively in trajectories from Rep9 and Rep14 (marked by red and orange stars in Figure S5 ). To understand the reason for the observed long lifetimes in these trajectories, we analyzed in more detail their modes of insertion into the lipid bilayer.

The extent of peptide penetration into the lipid bilayer was quantified using two complementary approaches. In one, we monitored z-coordinate of the C α atom of each residue in the SARS-CoV-2-FP and considered a residue inserted into the membrane if the z-distance between its C α atom and the second carbon atom in the tail of a POPC lipid (atom C22 in CHARMM36 notation) was <5Å. Using this protocol, we found that the simulations initiated from Rep9, Rep10, and Rep14 resulted in the highest frequency of insertion ( Figure Importantly, our analysis revealed that the trajectories in which the membrane-bound state is most persistent (see Figure S5 , runs marked by red and orange stars) also achieve the largest extent of membrane insertion ( Figure S2C , trajectories 13 and 29), but in two distinct modes of J o u r n a l P r e -p r o o f membrane penetration seen in Figure 3A and Figure S6 . In one (Mode 1), the peptide inserts the F833/I834 pair of hydrophobic residues. In the other (Mode 2), the bilayer is penetrated by a different pair of hydrophobic residues, L822/L823 in the N-terminal segment of the peptide.

To establish the specific depth of these insertions in relation to lipid head group and backbone regions, we calculated the number densities of all atoms of the peptide along the membrane-normal z-axis (i.e., the probability of finding the peptide atoms within rectangular slabs along the z-direction, see description in Figure S7 legend). These probability distributions

were then compared to those for atoms of the POPC lipids at different positions, e.g., the headgroup nitrogen and phosphorus atoms (N-and P-atoms, respectively), the lipid tail second carbon atoms (C22-atoms) to identify the depth of penetration relative to the membrane thickness. Figure S7 shows that in many trajectories the density of protein atoms overlaps with that of the lipid head group atoms, consistent with our results in Figure S2A that the peptide adsorbs on the lipid bilayer. But, in addition, the analysis shows that in the trajectories of replicas exhibiting deep penetrations and the longest lifetimes of bound states, the protein density overlaps with that of the C22-atoms (trajectories 13 and 29, marked by red and yellow stars in Figure S7 ). This indicates that the peptide penetrates relatively deeper into the hydrophobic region of the bilayer.

Investigation of the z-directional density profiles of each residue of the peptide confirmed the deep insertion of the same residues identified from the insertion count analysis described above, namely, F833/I834 in trajectory 13 and L822/F823 in trajectory 29. This is shown by the overlap of densities of the F833/I834 pair (in Figure 3A for trajectory 13) and the L822/F823 pair (in Figure 3C for trajectory 29) with those of the lipid N-, P-, and C22-atoms. Thus, we find that the inserted hydrophobic residues partition between the P and C22 atoms of the membrane, approximately equidistantly (i.e., ~3-4Å from each of them) along the membrane normal z-axis.

That these pairs of SARS-CoV-2-FP residues partition inside the membrane on the level of the C22-atoms (see also the structural snapshots in Figures 3B and 3D 

The Figure S9B for the 12 peptide construct structures illustrated in Figure S9A . Each of these starting conditions was simulated in 6 replicates as detailed in Table 1 and Methods.

The simulations of multiple (3, 6, 9, and 12) SARS-CoV-2-FPs interacting with the membrane revealed instances of at most 4 FPs simultaneously inserting into the lipid bilayer, but most frequently observed were concurrent insertions of 1-2 FPs ( Figure S10 ). Importantly, we found that the mode of membrane penetration by SARS-CoV-2-FP via its N-terminal segment (Mode 2 insertion described above) was the most prevalent in these sets of simulations. This is illustrated in Figure S11 which presents the fraction of trajectory time in which each residue of the peptide is inserted into the membrane in the combined trajectories from the 3, 6, 9, or 12 FP constructs. As shown in Figure S11 , these simulations captured mostly the same insertion modes observed for the single SARS-CoV-2-FP construct (Mode 1, Mode 2 and the C-terminus segment insertion which is possible only in the truncated FP construct). The highest frequency of insertions in all systems was found to involve the L822/F823 pair of residues (i.e., Mode 2).

To examine the relationship between the observed modes of membrane insertion and the initial structural models in the large number of trajectories from the multiple-PF constructs with randomly selected initial structure, we first quantified membrane insertions separately for those peptides that shared the same initial structure (irrespective of their initial placement on the grid). The histograms of membrane insertions for the 7 separate sets of data corresponding to the seven initial models are shown in Figure 4A . The striking observation from this comparison is that Mode 2 insertion for Rep14 model appears in all 4 sets of simulations (3, 6, 9, and 12 FPs) and results exclusively from Rep14 and Rep1 models, regardless of peptide concentration (see also snapshots in Figure 4B -C). Notably, out of the seven models in Figure 2 , 

To probe the arrangement of the individual membrane constituents around the peptide, we Figure 6 . For the lipids situated in the zones closest to the insertion we find higher S CD values indicating higher order in their hydrocarbon chains that propagates to ~30Å from the insertion (Zones 1 and 2) . Interestingly, we also find the PG headgroups (but not PC headgroups) in more vertical, "upright" orientation near the peptide (indicated by lower angle values with respect to membrane normal shown in panels 8F and 8G). This headgroup ordering effect is likely due to the observed electrostatic interactions between the PG head groups and K825 as it is confined to the first zone closest to the FP.

We next investigated if these FP-induced local structural perturbations in the membrane induce more global effects on bilayer properties, such as on the lateral pressure distribution 45 .

Pressure profiles were calculated (see Methods) from the single FP trajectories of Mode 2 insertion ( Figure 3C-D) , and from one of the replicates from 12 FPs construct in which 2 FPs were stably inserted via Mode 2 ( Figure S12 ). The calculated lateral pressure profiles as a function of the membrane-normal z coordinate in these two trajectories are shown in panels Figure 7A&B (red lines). For comparison, the black lines in these panels show the pressure distribution calculated for the peptide-free membrane system. To aid the interpretation of these plots in the structural context of the bilayer and the peptide, Figure 7C&D shows the position densities along the membrane normal for selected lipid atoms (P, N, and C) and FP atoms.

The pressure profile in the pure bilayer system follows the expected trends 46 : in the bulk solution, where the system is stress-free, the lateral pressure is near zero. Upon approaching the membrane, the lateral pressure deviates from zero and develops both positive and negative peaks in the lipid headgroup and backbone regions (10Å<|z|<30Å in Figure 7 ). The negative peaks are reflective of attractive forces at the membrane/water interface that shield the hydrophobic core of the membrane from solvent exposure and thus reduce the bilayer area, whereas the positive peaks are due to repulsive forces that increase the bilayer area. The additional positive peak in the bilayer center (z=0 in Figure 7 ) stems from repulsive forces that arise from entropic losses in dynamics of the lipid tails 46 .

The insertion of a single FP peptide has significant effects on the pressure distribution in the leaflet proximal to the peptide, and the profile becomes asymmetric between the two leaflets.

Indeed, as shown in Figure 7A 

The analysis of pressure profiles in the 12FP system with two FPs simultaneously inserted in the membrane ( Figure 7B ) reveals even more striking differences from the bare membrane system. All peptides, including the two FPs that interact with the membrane, are in the z>0 region of the simulation box, which makes the pressure in the bulk solution (z>30Å in Figure 7B) deviate strongly from zero. Importantly, while the pressure profile retains similar asymmetry as in the 1FP system, with the top leaflet exhibiting larger positive and negative peaks than the bottom one, the two inserted peptides affect the pressure distribution in the entire bilayer.

To understand the difference between the membrane perturbations in the single FP compared to the 12 FP construct, we examined the structural properties of the two leaflets separately in these two systems and compared them to those in the peptide-free bilayer. We found that the average order parameter values for POPC were similar in the two leaflets of the 12 FP construct, but lower than those in the bulk (pure) membrane system ( Figure S13 ). This change is accompanied by a slight increase in the average area per lipid ( Figure S14 ) and shows an overall deviation from bulk properties, indicative of the presence of stress in the membrane.

As noted earlier and shown in Figure 6 , Given its prevalence in our simulations and in light of the established role of the LLF motif in SARS-CoV membrane binding and infectivity 19, 31, 33 , we propose that the membrane-insertion mode involving the L822/F823 pair is likely to be functionally relevant in creating the fusioncompetent conditions. We note that as this manuscript was being finalized, we became aware of a complementary work in bioRxiv 49 Ebola virus [30] [31] [32] 34 . We find that FP binding modifies the molecular organization in the area of the lipid head groups and backbone atoms, as well as in most of the hydrocarbon chain layer; effects on the middle region of the bilayer hydrophobic core are minimal. Interestingly, we observed an enrichment of negatively charged PG lipids and cholesterol molecules near the inserted peptide. As described above, the electrostatic association of PG headgroups with the FP appears to provide additional stability for the LLF insertion mode, while segregation of cholesterol has an ordering effect on the lipids around FP that extends to ~30Å from the insertion site ( Figure 6 ). Given that the viral particle will bring multiple FPs to operate in close proximity to each other near the membrane, simultaneous interactions/penetration by multiple FPs will destabilize the bilayer as indicated by our observations for the 12FP system ( Figure 7 ).

Such bilayer perturbations may be essential for the fusion of the viral and host cell membranes.

Future mechanistic studies, involving more complete molecular models of the Spike as well as J o u r n a l P r e -p r o o f lipid compositions closer to those of physiological cell membranes will be necessary to address these mechanistic hypotheses and to test functional implications of membrane perturbations.

At this stage, however, the results offer a structure-specific platform to aid the ongoing efforts to design inhibitors of virus cell entry at a new and mechanistically important target.

Moreover, given the conservation of both sequence and cell-penetration mechanisms of specific CoVs known to be human pathogens, and the evidence that calcium binding is both essential and shared by FPs of such corona viruses, the known structural similarities and differences in the key regions of the mechanisms we describe should be very useful in seeking specific modes of blocking the penetration and infectivity of this larger set of CoVs. 

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The SARS-CoV Fusion Peptide Forms an Extended Bipartite Fusion Platform that Perturbs Membrane Order in a Calcium-Dependent Manner

Ca(2+) Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity

SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion

Calcium Ions Directly Interact with the Ebola Virus Fusion Peptide To Promote Structure-Function Changes That Enhance Infection

PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical pKa Predictions

VMD: visual molecular dynamics

Scalable molecular dynamics with NAMD

A Smooth Particle Mesh Ewald Method

The Nose-Hoover Thermostat

OpenMM 7: Rapid development of high performance algorithms for molecular dynamics

Membrane Builder for Mixed Bilayers and Its Application to Yeast Membranes

CHARMM36m: an improved force field for folded and intrinsically disordered proteins

Simulations of anionic lipid membranes: development of interaction-specific ion parameters and validation using NMR data

Accurate In Silico Modeling of Asymmetric Bilayers Based on Biophysical Principles

Molecular Simulations and Biomembranes: From Biophysics to Function

Physical properties of model biological lipid bilayers: insights from all-atom molecular dynamics simulations

Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine

Receptor-binding domain as a target for developing SARS vaccines

Binding Mode of SARS-CoV2 Fusion Peptide to Human Cellular Membrane

The authors thank members of the Weinstein lab, and in particular Drs. Giulia Morra andDerek Shore, for helpful discussions. H.W. and G.K. gratefully acknowledge support from the