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Binding of SARS-CoV-2 fusion peptide to host membranes

10 May 2021-bioRxiv (Cold Spring Harbor Laboratory)-
TL;DR: In this article, the authors use molecular dynamics simulations to study the two core functions of the SARS-CoV-2 peptide: to attach quickly to cellular membranes and to form an anchor strong enough to withstand the mechanical force during membrane fusion.
Abstract: During infection the SARS-CoV-2 virus fuses its viral envelope with cellular membranes of its human host. Initial contact with the host cell and membrane fusion are both mediated by the viral spike (S) protein. Proteolytic cleavage of S at the S2' site exposes its 40 amino acid long fusion peptide (FP). Binding of the FP to the host membrane anchors the S2 domain of S in both the viral and the host membrane. The reorganization of S2 then pulls the two membranes together. Here we use molecular dynamics (MD) simulations to study the two core functions of the SARS-CoV-2 FP: to attach quickly to cellular membranes and to form an anchor strong enough to withstand the mechanical force during membrane fusion. In eight 10 s-long MD simulations of FP in proximity to endosomal and plasma membranes, we find that FP binds spontaneously to the membranes and that binding proceeds predominantly by insertion of two short amphipathic helices into the membrane interface. Connected via a flexible linker, the two helices can bind the membrane independently, yet binding of one promotes the binding of the other by tethering it close to the target membrane. By simulating mechanical pulling forces acting on the C-terminus of the FP we then show that the bound FP can bear forces up to 250 pN before detaching from the membrane. This detachment force is more than ten-fold higher than an estimate of the force required to pull host and viral membranes together for fusion. We identify a fully conserved disulfide bridge in the FP as a major factor for the high mechanical stability of the FP membrane anchor. We conclude, first, that the sequential binding of two short amphipathic helices allows the SARS-CoV-2 FP to insert quickly into the target membrane, before the virion is swept away after shedding the S1 domain connecting it to the host cell receptor. Second, we conclude that the double attachment and the conserved disulfide bridge establish the strong anchoring required for subsequent membrane fusion. Multiple distinct membrane-anchoring elements ensure high avidity and high mechanical strength of FP-membrane binding.

Summary (3 min read)

Introduction

  • During infection, viruses first recognize and then enter their target cells.
  • The spike S1 subunit recognizes the human target cell by binding to the ACE2 receptor, and the S2 subunit then facilitates fusion of the viral membrane with host cellular membranes. [1] [2] [3].
  • As the membrane composition and pH of the endosome change, structural rearrangements may be induced in the S protein that facilitate membrane fusion.
  • Mutation studies by Madu et al. confirmed the importance of the LLF motif for the fusion activity of the FP.

The FP in solution forms two short amphipathic helices

  • Formed by consecutive residues, the LLF motif is spread across both faces of the NTH, as the two leucines are part of the predicted hydrophobic face and the phenylalanine is not.
  • In their simulation of FP in aqueous solution, the first segment expanded to the beginning of the second segment to form a single contiguous helix (AH2), the remainder of the second segment unfolded, and the third segment retained its helical structure.
  • These two distinct short helices (AH2 and CTH) are connected via a short loop.
  • Whereas the amino-acid sequence of CTH is highly conserved and shows no strong amphipathic properties, AH2 is less conserved but carries a strong hydrophobic moment .

NTH binds membranes with its amphipathic face

  • The authors reasoned that the two amphipathic helices NTH and AH2 may insert into the human membranes to anchor the S protein for membrane fusion.
  • In all three cases in which the NTH established the first stable contact, the binding followed a consistent path.
  • In the remaining insertion event into the endosomal membrane, the NTH established stable binding without F823 flipping in the simulated time and hence the helix remained on top of the membrane interface.
  • In the simulations with the mimetic of the outer leaflet of the plasma membrane, the authors observed one spontaneous NTH binding event, after the CTH and AH2 had already been inserted for more than 5 µs .
  • All three helices stayed bound to the membrane for ⇡ 0.5 µs until the NTH lost membrane contact.

The C-terminus of the FP binds via flexible elements

  • In all their simulations, the authors observed membrane binding also with the C-terminal end of the FP.
  • Binding involved AH2, the CTH and flexible elements flanking AH2 at both ends.
  • In some cases, these membrane interactions were relatively short lived , in other cases binding was stable for more than 6 µs.
  • The short hydrophobic stretches that inserted most frequently are centered around residues I834, L841 and I844.
  • Notably however, the short AH2 and to an even larger extent the CTH, in some cases partially unfolded to flexible amphipathic structures when bound to the membranes .

The inserted NTH can withstand high pulling forces

  • This process mimics the forces experienced by the FP during its presumed primary function of pulling host and viral membrane into proximity.
  • By pulling the C-terminal end up via a harmonic spring moving at constant velocity, the force applied to the peptide increases more or less linearly in time, until peptide segments, and ultimately the entire peptide, are pulled out of the membrane.
  • By pulling the bound NTH out of the endosomal membrane, the authors found that the binding of the NTH alone can withstand pulling forces between 40 and 65 pN.
  • Higher forces were needed to pull the NTH out of the deeply bound state with inserted F823 .
  • Interestingly, even though F823 was pulled out of the membrane along this path , the shallow state did not appear as a distinct intermediate in the pulling traces.

Additional insertion of AH2 and CTH dramatically stabilize membrane anchoring

  • The authors performed additional pulling simulations with the full length FP, starting from a binding mode with all three helices inserted into the membrane interface.
  • One might assume that these peaks were the result of the CTH and AH2 detaching individually.
  • Visual inspection revealed that this is not the case.
  • In 35% of the simulations the entire peptide was detached from the membrane after the second force peak, meaning that all three helices detached nearly simultaneously with the detachment of I844.
  • This then resulted in a series of additional force peaks, with rupture forces in the same regime as for the isolated NTH fragment.

Discussion

  • Two separate binding regions increase the likelihood to stay bound under load Due to the connection via a long disordered linker, the NTH and the two more C-terminal helices, AH2 and CTH, act relatively independent from one another.
  • For SARS-CoV-2, the concept of avidity -with multiple spread-out interactions maintaining a bound statehas emerged at multiple levels: in the ACE2-S interaction, 24 in the virion-host interaction, 14 and here in the FP-membrane interaction.
  • This underlines their idea of the two sides of the FP acting cooperatively, promoting each other's membrane binding and stabilizing the anchoring overall.
  • 25, 26 Gorgun et al. used a truncated version of the FP where they cut it behind L841, right where the authors observe the AH2.
  • They identified three binding modes: one with the loop inserted into the membrane, one with the NTH inserted and a last binding mode where the whole FP acquires helical structure and inserts on top of the membrane.

Di↵erences in lipid density may alter preferred binding mode

  • The binding modes the authors described seem to loosely group into the NTH binding to the endosomal membrane and C-terminal regions binding to the outer plasma membrane mimetic.
  • By contrast, the high density of the outer plasma membrane may favor the insertion of smaller, often disordered hydrophobic stretches such as the ones around I844.
  • This creates an artificial energetic penalty that competes with the binding free energy of the amphipathic peptide.
  • Unfortunately, this effect is di cult to correct for, short of performing simulations with prohibitively large boxes or with preemptively removed lipids from one leaflet.
  • Furthermore, the lower penalty from the lateral pressure would have likely led to the simultaneous binding of two or all three helices.

Binding of few FPs may be strong enough to facilitate membrane fusion

  • 21 Whereas already the bound NTH alone can sustain such forces, the full FP is anchored even more strongly by its three helices.
  • The structure with a disulfide bridge thus endows the FP with high anchoring strength that is reminiscent of the catch bonds giving cell-cell contacts high mechanostability.
  • The observed stability of the binding raises the question of how many bound FPs are required to be engaged for successful fusion.
  • 21 This may ultimately increase the infection success of the virus, as it would reduce one source of failure.

Conclusions

  • From atomistic molecular dynamics simulations, the authors gained a detailed view of the interactions between the SARS-CoV-2 FP with lipid bilayers mimicking the endosomal membrane and the outer leaflet of the plasma membrane.
  • In all four runs with the more flexible and less packed endosomal membrane, the FP eventually bound into the lipid bilayer with its NTH.
  • Insertion of all three helices at the same time was observed rarely in their simulations.
  • The Cys-Cys disulfide bond linking the centers of AH2 and CTH emerged as an important stabilizer.
  • The authors speculate that by spreading the membrane interaction across multiple distinct elements, with NTH, AH2, CTH and the intervening amphipathic loops all connecting to the membrane, the virus achieves a trade-o↵ between rapid insertion of individually small elements into the membrane and their firm membrane anchoring.

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Binding of SARS-CoV-2 fusion peptide to host
membranes
Stefan L. Schaefer,
Hendrik Jung,
and Gerhard Hummer
,,
Department of Theoretical Biophysics, Max Planck Institute of Biophysics, 60438
Frankfurt am Main, Germany
Institute of Biophysics, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany
E-mail: ger hard.hummer@biophys.mpg.de
Abstract
During infection the SARS-CoV-2 virus fuses its viral envelope with cellular mem-
branes of its human host. Initial contact with the host cell and membrane fusion are
both medi ate d by the viral spike (S) protein. Proteolytic cleavage of S at the S2
0
site
exposes its 40 amino acid long fusion peptide (FP). Binding of the FP to the host
membrane anchors the S2 domain of S in both the viral and the host membrane. The
reorganization of S2 then pulls the two membranes together. Here we use molecu-
lar dynamics (MD) simulations to study the two core functions of the SARS-CoV-2
FP: to at t ach quickly to cellul ar membranes and to form an anchor strong enough
to withstand the mechanical for ce during membrane fusion. In eight 10 µs-long MD
simulations of FP in proximity to endosomal and plasma membranes, we find that FP
binds spontaneously to the membranes and that binding proceeds pr e domi n antly by
insertion of two short amphipathic helices into the membrane interface. Conn ect e d via
a flexible linker, the two helices can bind the membrane independently, yet bind in g of
one promotes the binding of the other by tet he ri ng it close to the t ar get membrane.
By simulating mechanical pulling forces acting on the C-terminus of the FP we then
1
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted May 10, 2021. ; https://doi.org/10.1101/2021.05.10.443474doi: bioRxiv preprint

show that the bound FP can bear forces up to 250 pN before detaching from the mem-
brane. This detachment force is m ore than ten-fold higher than an estimate of the
force required to pull host and viral membranes together for fusion. We identify a fully
conserved disulfide bridge in the FP as a major factor for the high mechanical stability
of t he FP membrane anchor. We concl ud e, first, that the sequential bind i ng of two
short amphipathic helices allows the SARS-CoV-2 FP to i ns er t quickly into the target
membrane, before the virion is swept away after shedding the S1 domain connecting
it to the host cell receptor. Second, we conclude that the double attachment and the
conserved disulfide bridge establish the strong anchoring required for subsequent mem-
brane fusion. Multiple distinct membrane-anchorin g elements ensure high avidity an d
high mechanical strength of FP-membrane binding.
Introduction
During infection, viruses first recognize and then enter their target cells. Coronaviruses
such as SARS-CoV-2, the virus responsible for the ongoing COVID-19 pandemic, use their
trimeric spike (S) glycoprotein for both tasks. The spike S1 subunit recognizes the human
target cell by binding to the ACE2 receptor, and the S2 subunit then facilitates fusion of
the viral membrane with host cellular membranes.
1–3
To initiate fusion, in analogy to the
hemagglutinin (HA) fusion protein of influenza, the SARS-CoV-2 S2 subunit is expected
to first form one long t ri m e ri c coiled coil.
4
This elongation would bring the fusion peptides
(one per monomer) into the proximity of the membrane of the target cell. Binding to t h i s
membrane simultaneously anchors the S2 subunit in both the viral membrane (via its stalk)
and the host membrane (vi a the FP). Wh en the S2 su b u n i t subsequently collapses to form a
six-helix bundle in a proposed jack-knife m e chanism, this pull s the host membrane and the
viral membrane into proximity for eventual fusion.
4–8
SARS-CoV-2 has two dierent routes of entry into the human host cell, either directly
by fusion with the plasma membrane or by endosomal escape.
2,3,9
In the latter pathway, the
2
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted May 10, 2021. ; https://doi.org/10.1101/2021.05.10.443474doi: bioRxiv preprint

SARS-CoV-2 virion is endocytosed by the host cel l after binding to the ACE2 receptor. As
the membrane composition and pH of the endosome change, structural rearrangements may
be induced in the S protein that facilitate membrane fusion. The virus then escapes the
endosome before reaching the lysoso m e, rel ea si n g i t s RNA into the cytoplasm of the host.
9
The FP of SARS-CoV-2 spike was identified as the 40 amino-acid long sequence just C-
terminal of the S2
0
-cleavage si te.
2,10,11
Upon proteolytic cleavage, S sheds i t s S1 sub u n i t and
releases the FP as the n ew N-terminus of its S2 subu n i t .
3,12,13
Despite the concerted eorts to
study the structure and flexibility of the S protein,
8,14–18
the structure of the FP after contact
with t h e membrane has so far remained elusive. Nonetheless, mutagenic studies and electron
spin resonance (ES R) experiments have provided some insight into the structure-function
relationship of the SARS-CoV-1 FP. Using ESR, Lai et a l . observed that both e n d s of the
SARS-CoV-1 FP increased the order parameter of lipids that were spin la beled in their
membrane interface region.
19
N- and C-terminal fragments of the FP induced this e ect
individually; however, the intact FP showed the strongest eect on lipid order. Notably, no
such ordering eect was observed after mutating the LLF motif in the N-termi n a l region of
the FP to AAA. Mutation studies by Madu et al. confirmed th e im portance of the LLF
motif for the fusion acti v i ty of th e FP.
10
Together, these experiments resulted in the idea
of a bi p a r ti t e fusion platform , with the LLF motif close to the N-terminus playing a crucial
role.
19
The ability to in cr ease the order parameter of spin labeled lipids was also confirmed
for the SARS-CoV-2 FP.
20
Given the lack of experimental structural data, we performed atomistic molecular dynam-
ics simulations (MD) to eluci d at e the binding modes of the SARS-CoV-2 FP to the dierent
host membranes it can encounter during infection. In a first set of simulations, we placed the
FP in proximity to membranes mimicking the endosome and the outer leaflet of the plasma
membrane, respectively. In this way, we could probe th e spontaneous binding of the FP to
these membranes. In a second set of simulations, we studied the mechanical strength of the
FP membrane anchor. Starting wi t h membrane-bound FP, we pulled the FP away from the
3
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted May 10, 2021. ; https://doi.org/10.1101/2021.05.10.443474doi: bioRxiv preprint

membrane until it detached. Theoretical results suggest th at i n al l bi n d i n g m odes observed
here the FP anchoring is strong enough to support the complete fusion p r ocess.
21
Results
The FP in solution forms two short amphipathic helices
To explore the dynamics of the SARS-CoV-2 FP after S1 shedding and upon exposure to the
surrounding medium, we performed MD simulations in aqueous solution, starting from the
structure of the FP in intact S. Sequence and structural evi d en ce suggests that the FPs of
human infectious coronaviruses contain on e highly conserved N-terminal amphipathic helix
(NTH), a less conserved second amphipathic helix (AH2), and the C-termina l helix (CTH)
(Figure 1a, c). The NTH is folded in the prefusion cryo-EM structure of the SARS-C oV- 2
SproteinresolvedbyCaietal.
8
During a 1 µs simulation of the FP in water, the two
C-terminal resid u es of the NTH (N824 and K825) quickly unfold ed and the shorter NTH
stabilized (Figure 1a, b). Formed by co n secu t i ve residues, the LLF motif is spread across
both faces of the NTH, as the two leucines are part of the predicted hydrophobic face and
the phenylalanine is not. At the C-terminal end of the FP seg m ent, the EM structure of S
shows thr ee helical segments interrupted by short disordered regions. In our simulation of
FP in aqueous solution, the first segment expanded to the beginning of the second segment
to form a single contiguous helix (AH2), the remainder of the second segment unfolded, and
the third segment retained its helical structure. These two d i st i n ct sho rt heli c es (AH2 and
CTH) are connected via a short loop. Whereas the amino-acid sequence of CTH is highly
conserved and shows no strong am p h i p a th i c properties, AH2 is less conserved but carries
a strong hydrophobic moment (Figure 1c). Notably, AH2 and the CTH are additionall y
connected via a fully conserved disulfide-bridge.
4
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted May 10, 2021. ; https://doi.org/10.1101/2021.05.10.443474doi: bioRxiv preprint

Figure 1: Amphipathic helices in SARS-CoV-2 fusion peptide. (a) Alignment of
the FP region o f human infectious cor o n aviruses and helix a ssi g n m ent from the cryo-EM
structure (PDB ID: 6XR8, top)
8
and from our simulation of the peptide without a membrane
present (bottom). The red box marks the LLF motif in th e N-terminal region. The fully
conserved cysteines are at positions 840 and 851. The alignment was calculated using Clustal
Omega.
22
See SI Figure S1 for a larger alignment that includes other betacoronaviruses. (b)
Structural change of the FP in a 1 µssimulationinwaterandNaClincartoonrepresentation
(NTH in blue, AH2 in beige, CTH in green). (c) Amphipathic profiles of the NTH (top),
the AH2 (middle) and the CTH (bottom) from SARS CoV-2 with hydrophobicity H and
hydrophobic moment µH calculated with HeliQuest.
23
5
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted May 10, 2021. ; https://doi.org/10.1101/2021.05.10.443474doi: bioRxiv preprint

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References
More filters
Journal ArticleDOI
TL;DR: In this paper, a method is described to realize coupling to an external bath with constant temperature or pressure with adjustable time constants for the coupling, which can be easily extendable to other variables and to gradients, and can be applied also to polyatomic molecules involving internal constraints.
Abstract: In molecular dynamics (MD) simulations the need often arises to maintain such parameters as temperature or pressure rather than energy and volume, or to impose gradients for studying transport properties in nonequilibrium MD A method is described to realize coupling to an external bath with constant temperature or pressure with adjustable time constants for the coupling The method is easily extendable to other variables and to gradients, and can be applied also to polyatomic molecules involving internal constraints The influence of coupling time constants on dynamical variables is evaluated A leap‐frog algorithm is presented for the general case involving constraints with coupling to both a constant temperature and a constant pressure bath

25,256 citations

Journal ArticleDOI
TL;DR: The dynamical steady-state probability density is found in an extended phase space with variables x, p/sub x/, V, epsilon-dot, and zeta, where the x are reduced distances and the two variables epsilus-dot andZeta act as thermodynamic friction coefficients.
Abstract: Nos\'e has modified Newtonian dynamics so as to reproduce both the canonical and the isothermal-isobaric probability densities in the phase space of an N-body system. He did this by scaling time (with s) and distance (with ${V}^{1/D}$ in D dimensions) through Lagrangian equations of motion. The dynamical equations describe the evolution of these two scaling variables and their two conjugate momenta ${p}_{s}$ and ${p}_{v}$. Here we develop a slightly different set of equations, free of time scaling. We find the dynamical steady-state probability density in an extended phase space with variables x, ${p}_{x}$, V, \ensuremath{\epsilon}\ifmmode \dot{}\else \.{}\fi{}, and \ensuremath{\zeta}, where the x are reduced distances and the two variables \ensuremath{\epsilon}\ifmmode \dot{}\else \.{}\fi{} and \ensuremath{\zeta} act as thermodynamic friction coefficients. We find that these friction coefficients have Gaussian distributions. From the distributions the extent of small-system non-Newtonian behavior can be estimated. We illustrate the dynamical equations by considering their application to the simplest possible case, a one-dimensional classical harmonic oscillator.

17,939 citations

Journal ArticleDOI
03 Feb 2020-Nature
TL;DR: Identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China, and it is shown that this virus belongs to the species of SARSr-CoV, indicates that the virus is related to a bat coronav virus.
Abstract: Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large number of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their natural reservoir host, bats1–4. Previous studies have shown that some bat SARSr-CoVs have the potential to infect humans5–7. Here we report the identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started on 12 December 2019, had caused 2,794 laboratory-confirmed infections including 80 deaths by 26 January 2020. Full-length genome sequences were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. Pairwise protein sequence analysis of seven conserved non-structural proteins domains show that this virus belongs to the species of SARSr-CoV. In addition, 2019-nCoV virus isolated from the bronchoalveolar lavage fluid of a critically ill patient could be neutralized by sera from several patients. Notably, we confirmed that 2019-nCoV uses the same cell entry receptor—angiotensin converting enzyme II (ACE2)—as SARS-CoV. Characterization of full-length genome sequences from patients infected with a new coronavirus (2019-nCoV) shows that the sequences are nearly identical and indicates that the virus is related to a bat coronavirus.

16,857 citations

Journal ArticleDOI
16 Apr 2020-Cell
TL;DR: It is demonstrated that SARS-CoV-2 uses the SARS -CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming, and it is shown that the sera from convalescent SARS patients cross-neutralized Sars-2-S-driven entry.

15,362 citations

Journal ArticleDOI
TL;DR: In this paper, a new Lagrangian formulation is introduced to make molecular dynamics (MD) calculations on systems under the most general externally applied, conditions of stress, which is well suited to the study of structural transformations in solids under external stress and at finite temperature.
Abstract: A new Lagrangian formulation is introduced. It can be used to make molecular dynamics (MD) calculations on systems under the most general, externally applied, conditions of stress. In this formulation the MD cell shape and size can change according to dynamical equations given by this Lagrangian. This new MD technique is well suited to the study of structural transformations in solids under external stress and at finite temperature. As an example of the use of this technique we show how a single crystal of Ni behaves under uniform uniaxial compressive and tensile loads. This work confirms some of the results of static (i.e., zero temperature) calculations reported in the literature. We also show that some results regarding the stress‐strain relation obtained by static calculations are invalid at finite temperature. We find that, under compressive loading, our model of Ni shows a bifurcation in its stress‐strain relation; this bifurcation provides a link in configuration space between cubic and hexagonal close packing. It is suggested that such a transformation could perhaps be observed experimentally under extreme conditions of shock.

13,937 citations

Frequently Asked Questions (17)
Q1. What have the authors contributed in "Binding of sars-cov-2 fusion peptide to host membranes" ?

Here the authors use molecular dynamics ( MD ) simulations to study the two core functions of the SARS-CoV-2 FP: to attach quickly to cellular membranes and to form an anchor strong enough to withstand the mechanical force during membrane fusion. CC-BY-NC-ND 4. 0 International license made available under a ( which was not certified by peer review ) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 

The inserted NTH can withstand high pulling forcesThe authors determined the strength of the membrane anchoring by subjecting the the C-terminus of the FP to mechanical force. 

As the membrane composition and pH of the endosome change, structural rearrangements may be induced in the S protein that facilitate membrane fusion. 

In 35% of the simulations the entire peptide was detached from the membrane after the second force peak, meaning that all three helices detached nearly simultaneously with the detachment of I844. 

In eight independent MD simulations of 10 µs each, the authors observed five spontaneous insertion events of the NTH into the membrane interface. 

By pulling the bound NTH out of the endosomal membrane, the authors found that the binding of the NTH alone can withstand pulling forces between 40 and 65 pN. 

26Di↵erences in lipid density may alter preferred binding modeThe binding modes the authors described seem to loosely group into the NTH binding to the endosomal membrane and C-terminal regions binding to the outer plasma membrane mimetic. 

the authors could show that by relieving lateral pressure in the exposed leaflet, the authors could stabilize a binding mode with all three helices inserted fully. 

As the authors showed, the deep binding state associated with F823 insertion increases the pulling force that the NTH can withstand, which in turn supports the fusion activity of the FP. 

the forces required to completely detach the NTH from the deep state are 10-15 pN higher compared to the shallow binding mode. 

In the remaining 65% of pulls, parts of the long linker and the NTH stayed bound independently of the AH2 and the CTH and only detached later. 

The initial structure for the 20 replica pulling simulations was taken from an unbiased simulation of FP on the outer plasma membrane (for details see methods). 

In three simulations (Figure 2; runs 1, 2, 4), F823 flipped its orientation after being bound to the endosomal membrane for ⇡ 0.7, 2, and 3 µs, respectively, so that its aromatic sidechain became completely buried under the lipid headgroup region. 

In addition, the authors can directly link the described crucial role of the LLF motif to their observed NTH binding as it consistently includes the insertion of both leucines (L821 and L822) into the glycerol backbone region of the membrane lipids. 

The authors therefore expect that FP binding will eventually converge toall three helices bound to the membrane in the course of a real infection event. 

The authors could show that the bound FP — even though it is bound only to the interface of the membrane — can withstand large pulling forces exceeding 200 pN. 

To relieve the lateral-pressure asymmetry between the two leaflets caused by inserting a large structure into only the top leaflet of a finite-size membrane patch, the authors selected this relatively short lived state with all three helices bound, removed 5 lipid molecules of the over-compressed leaflet and restarted the simulation. 

Trending Questions (1)
How does the SARSCoV2 virus bind to host cell?

The SARS-CoV-2 virus binds to host cells through the viral spike (S) protein, specifically through its fusion peptide (FP) region.