Article

Cite This: Langmuir 2017, 33, 11496-11510 pubs.acs.org/Langmuir

Assessing the Structure and Stability of Transmembrane Oligomeric

Intermediates of an α‑Helical Toxin
Rajat Desikan,† Prabal K. Maiti,‡ and K. Ganapathy Ayappa*,†,§
†
Department of Chemical Engineering, ‡Centre for Condensed Matter Theory, Department of Physics, and §Centre for Biosystems
Science and Engineering, Indian Institute of Science, Bengaluru, India 560012
*
S Supporting Information
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ABSTRACT: Protein membrane interactions play an important role in our understanding of diverse phenomena ranging from
membrane-assisted protein aggregation to oligomerization and folding. Pore-forming toxins (PFTs) are the primary vehicle for
infection by several strains of bacteria. These proteins which are expressed in a water-soluble form (monomers) bind to the target
membrane and conformationally transform (protomers) and self-assemble to form a multimer transmembrane pore complex
through a process of oligomerization. On the basis of the structure of the transmembrane domains, PFTs are broadly classified
into β or α toxins. In contrast to β-PFTs, the paucity of available crystal structures coupled with the amphipathic nature of the
transmembrane domains has hindered our understanding of α-PFT pore formation. In this article, we use molecular dynamics
(MD) simulations to examine the process of pore formation of the bacterial α-PFT, cytolysin A from Escherichia coli (ClyA) in
lipid bilayer membranes. Using atomistic MD simulations ranging from 50 to 500 ns, we show that transmembrane oligomeric
intermediates or “arcs” form stable proteolipidic complexes consisting of protein arcs with toroidal lipids lining the free edges. By
creating initial conditions where the lipids are contained within the arcs, we study the dynamics of spontaneous lipid evacuation
and toroidal edge formation. This process occurs on the time scale of tens of nanoseconds, suggesting that once protomers
oligomerize, transmembrane arcs are rapidly stabilized to form functional water channels capable of leakage. Using umbrella
sampling with a coarse-grained molecular model, we obtain the free energy of insertion of a single protomer into the membrane.
A single inserted protomer has a stabilization free energy of −52.9 ± 1.2 kJ/mol and forms a stable transmembrane water channel
capable of leakage. Our simulations reveal that arcs are stable and viable intermediates that can occur during the pore-formation
pathway for ClyA.

■ INTRODUCTION
The eukaryotic cellular plasma membrane, composed mainly of
amphipathic lipid molecules, serves as a semipermeable barrier
compartmentalizing the cytosolic cellular contents from the
extracellular environment and is vital for life. Pore-forming
toxins (PFTs) are an ubiquitous class of lytic proteins that
primarily undermine the structural integrity of the plasma
membrane and cause cell death by disrupting the osmotic and
ionic gradients across the cellular plasma membrane.1−5 The
pathogenesis of several bacterial infections in humans caused by
Vibrio cholerae, Listeria monocytogenes, Bacillus anthracis,
Escherica coli, and Staphylococcus aureus is primarily PFT-
mediated; transmembrane pore formation causes unregulated
transport across the cell membrane, thus leading to cell death
and infection propagation.5,6 The water-soluble PFT proteins
expressed by the organism (referred to as monomers)
spontaneously recognize and bind to the target plasma
membrane or to specific protein or lipid receptors.2,5 Upon
binding to the membrane, monomers undergo a conforma-
tional change into a membrane-bound form (termed a
protomer). The assembly-competent membrane-bound proto-
mer spontaneously undergoes membrane-assisted oligomeriza-
Special Issue: Tribute to Keith Gubbins, Pioneer in the Theory of
Liquids
Received: July 1, 2017
Revised: September 19, 2017
Published: September 20, 2017

© 2017 American Chemical Society 11496 DOI: 10.1021/acs.langmuir.7b02277

Langmuir 2017, 33, 11496−11510
Langmuir
Figure 1. Schematics of the widely accepted prepore pathway and the growing pore pathway for PFTs, using ClyA as the model PFT, are illustrated
(top view of the membrane). The various membrane-bound pore-forming intermediates of the dodecameric ClyA pore (single protomers, trimers,
hexamers, and nonamers) are shown in red and are derived from the crystal structure of ClyA (PDB ID 2WCD), the membrane lipids are
represented as green circles, and water molecules in the pore lumen are represented as blue circles. In the prepore pathway, none of the
intermediates apart from the fully formed pore are capable of leakage.
tion with other protomers to form higher-order oligomeric pore
intermediates. The process of oligomerization is terminated
upon formation of the final multimeric transmembrane pore
complex, whose diameters typically range from a few to tens of
nanometers.2 PFTs are primarily classified as α- and β-PFTs,
depending on whether the transmembrane domains of the pore
assume amphiphilic α-helices or β-sheets as the dominant
secondary structures.2 Unlike protein aggregation, which is
driven primarily by misfolded states, the transmembrane pores
formed by the PFTs have a definite oligomeric structure
implying the underlying stability and robustness associated with
this process of membrane-assisted self-assembly. In contrast to
PFTs, integral host membrane proteins (especially those with
α-helical transmembrane domains) are partitioned into the lipid
membrane by the translocon protein complex, an active process
that requires energy supply by GDP−GTP exchange and
multiple cellular machineries to work in tandem.7,8 Therefore,
PFTs are unique from a membrane−protein perspective
because the toxins are originally secreted in a water-soluble
form but eventually sample the membrane environment to
form multimeric integral transmembrane channels.1,2
Many mechanisms for understanding protomer oligomeriza-
tion on the target membrane have been proposed. According to
the widely accepted prepore paradigm (illustrated in Figure 1),
protomers oligomerize on an intact membrane into a fully
formed prepore, and transmembrane pore formation occurs
with the insertion of the prepore assembly into the plasma
membrane. This transition can be accompanied by a large
conformational change as in the case of cholesterol-dependent
cytolysins such as pneumolysin.9 The prepore pathway has
been experimentally verified for many members of the class of
β-PFTs such as α-hemolysin and γ-hemolysin or the
cholesterol-dependent cytolysins such as perfringolysin O.10
Alternately, in the growing pore paradigm (illustrated in Figure
1), membrane-inserted pore intermediates also known as arcs
grow by oligomerization to form the pore complex. This
pathway is supported for some PFTs by the observation of
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transmembrane partially oligomerized pores or arcs that are
lined by a lipid edge.1,11,12 The paucity of high-resolution
structures of membrane-inserted PFT pores and pore-forming
intermediates impedes our understanding of pore-forming
mechanisms. Although water-soluble monomeric structures of
several PFTs have been resolved, the resolution of membrane-
bound intermediates, such as the protomeric and intermediate
oligomeric states, and of the final transmembrane pores is
challenging. In addition, tracking PFT oligomerization on
membranes is a formidable challenge since pore-forming
proteins lie well below the optical resolution limit, and the
dynamics are typically complete on time scales of hundreds of
microseconds.13 Ionic conductance and vesicle leakage experi-
ments provide indirect evidence of the underlying pore kinetics
and morphology.14,15 Hence, proposed mechanisms are either
derived from indirect observations of pore assembly or
reconstructed from the few available crystal structures of the
PFT monomers and the membrane-inserted pores.1,16
While pore structures and assembly mechanisms of β-PFTs
such as Staphylococcus aureus α-hemolysin,17 Staphylococcus
aureus γ-hemolysin,18 and Vibrio cholerae cytolysin19 have been
extensively studied, molecular mechanisms of α-PFT oligome-
rization and lipid reorganization during pore formation are
poorly understood.1,2,4,14,20−23 Intrinsic participation of the
membrane lipids along with the inserted transmembrane helices
to form the pore complex is a recurring theme for α-PFTs.14,24
Escherichia coli cytolsin A (ClyA) is currently one of only two
α-helical PFTs with available crystal structures of both the
water-soluble monomer25 and the membrane-inserted pore
complex.16 Thus, ClyA can be conveniently used to decipher
the assembly mechanism for the class of α-helical PFTs.16,20,26
The crystal structure of the pore reveals the formation of a
dodecameric homo-oligomeric complex having a total length of
13 nm, with the inner channel diameter varying from 7 nm at
the extracellular end to 4 nm at the transmembrane or cytosolic
end.16 The current understanding and challenges regarding the
pore-forming pathway of ClyA are detailed below.
DOI: 10.1021/acs.langmuir.7b02277
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Figure 2. Top and front views of starting configurations of the ClyA arcs (6-mer to 10-mer and the 12-mer) are illustrated here. The pink portion of
the arcs corresponds to the solvent-exposed domain, and cyan represents the transmembrane domain. The n-mer complexes were formed by simply
removing the required number of pore subunits from the 12-mer. During the course of all atomistic simulations, the arc structures do not deviate
significantly from the initial configurations shown here (discussed later).
The water-soluble ClyA monomer initially binds to the target
membrane, which contains cholesterol that acts as a lipid
receptor for ClyA,5 and subsequent membrane-assisted
oligomerization results in the formation of the transmembrane
pore complex. Surprisingly little is known about ClyA’s
oligomerization pathways, the molecular role for cholesterol,
and the fate of lipids during pore formation.20,21 ClyA pore
intermediates (termed arcs) in electron micrographs have been
observed upon inducing pore formation in detergent,27,28 in
planar lipid monolayer films,29 and in single-molecule experi-
ments in detergent.30 Combined experimental and modeling
studies have shown that these ClyA arcs (1-mer to 11-mer)
mostly oligomerize in a reversible and sequential manner on the
membrane,15,31 though nonsequential oligomerization has been
shown in detergents.30,32 While ClyA was previously assumed
to follow the prepore pathway16 where only the 12-mer causes
leakage, an experimental and modeling study (performed in our
laboratory) of ClyA-induced calcein dye leakage from small
unilamellar vesicles15 has shown that 5-mer to 12-mer ClyA
arcs cause leakage. An unlikely noncanonical pathway, where
intact ClyA prepores oligomerize in the absence of membranes
and directly insert into intact target membranes upon contact,
had previously been proposed,33 but this mechanism has been
challenged34 and is not considered in this study.
Several questions which require a molecular understanding of
pore formation remain unanswered in this extended paradigm
of ClyA pore formation. Two of them are the following: (i) Is a
membrane-inserted protomer a favorable state, and can it form
a water channel? (ii) How do the membrane-inserted arcs clear
the central lipids corresponding to the pore lumen to form
stable water channels capable of leakage? Because experiments
are often resolution-limited on spatiotemporal scales, we
attempt to answer these questions in this article with evidence
from fully atomistic and coarse-grained molecular dynamics
simulations. Fully atomistic MD simulations of PFTs enable us
to observe equilibrium configurations of these large mem-
brane−protein complexes at molecular resolution and have
been used to study the structure and properties of the ClyA
pore,35−37 the mechanism of conformational transition from
the soluble ClyA monomer to the membrane-bound proto-
mer,38 and transport properties of various molecules through
the pore lumen of the β-PFT, α-hemolysin.39−43
In this article, we carry out MD simulations of the single
transmembrane ClyA protomer (1-mer) and the full 12-mer
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ClyA pore as well as ClyA arcs at various levels of
oligomerization (6-mer to 10-mer). In both atomistic and
coarse-grained simulations, we observe that the 1-mer is stable
and creates a transmembrane water channel. Coarse-grained
simulations using the MARTINI force field (with polarizable
water44 and PME electrostatics) and umbrella sampling are
used to obtain the free energy of protomer−membrane binding.
These simulations show that a single membrane-inserted
protomer is a thermodynamically favorable state. To address
the issue of partially oligomerized intermediates, we performed
to our knowledge for the first time extensive all-atom molecular
dynamics simulations of 6-mer to 10-mer arcs in both saturated
DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and un-
saturated POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-
choline) lipid membranes. In all simulations carried out over
50−500 ns, the proteolipid arclike structures retain their
structural integrity, indicating that these purported structures
are stable. We observe that lipids initially present in the arc
spontaneously evacuate the arc interior, leading to the
formation of a transmembrane water channel, and we discuss
the possible biological implications of our observations.
■ COMPUTATIONAL METHODS
All-Atom MD Simulations. The dodecameric crystal structure of
the ClyA pore channel (PDB ID 2WCD) has unresolved N-terminal
residues 1−7 and C-terminal residues 293−303, and these residues
were added to the crystal structure as described previously.35 Standard
residue protonation states according to the force field definitions have
been assumed for all protein molecules in this study. Coordinates of
the ClyA protomer as well as that of partial n-mer pore intermediates
or arcs were obtained from the reconstructed crystal structure. The
implicit assumption here is that in the absence of further structural
information on these elusive pore intermediates it is reasonable to
assume that the protomers’ structure and assembly in the arcs are not
drastically different from those of the protomers in the crystal structure
of the ClyA pore. As mentioned in the Introduction, multiple studies
have observed ClyA arcs, and combined experimental and kinetic
modeling studies have predicted their existence.15,27−32,34 Even if the
structures were slightly different, molecular dynamics simulations
enable conformational sampling and thus often yield energetically
relaxed equilibrium structures at molecular resolution, which can be
utilized to deduce the mechanism and energetics of pore formation.
The 6-mer to 10-mer complexes and the 12-mer are illustrated in
Figure 2 and are obtained by deleting the requisite number of
protomers from the pore crystal structure. All of the MD simulations
presented in the main text of this article were performed using
DOI: 10.1021/acs.langmuir.7b02277
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GROMACS software version 4.6.4 (www.gromacs.org),45 and some Table 1. Complete List of All of the Production Simulations
simulations in the Supporting Information were performed using Performeda
GROMACS software version 2016.3 (see the Supporting Information
for details). Visualization of the initial and final configurations obtained NaCl
from the simulations was performed with VMD version 1.9.3.46 simulation concentration
protein membrane ensemble time (ns) (M)
Additional analyses were carried out using self-written tools in
MATLAB version R2017a. ClyA protomer (1- DMPC NPT 500 0.15
The single ClyA protomer as well as the n-mer arcs were inserted mer)
into pre-equilibrated DMPC (1,2-dimyristoyl-sn-glycero-3-phospho- solvated ClyA NVT 300 0.15
protomer
choline) or POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)
membranes by deleting the overlapping lipids between the protein and ClyA arc (6- DMPC NPT 224 charge-neutral
mer)
membrane. The lipid−protein complex was solvated using TIP3P47
ClyA arc (6- POPC NPT 50 charge-neutral
water molecules, with adequate solvent buffers on both the solvent- mer)
exposed side and the cytosolic side of the ClyA arcs. Each ClyA
ClyA arc (6-mer, POPC NPT 20 0.15
protomer carries a positive charge of 7, hence a sufficient number of CHARMM36, SI)
Na+ counterions (42, 49, 56, 63, 70 for the 6-mer, 7-mer, 8-mer, 9-mer, ClyA arc (7- DMPC NPT 50 charge-neutral
and 10-mer, respectively) were added to create charge-neutral initial mer)
configurations. The 12-mer simulations performed previously35 and ClyA arc (7- POPC NPT 50 charge-neutral
the 1-mer simulations were carried out at 0.15 M NaCl concentration. mer)
Upon solvent and ion addition, the solvent molecules in contact with ClyA arc (8- POPC NPT 50 charge-neutral
the lipid core in the bilayer−protein interstices were removed. The mer)
AMBER 99SB-ILDN force field48 with ϕ corrections,49 which was ClyA arc (9- DMPC NPT 50 charge-neutral
shown to perform well for proteins,50 was used to describe the intra- mer)
and intermolecular interactions of the protein and ions. The AMBER ClyA arc (9- POPC NPT 50 charge-neutral
force field-compatible Slipid parameters51 were used to model the mer)
DMPC and POPC lipids. ClyA arc (9-mer, POPC NPT 20 0.15
Electrostatic interactions were computed using the particle mesh CHARMM36, SI)
Ewald method52 with a 1.0 nm real space cutoff, and the van der ClyA arc (10- DMPC NPT 289 charge-neutral
mer)
Waal’s interactions were computed with a 1.0 nm cutoff. All bonds
were constrained using the LINCS algorithm.53 A leapfrog integrator ClyA arc (10- POPC NPT 50 charge-neutral
mer)
with a 2 fs integration time step was used along with Verlet buffered
ClyA pore DMPC NPT 100 0.15
lists (target energy drift of 0.005 kJ/mol/ns per atom). Neighbor lists (12-mer)
were updated every 10 steps; three-dimensional periodic boundary
ClyA protomer DMPC NPT 2400 charge-neutral
conditions were used. For the single solvated protomer simulations in (MARTINI, PMF)
the absence of a membrane, simulations were carried out with the ClyA protomer DMPC NPT 200 charge-neutral
above parameters but in the NVT ensemble without pressure coupling. (MARTINI, Figure
A synopsis of all simulations performed along with relevant detail is S2)
shown in Table 1. a
All of the simulations were carried out with explicit solvent and at 310
All systems were energy minimized for 10 000−100 000 steps using K. For charge-neutral simulations, only Na+ counterions are present to
the steepest-descent method (step size 0.01 nm). Initially, 200−500 ps ensure charge neutrality.
runs in the NPT ensemble with harmonic restraints on the protein
atoms were performed. Subsequently, 50- to 500-ns-long runs in the
NPT ensemble were performed for the ClyA protomer and all arcs in with spring constants of 100 kJ mol−1 nm−2. This is because windows
DMPC and POPC membranes as well the 12-mer pore in a DMPC 11 and 12 correspond to the water−bilayer interface and exhibit large
membrane (Table 1). We used the Nosé−Hoover thermostat54,55 or fluctuations in ζ for weak harmonic restraints. All simulations were
the velocity-rescale thermostat56 to control the system temperature at performed in the NPT ensemble, and the simulation parameters and
310 K by using a time constant of 0.5 or 0.1 ps, respectively. System choice of algorithms are similar to that used in the all-atom simulations
pressure corresponding to 1 bar was maintained in the plane of the (described above). Briefly, a leapfrog integrator with a 20 fs integration
membrane as well as in the direction normal to the membrane by time step was used, PME electrostatics were employed with an
using the semi-isotropic Parrinello−Rahman pressure-coupling effective dielectric constant of ϵr = 2.5 and a cutoff of 1.2 nm, the
scheme57 (isothermal compressibilities of κxy = κz = 4.5 × 10−5 velocity-rescale thermostat56 was used to set the system temperature at
bar−1, time constant of 10 ps). 310 K with a time constant of 0.1 ps, and the semi-isotropic
PMF of ClyA Protomer Membrane Binding from MARTINI Parrinello−Rahman pressure coupling scheme57 with a time constant
Simulations with Polarizable Water. The single ClyA protomer in of 12 ps was utilized to equilibrate the unit cell.
a DMPC membrane was set up using the MARTINI force field To ensure that the protein’s internal conformation is at equilibrium
(Elnedyn 2.2) with a polarizable water model44 and PME in order to at each window during umbrella sampling, we examine the RMSD of
capture the electrostatics accurately, especially at the complex the full protein and helix αA1 from the 100-ns-long unrestrained
protein−membrane−water interfaces. Upon equilibrating the ClyA equilibrium simulations of window 24 (described in Figure S2). We
protomer in the DMPC membrane for 100 ns, the steered MD feature find that the conformational change of the N-terminus is complete by
of GROMACS was used to create 24 initial configurations along the 20 ns (Figure S3), and a final snapshot after 100 ns is illustrated in
reaction coordinate ζ (defined as the distance between the centers of Figure S4. Hence, the first 20 ns of each window is censored, and the
mass of the membrane and protomer) such that each window was resulting PMF and associated binding energy are reported in Figure 5.
separated at intervals of Δζ = 0.2 nm. The phosphate atoms of the The effect of varying the censoring threshold on the PMF is reported
membrane were restrained in the Z coordinate using harmonic in Figure S5, where we find that the PMF is relatively insensitive to the
restraints of 100 000 kJ mol−1 nm−2. Solvent and unit-cell equilibration censoring threshold. The potential of mean force along the reaction
simulations for 2 ns and umbrella sampling simulations for 100 ns per coordinate was obtained by using the weighted histogram analysis
window were carried out with a weak restraining harmonic force at method (WHAM; implemented in the g_wham58 tool in GROMACS
that particular value of ζ. A spring constant of 10 kJ mol−1 nm−2 was version 4.6.4.) on histograms from the umbrella sampling trajectories,
used for all windows (see Figure S2 for the rationale behind choosing and the final PMF was generated by dividing histograms along the
this value), except windows 11 and 12 which were simulated again reaction coordinate into 200 bins. The error bars on the PMF were

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Figure 3. (A) Initial and final snapshots of a single ClyA protomer and the ClyA pore inserted into DMPC membranes (the N-terminal helix αA1 is
colored green, the C-terminal helix αG is colored blue, and the β-tongue consisting of residues 177−203 is colored magenta). (B) RMSD traces of
the various conformations of ClyA show structural differences. (C) Comparison of the equilibrium RMSFs of the single transmembrane ClyA
protomer (averaged over various blocks of time) with a protomer subunit in the transmembrane ClyA pore. (D) Axial tilts of helices αA1 and αB
with respect to the Z axis are plotted as a function of simulation time for the single protomer and are compared with the tilts averaged over all
protomer subunits in the ClyA pore. The axes for helices αA1 and αB are defined as vectors passing through the center of mass of residues 11 and 33
and residues 38 and 99 (shown as labeled yellow beads in the protomer on the right), respectively. (E) Temporal trend in the radius of gyration (Rg)
of the single protomer.

computed by generating 2000 bootstrapped trajectories from the
obtained histogram such that the new random trajectories had similar
converged histograms and autocorrelation times.
Spring Constant and Force between the Edge Protomers
from Atomistic MD Simulations. If the distance between the edge
protomers in the arcs is normally distributed around a mean, then
these edge protomers can be assumed to be connected by a virtual
harmonic spring with a spring constant k and a restoring force F.
Assuming a classical Hamiltonian for the spring in the canonical
ensemble, the equation for the spring constant (in kJ mol−1 nm−2) at a
temperature of 310 K can be derived as shown below.
time of λCOM(t), estimated by fitting a single exponential to the 6-mer
and 10-mer trajectories, was found to be τ6 = 1.13 ns and τ10 = 2.04 ns,
respectively. Subsequently, λCOM(t) from the 6-mer and 10-mer
trajectories was divided into blocks of length τ6 and τ10 after discarding
the first 50 ns of both trajectories, and k and F were computed for
every block from eq 5. The mean k and F values were computed by
averaging over all of the blocks, and error bars were computed by 1-
deletion block-jackknife resampling.59,60 From this method, the mean
values of k and F for the 6-mer were calculated to be 39.68 ± 10.56
pN/nm and 306.06 ± 81.42 pN, respectively, and for the 10-mer,
11.39 ± 3.78 pN/nm and 76.61 ± 25.03 pN, respectively.

■
The Hamiltonian of a one-dimensional harmonic oscillator is given
as RESULTS
2 2
p kr Membrane-Bound ClyA Protomer Retains Structural
/(p , r ) = +
2m 2 (1) Integrity and Stabilizes a Continuous Transmembrane
where r is defined as the normally distributed Euclidean distance Water Channel. A key factor in the pore-formation process of
between the center of mass of the two edge protomers. The variance in PFTs such as ClyA is the conformational transformation from a
the mean position can be calculated as water-soluble monomer to an assembly-competent protomer.
To the best of our knowledge, experimental data pertaining to
∬ r 2e(−β /(p , r)) dp dr ⎜⎛ ∬ r e(−β /(p , r)) dp dr ⎞⎟
2
the energetics of membrane binding of ClyA is absent in the
⟨r 2⟩ − ⟨r ⟩2 = −⎜ (−β /(p , r )) dp dr ⎟ literature. First, we determine if the single transmembrane
∬ e(−β /(p , r))dp dr ⎝ ∬e ⎠ protomer is structurally stable relative to a protomer bound in
(2) the fully oligomeric pore. To contrast the structural attributes
2 2 of ClyA in these two conformations (single transmembrane
∫ e(−βp /2m)
dp ∫ r 2e(−βkr /2)
dr
protomer and bound protomer in the pore complex), a 500-ns-
⟨r 2⟩ − ⟨r ⟩2 = 2 2
(−βp /2m) (−βkr /2) long all-atom MD simulation of a single transmembrane ClyA
∫e dp ∫ e dr
protomer and a 100-ns-long all-atom simulation of the
⎛ (−βp2 /2m) 2 ⎞ 2
∫e dp ∫ r e(−βkr /2) dr ⎟ transmembrane ClyA pore were performed. The initial and
−⎜ final simulation snapshots are illustrated in Figure 3A.
⎜ (−βp2 /2m) 2 ⎟
⎝∫e dp ∫ e(−βkr /2) dr ⎠ (3) The RMSD traces with respect to the initial simulation
Because these are Gaussian integrals, we get structures of the single membrane-inserted protomer and the
pore are contrasted (illustrated in Figure 3B). The single
2

⟨r 2⟩ − ⟨r ⟩2 =
(
κBT 1 − π ) membrane-inserted protomer has a RMSD with larger
k (4) fluctuations when compared to that of the pore. A comparison
of the per-residue root-mean-square fluctuations (RMSFs)
Hence, between the single transmembrane ClyA protomer in
2 2 comparison to protomer subunits in the dodecameric pore
k=
(
κBT 1 − π) and F = (
κBT 1 − π )⟨r⟩ (RMSF averaged over all 12 protomer subunits) is illustrated in
2 2
⟨r ⟩ − ⟨r ⟩ ⟨r ⟩ − ⟨r ⟩2
2
(5) Figure 3C. The fluctuations of all of the residues in the single
where magnitude of the force (F) is in pN molecule . Because we −1 protomer are consistently higher than the residues in the pore,
report only single-molecule results, we write the units of force simply and this is attributed to the absence of stabilizing protein−
as pN. From now onward, r(t) is referred to as λCOM(t). Results for the protein interactions for the single protomer. Changes in the
6-mer and the 10-mer are calculated as follows. The autocorrelation orientation of the single transmembrane protomer with respect
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to the membrane are illustrated by a comparison of the helix
tilts of helices αA1 and αB, as shown in Figure 3D. It can be
seen that the helix tilts of the single protomer exhibits drift as
well as large fluctuations from the initial value, while a protomer
in the pore is stable. This is consistent with previous Go-model
simulations of the ClyA protomer in the presence of an explicit
model membrane,38 where the protomer samples a large
number of helix tilt angels in the membrane-inserted state. The
temporal trend of the radius of gyration (Rg) of the single
protomer is shown in Figure 3E, and we observe stable trends
by the end of the 500-ns-long simulation. Overall, these results
confirm that the protomer in the pore is a more favorable state
because of protein−protein interactions with neighboring
protomers, but the single protomer does not lose its structural
integrity in the absence of the other protomers.
The all-atom MD simulations of the single membrane-
inserted protomer (described above) also show for the first
time to our knowledge that a stable and continuous membrane-
spanning water column is stabilized by the hydrophilic face of
the amphipathic transmembrane N-terminal helix (Figure 4A;
also see Figure S1). Transmembrane amphipathic helix αA1 is
cleanly separated into hydrophobic and hydrophilic surfaces,
thus resembling a Janus surface (illustrated in Figure 4A). The
Figure 4. (A) Instantaneous snapshot at 300 ns of the transmembrane
protomer from an all-atom MD simulation shows the transmembrane
water channel. Final snapshots at 500 ns are shown in Figure S1.
Transmembrane residues 1 to 36, which comprise the N-terminus and
the amphipathic helix αA1, are depicted below with the hydrophobic
residues colored green, hydrophilic residues colored red, and proline
36 colored purple. (B) Snapshots (100 ns each) of the transmembrane
protomer from the first window of the MARTINI umbrella sampling
simulations (also illustrated in Figure 5) show that the single protomer
forms a water channel in coarse-grained simulations as well. Note that
the center-of-mass distances between the protomer and the membrane
are restrained by a weak harmonic force in MARTINI, but all-atom
simulations are not.
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hydrophobic surface interacts with the hydrophobic lipid acyl
chains, whereas the hydrophilic surface stabilizes the sponta-
neously formed transmembrane water channel.
Because the equilibration of lipids and solvent around the
protein is crucial, we carried out simulations with an alternate
equilibration protocol and a completely different force field,
CHARMM36.61,62 The single protomer was inserted into a
POPC membrane using the CHARMM-GUI membrane
builder63−65 (http://www.charmm-gui.org/), ensuring that
lipids were completely packed around the protein, with
extensive unit cell and solvent equilibration simulations (details
in Supporting Information). Using this initial configuration, 20
ns simulations revealed the formation of the water channel as
discussed above, indicating that the formation of the water
channel is a robust phenomenon (Figure S6).
From this finding, it is conceivable that the oligomerization
of the protomers in the noninserted state extends this
transmembrane domain into a Janus surface, with the alkyl
lipid tails stabilized by the hydrophobic outer surface and the
aqueous channel in the pore lumen stabilized by the inner
hydrophilic surface, as described in the growing pore
mechanism (Figure 1). Another α-PFT fragaceatoxin C also
possesses an amphipathic N-terminal helix with a Janus surface
topography which forms the transmembrane domain of the
membrane-inserted pore.24 These findings for ClyA indicate
that oligomerization and membrane leakage can be initiated by
a single membrane-inserted protomer.
We next assess the binding free energy of a single ClyA
protomer to a DMPC membrane by employing coarse-grained
simulations (MARTINI force field with polarizable water and
full electrostatics) with the umbrella sampling technique. We
point out that although it is desirable to obtain the PMF using
an all-atom description, complete umbrella sampling, given the
size of the protein and the corresponding simulation box, is
computationally prohibitive. Furthermore, MARTINI force
fields have been know to capture protein membrane
interactions quite accurately.66,67 The free-energy profile is
shown in Figure 5. It can be clearly seen that membrane
binding by ClyA is thermodynamically favorable, with a free-
energy gain of ΔE = −52.9 ± 1.2 kJ/mol, or ∼ −12.6 ± 0.3
kcal/mol, at ζ ≈ 6.0 nm for membrane-inserted conformations
relative to completely solvated conformations (configurations
illustrated in Figure 5). This is similar to a previous estimate of
−46 kJ/mol for ClyA.38
The membrane-inserted protomer (from the initial window)
of the coarse-grained simulations also stabilizes a trans-
membrane water channel as shown in Figure 4B. In addition
to this, the protomer is completely solvated in the final
windows of the umbrella sampling simulations and is seen to
undergo significant structural distortions in the N-terminus and
helix αA1. The final snapshot after 100 ns from umbrella
sampling simulations of configuration 24 is magnified in Figure
S4, and structural distortions such as unfolding of the N-
terminus and helix αA1 (colored orange) can be observed.
Fully Solvated Protomer Is Structurally Unstable
Compared to a Membrane-Inserted Protomer. To
analyze the structural distortions of the fully solvated protomer
in detail, fully atomistic simulations of the transmembrane vs
solvated protomer conformations were compared, and the
initial and final snapshots of the protomer in both solvated and
transmembrane configurations are shown in Figure 6A−F. The
evident distortion in the N-terminus and helix αA1 (residues
1−34) is due to the exposure of hydrophobic residues in this
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Figure 5. Free energy of the protomer binding to the membrane is
shown using the potential of mean force (PMF) along reaction
coordinate ζ (distance between the center of mass of the membrane
and the center of mass of the protomer). The PMF was obtained by
simulating 24 configurations of the transmembrane protomer, with
overlapping histograms at intervals of Δζ = 0.2 nm for 100 ns each
(Methods). These simulations were carried out using the MARTINI
coarse-grained force field with polarizable water and full electrostatics.
Snapshots (100 ns) from the 1st window, 13th window, and last
window are illustrated along with the value of ζ at 100 ns. The protein
residues corresponding to the N-terminus and helix αA1 and the rest
of the protein are represented by orange and red vdW spheres,
respectively. PO4 membrane beads are shown as green vdW spheres.
amphipathic domain to the aqueous environment (also see
Figure S4). This is corroborated by the time traces of the
backbone root-mean-square deviations (RMSD) of this domain
(shown in Figure 6G) which plateau at a higher value for the
solvated protomer as well as the decreased helicity of these
residues in the solvated protomer as compared to that of the
membrane-inserted protomer. The loss of structural integrity in
the putative transmembrane domain of the solvated protomer
(illustrated in Figure 6G) suggests that the membrane-bound
protomer conformation with the N-terminus residues bound to
the membrane is structurally favorable. The protomer can be in
a noninserted state with the amphipathic N-terminus and helix
αA1 bound to the upper leaflet, or the protomer may span the
membrane and create a membrane channel as shown in Figure
4. Similar evidence for membrane insertion of the N-terminal
helix prior to oligomerization has been shown for other α-
helical pore-forming proteins such as Equinatoxin II68 and
Bax.69
Spontaneous Formation of Membrane-Permeabiliz-
ing Proteolipid ClyA Arcs. In the previous section (Figure
4), we observed that a single membrane-inserted protomer
stabilizes a transmembrane water channel. In this section, we
investigate the stability of larger transmembrane arcs to displace
central lipids and form stable water channels in the membrane.
To observe the spontaneous formation of water channels, the
transmembrane arcs were initially inserted into previously well-
equilibrated DMPC lipid bilayers with the lipids initially
present in and around ClyA arcs. No water defect was present
at the start of simulations, and the membrane was intact except
for the inserted protein complexes. Representative simulation
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Figure 6. (A−F) Initial and final simulation snapshots of the fully
solvated protomer compared to the transmembrane protomer show
that the structure of residues 1−34 comprising the N-terminus and
helix αA1 is significantly distorted when the protomer is away from the
membrane, and these residues are completely exposed to the solvent.
The ClyA protomer is colored red except for transmembrane residues
1−34, which are colored orange, and phosphate beads in the
membrane colored green. The solvent is omitted for clarity. (G)
Final snapshots (all-atom) of the membrane-inserted protomer and
the completely solvated protomer without a membrane highlight the
structural distortions at the N-terminus. Corroboratively, the RMSD of
residues 1−34 is significantly higher for the fully solvated protomer
than for the transmembrane protomer, indicating the greater stability
of the transmembrane state. Secondary structural analysis (% helicity)
shows the loss of helical structure for the stretch of residues 1−34 in
the solvated protomer but not for the membrane-inserted protomer.
snapshots of the initial and final configurations (50 ns) of the 6-
mer and 9-mer ClyA arcs in DMPC bilayers and 6-mer to 10-
mer arcs in POPC bilayers are shown in Figure 7A,C. By 50 ns
for all arcs, the lipids initially present in the hydrophilic arc
interior were spontaneously displaced into the surrounding
membrane, with a transmembrane water channel replacing the
displaced lipids. The displacement sequence for the lipids over
the 50-ns-long simulation for the 7-mer arc in the DMPC
membrane is illustrated in Figure 7B. This indicates that the
favorable configuration for transmembrane ClyA arcs is the
formation of a transmembrane water channel that solvates the
inner hydrophilic surface of the transmembrane domain.
Results from all-atom simulations of the 6-mer and 10-mer
arcs in DMPC with longer run times (up to 289 ns) show that
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Figure 7. (A) Rapid lipid evacuation from the interior of the transmembrane ClyA pore intermediates (6-mer and 9-mer; water and ions removed for
clarity) into the plane of the surrounding DMPC membrane occurs within 50 ns. (B) A timeline of lipid evacuation is shown for the 7-mer. (C) Fully
atomistic simulations of ClyA arcs inserted into POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membranes show rapid lipid evacuation
within 50 ns for the 6-mer to the 10-mer (water and ions removed for clarity). Corresponding lipid survival probabilities for all n-mer arcs in DMPC
and POPC membranes are shown in Figure 8.

Figure 8. A line connecting residue 189 on chain A and residue 11 on chain G of a 7-mer (colored green) defines the boundary between the interior
and the exterior of the pore. The continuous survival probability (CSP) of DMPC and POPC lipids within the 6-mer, 7-mer, 9-mer, and 10-mer
ClyA arcs captures the probability that a lipid resides in the interior of an arc for a specific length of time.

the water channel and the proteolipid arc complexes on longer the lipids within the arc. The definition of the arc boundary is
time scales are stable (discussed subsequently). Using a similar assumed to be a line joining residues 11 and 189 in the edge
CHARMM-GUI protocol for lipid packing in the arc interior protomers of the arc as shown in Figure 8. For the ith lipid, the
(without water) as implemented for the single protomer, 20 ns instantaneous probability Pi(t) is either 1 if the lipid is inside
simulations with the CHARMM36 force field for the 7-mer arc
the arc or 0 otherwise. The lipid position is tracked by the
revealed rapid lipid evacuation and the formation of a
transmembrane water channel, similar to the results presented coordinates of its phosphorus atom. The survival probability for
above, reinforcing the observation of lipid evacuation and water N lipids at time t is given as
channel formation (Figure S7).
N to + t
Kinetics of Lipid Evacuation from the Atomistic MD
Simulations. Because lipids are displaced from the hydrophilic S(t ) = ∑ ∏ Pi(tk)
arc interior within a period of 50 ns, we computed the i=1 tk = to (6)
continuous survival probability (CSP) of the lipids originally
present in the arcs (illustrated in Figure 7) to quantify the where the angular brackets represent shifted time averages. The
kinetics of lipid evacuation. CSP defines the residence time of normalized CSP, ⟨S(t)/S(0)⟩, is calculated over 50 ns
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Figure 9. (A) Front view of a 9-mer (red) with the stable transmembrane water channel (blue) in a membrane (transparent green slab) illustrating
that the stable ClyA arcs are functional membrane-permeabilizing entities. A cross-section across the bilayer of the 9-mer arc in DMPC reveals the
toroidal orientation of the lipids at the edge of the bilayer facing the arc interior (lipid choline headgroups colored red and phosphate groups colored
green). (B) An analysis of the lipid tilts in the arc vicinity: the distribution of the angle subtended by the lipid vector with the z axis is shown. Two
peaks corresponding to the upper and lower lipid leaflets can be seen for lipids in the bulk membrane (blue). However, in the lipid shell immediately
surrounding the 9-mer (green), θ assumes intermediate values which correspond to toroidal lipids lying nearly perpendicular to the z axis. (C)
Toroidal lipids participate in a larger number of hydrogen bonds in both 7-mer and 9-mer arcs in comparison with lipids away from the proteolipidic
edge. (D) Water radial distribution functions (g(r)) around the phosphorus atoms of the toroidal and free lipids for 7-mer and 9-mer arcs are shown.

(sampling frequency of 10 ps, 5000 snapshots over each 50 ns
trajectory) and fit to the following biexponential function:
S (t ) ⎛ t⎞ ⎛ t⎞
= A exp⎜ − ⎟ + B exp⎜ − ⎟ + k
S(0) ⎝ τf ⎠ ⎝ τs ⎠
In the above equation, constant B = 1 − A − k from initial
conditions. Two distinct time constants are observed in the
CSP fits: τf describes the fast component of lipid evacuation,
and τs describes the slower component. Because the survival
probabilities have decayed to almost zero for most of the cases
as shown in Figure 8, τs describes the residence time of the
lipids inside the arc. We attribute the fast component (<1 ns)
to the rapid response of the lipids whose hydrophobic acyl tails
are abruptly exposed to the inner hydrophilic arc interface. The
longer time constant (7.9−16.9 ns) represents the gradual
reorganization and formation of the toroidal lipid edge
(discussed in the subsequent section). Thus, we obtain 7.9−
16.9 ns from the CSP of lipids inside the arcs as quantitative
estimates of the lipid survival time scales depending on the arc
size and the lipid type (DMPC/POPC). We also observe that
POPC has a relatively shorter survival time when compared to
DMPC, indicating that unsaturated lipids may undergo faster
expulsion from the arcs than fully saturated lipids such as
DMPC, despite the presence of longer acyl chains in POPC.
The biological significance of the calculated lipid evacuation
time scale is that because spontaneous channel formation upon
inserting ClyA arcs occurs within a few tens of nanoseconds on
pure lipid bilayer membranes, this process is not predicted to
be a rate-limiting step in pore formation. We point out that
pore formation and the ensuing lipid dynamics are dependent
on the pore-formation pathway. In the case of the prepore
mechanism, assembly first occurs on the membrane surface to
form a prepore which subsequently inserts into the membrane.
The fate of the lipids during this stage is largely unknown.
Recent AFM images70−72 show the presence of inserted arcs at
different stages of oligomerization for the cholesterol-depend-
(7)
ent toxins, listeriolysin O and suilysin, which are known to
follow a prepore pathway. In this scenario, partially assembled
pores (or prearcs) on the membrane surface undergo a
conformational change resulting in membrane insertion and the
formation of a transmembrane pore. Our simulations shed light
on a possible lipid displacement pathway to stabilize the
transmembrane arcs. Recent work in our laboratory15 suggests
that ClyA leakage on small unilamellar vesicles occurs
predominantly through membrane-inserted arcs formed by
the stochastic insertion of prearcs. Here again, rapid lipid
displacement dynamics would lead to the formation of a stable
arc. However, it must be noted that while the lipid
reorientational dynamics has been observed in our simulations
to be typically complete within tens of nanoseconds, the actual
displacement of lipids from the arc interior on complex cellular
membranes with an underlying cytoskeleton may be signifi-
cantly slower.
Molecular Analysis of the Toroidal Lipid Edge. An
important property of the lipid bilayer membranes is the
propensity to form an edge defect, characterized by the line
tension of the edge (Λ). This tension causes the reorientation
of lipids at the edge of membranes in ribbon configurations into
configurations similar to the reorientation of lipids observed at
the mouth of the arcs.73 The formation of the toroidal lipid
edge shields the hydrophobic lipid acyl tails from solvent
exposure, locally offsets the energy penalty due to missing
protomers, and stabilizes the partially formed arcs and may
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relieve curvature stress.74,75 Lipid edges configured to form
stable water channels lined by proteolipid walls have also been
observed in electron micrographs,11,76,77 AFM images of stable
partially oligomerized arcs of cholesterol-dependent PFTs in
membranes,78 X-ray diffraction studies of Bax-derived pep-
tides,79 and MD simulations of protegrin arcs in lipid bilayers.80
Several studies have hypothesized the presence of proteolipidic
oligomerization intermediates during pore formation.1,11
However, the experimental observation of lipid reorientation
to form the toroidal edge during pore formation and its
structural characterization is yet to be reported.75
MD simulations of the proteolipid ClyA arcs show the
toroidal lipid edge in molecular detail, as illustrated in Figure
9A for the 9-mer arc in the DMPC membrane. The toroidal
lipids at the water−lipid interface of the arcs where protomers
are missing form an incomplete toroidal edge with the lipids
orientated perpendicular to the membrane normal at the mouth
of the arcs. The polar lipid headgroups (shown in red and
green) are shown facing the continuous water channel enclosed
by the proteolipid complex (cyan). An analysis of lipid tilts in
the arc vicinity show distributions of the angle (θ) subtended
by the lipid vector with the membrane normal (Figure 9B). The
lipid vector is defined as a vector passing from the center of
mass of the last carbon atoms in the lipid alkyl tails to the
phosphate bead, and the membrane normal is oriented toward
the lower leaflet. Two distinct well-formed peaks corresponding
to the lower leaflet (0° < θ < 90°) and the upper leaflet (90° <
θ < 180°) can be seen in the second lipid shell (blue), which
represents the free lipids in the bulk membrane. However, in
the lipids which form the toroid edge of the arc (green), θ
assumes intermediate values around 90°, which correspond to
toroidal lipids lying nearly perpendicular to the z axis. The lipid
shells are chosen such that shell 1 (green) consists of lipids
situated in an angular cross section at a distance less than R
away from the arc center (where R is the radius of the arc), and
shell 2 (blue) consists of lipids situated in an angular cross
section at a distance more than 2R away from the arc center.
Additionally, the distribution corresponding to the upper leaflet
shows greater broadening compared to lipids in the lower
leaflet, indicative of an induced leaflet asymmetry due to the
presence of the arc.
The toroidally reoriented lipids facing the water channel in
the arc interior participate in a larger number of hydrogen
bonds in comparison to free lipids (membrane lipids away from
the influence of the arc) in both 7-mer and 9-mer arcs as shown
in Figure 9C. To find the water structuring around these
toroidal lipids, an analysis of the water radial distribution
functions (g(r)) around the phosphorus atoms of the toroidal
and free lipids in the membrane was carried out (Figure 9D).
The water g(r) for the free lipids is nearly identical for both 7-
mer and 9-mer arcs. The water g(r) for toroidal lipids present in
the lipid edge shows minor enhancements mostly in the second
solvation shell as compared to free lipids in the membrane but
equilibrate to bulk densities with increasing r. This may be due
to suboptimal packing of lipids in the toroidal edge. The higher
coordination number of water around the toroidal lipids may
explain the greater hydrogen bonding observed in both 7-mer
and 9-mer arcs, as shown in Figure 9C.
Energetics of the Toroidal Lipid Edge. Because toroidal
lipids are created at an energetic cost, we use a simple model to
estimate the penalty incurred as a result of the presence of the
toroidal lipid edge. For each arc, the free energy of the edge is
simply given as
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[Fn]toroidal lipid = ΛL(n); n = 1, ..., nmax (8)
where L(n) is the length of the lipid edge defined by the
oligomeric status of the arc and Λ is the line tension associated
with the edge. The chord length, L(n), can be derived using
simplified geometric arguments as follows. If there are nmax
protomers in a fully formed pore, then each protomer subtends
an angle of 2π . Therefore, the angle subtended by the arc in
nmax
the center of the pore is θ(n) = ( )2π when
n
nmax
n
nmax
≤ 0.5 and
(
θ(n) = 1 −
n
nmax )2π when n
nmax
> 0.5, as illustrated in Figure
10. Assuming that the curvature of the arcs is similar to that of
Figure 10. Top-view schematics of two arc complexes where
n n
n
≤ 0.5 and n > 0.5 are illustrated (protomers illustrated in
max max
blue). The length of the lipid edge, L(n), the angle subtended by the n-
mer arcs, θ(n), and the radius of the pore, Rpore, are also depicted. The
energetic penalty for the formation of the lipid edge given by eq 9 is
plotted for the 1-mer to 12-mer arcs for Λ = 10 and 46.1 pN. Λ
depends on the length of the lipid acyl chains and whether the lipid is
saturated or unsaturated, and the above range encompasses various
lipid bilayers as reported previously.73
the fully formed pore, we can extend the argument that the
protomers in a partially oligomerized arc also subtend the same
angle in the center of the pore. Because the ClyA pore has a
tapering transmembrane region, we use an average value of the
upper and lower pore radii (Ru and Rl, respectively) to obtain
the edge length L(n). Figure 9A depicts the upper and lower
leaflets in the context of the arcs. The edge penalty for the
transmembrane n-mer arc complex can be expressed as
⎛ θ(n) ⎞
[Fn]toroidal lipid = Λ(Ru + Rl)sin⎜ ⎟ ; n = 1, ..., nmax
⎝ 2 ⎠
(9)
The typical value of Λ for the lipid membrane edge formation
lies in the range of 10−46.1 pN,73 and for the DMPC lipids, the
value is 19.2 ± 2.8 pN. Using this range for Λ and values of Ru
= 7 nm and Rl = 4 nm,16,37 we obtain the variation in
[Fn]toroidal lipid as illustrated in Figure 10. The energetics are
proportional to L(n), reaching a maximum at θ = 90 or n = 6.
For the dodecameric ClyA pore where n = nmax = 12, the
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Figure 11. (A) RMSD time traces and corresponding histograms for the 6-mer and 10-mer arcs in DMPC membranes, simulated for 224 and 289 ns,
respectively. (B) Superposition of the initial (cyan) and final (red) snapshots of the 6-mer and 10-mer arcs shows that the arc geometry is
undistorted as the simulation proceeds. (C) Stable Rg (radius of gyration) time traces and histograms (inset) of the 6-mer and 10-mer arcs further
corroborate the notion of arc stability. (D) Time traces and distributions of the axial tilts of helices αA1 and αB (as illustrated in Figure 3D) averaged
over all of the protomer subunits in the 6-mer and 10-mer arcs are shown. (E) The time trace of the center-of-mass distance between the edge
protomers (λCOM(t)) is shown; the inset illustratates λCOM for the 6-mer.

penalty is zero when pore formation is complete. The estimate
for forming the toroidal edge which lies in the range of 10−150
kJ/mol must be overcome for the n-mer arcs to be stable in the
membrane as our MD simulations suggest. Thus, stabilization
of the arc must be driven by protomer−protomer and
protomer−membrane interactions which offset the cost of
forming the edge. Evaluating these energetic contributions to
derive a complete free-energy landscape for the oligomerization
process is outside the scope of this article, and we are currently
pursuing these computations.
Structural Stability of the 6-mer and 10-mer
Proteolipidic ClyA Arcs from Longer Atomistic MD
Simulations. The fully atomistic 50-ns-long 6-mer and 10-mer
ClyA arc simulations in DMPC membranes were extended to
224 and 289 ns, respectively, in order to verify the structural
stability of the arcs at longer time scales (Figure 11). The
RMSDs of both arcs (Figure 11A) show small deviations from
initial structures, as confirmed by the superposition of initial
and final structures (Figure 11B) and the mostly constant
radius of gyration (Rg, Figure 11C). These observations imply
that the 6-mer and 10-mer arcs are stable and reveal no
propensity to disassociate over simulation time scales of
hundreds of nanoseconds. RMSD and Rg histograms appear
to be normally distributed as shown in Figure 11A,C. The
orientation of the arcs measured by averaging helix tilts of
helices αA1 and αB over all protomer subunits in the arcs is
shown to be stable over the simulation time scales in Figure
11D and similar to the observations of the 12-mer (Figure 3D).
Fluctuations in the orientation of the 6-mer are marginally
higher than that of the 10-mer. The time trace of the length of
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the simulated unit cell is illustrated for both the 6-mer and the
10-mer in Figure S8, and the trends are observed to be stable.
Time traces of the Euclidean center-of-mass distance
between the edge protomers (λCOM(t), illustrated schematically
for a 6-mer in the inset of Figure 11E) are analyzed for both the
6-mer and 10-mer arcs (Figure 11E). Stable λCOM(t) trends can
be used to evaluate the tendency of the arcs to close and form
n-mer pores where n < nmax. Furthermore, the edge-protomer
center-of-mass distances can be used to compute the spring
constant (k) between the edge protomers (which gives a rough
measure of the arc stiffness) and the equilibrium harmonic
force (F) between the arc edges (eq 5 of Methods).
The mean values of k and F for the 6-mer are 39.68 ± 10.56
pN/nm and 306.06 ± 81.42 pN, respectively, which are ∼4
times greater than that of the 10-mer (11.39 ± 3.78 pN/nm
and 76.61 ± 25.03 pN, respectively). Intuitively, smaller
oligomers have a higher spring constant due to lower arc-
backbone fluctuations. However, as oligomerization proceeds
and additional protomers are added to the arcs, the flexibility of
n-mer arc-backbones increases as a result of the increasing arc
length and attractive forces between the edge protomers, which
also become progressively proximal as oligomerization
proceeds. This renders flexibility to the arcs and increases
edge-protomer fluctuations (Figure 11E), thus leading to a net
decrease in arc stiffness as evidenced by lower spring constants
and forces for the 10-mer. However, the 10-mer arc does not
close and form a full pore on the simulated time scales, and this
is consistent with the notion that the 12-mer is the
predominant pore stoichiometry observed experimentally.16,32
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■ DISCUSSION AND CONCLUSION
We have carried out extensive molecular dynamics simulations
in an effort to shed light on the state of the oligomeric
intermediates that could form during pore formation for the α-
PFT, ClyA. Fully atomistic MD simulations show that the
transmembrane ClyA pore intermediates or arcs are structurally
stable with toroidal lipids forming the part of the water channel
where proteins are missing. Initial conditions with lipids present
within the arc interior illustrate the dynamics of lipid evacuation
and reorientation to form toroidal lipid edges bordering the
water channel. While the lipid reorientational dynamics is
typically complete within tens of nanoseconds, it must be noted
that lipid evacuation time scales on actual cellular membranes
with an underlying cytoskeleton may be significantly slower.
Other studies on melittin81 and actinoporins14,24 have
suggested that lipid participation is essential in stabilizing the
transmembrane pore. Toroidal lipid topologies observed in our
MD simulations, lining the free edges of the ClyA arcs, are
similar to the reoriented lipids observed during electro-
poration82 and X-ray reconstructions of lipid toroids in
transmembrane pores of α-PFTs.79 Our study suggests that
ClyA proteolipidic arcs (Figure 7) could potentially form the
oligomerization intermediates in the pore-forming pathway of
ClyA, similar to the growing-pore paradigm proposed for PFTs
(illustrated in Figure 1). We provide molecular details about
the single transmembrane protomer conformation, about which
no experimental details are currently known. Recent structure-
based models have been used to study the conformational
change from a water-soluble monomer to a membrane-inserted
protomer, 38 where membrane-bound intermediates are
sampled during the transition into the membrane. Here we
show that the membrane-inserted protomer is a relatively stable
state with a favorable free energy of binding to the membrane
(estimated using umbrella sampling). Surprisingly, the existence
of a stable water channel seen in both the atomistic and
MARTINI simulations of the single membrane-inserted
protomer shows its membrane-permeabilizing ability (Figure
4).
We briefly comment on the existence of arcs within the
framework of two pore-forming paradigms. In the prepore
model, protein oligomerization occurs at the membrane
interface until a prepore complex is assembled. Subsequently
concerted membrane penetration of the prepore completes the
formation of the transmembrane pore complex (Figure 1). In
this regard, β-PFTs have been experimentally studied and pore
formation is found to occur with the formation of a prepore in
several cases.9,17,78,83,84 In the growing pore model, as
oligomerization proceeds, lipid evacuation and the formation
of transmembrane water channels are assumed to occur
simultaneously. This phenomenon has not been observed or
characterized at molecular resolution and remains an open
problem. In addition, lipid evacuation during the formation of
the transmembrane α-helical barrel in α-PFTs such as ClyA
remains unanswered20 because the oligomerization pathways
and kinetics are not fully determined. Multiple models have
been proposed where arcs have been implicated to play a
critical role in PFT-mediated lysis pathways,1,75 specifically in
the case of ClyA.15,30 Semicircularized arcs have been shown to
cause ion leakage in various membranes.4,12 The release of
calcium and potassium ions by these arcs can instigate several
downstream cellular processes that can enhance the lytic ability
of PFTs even at sublytic concentrations,4,85−88 and our study
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supports this view. Identifying the presence of partially
oligomerized intermediates akin to the arclike structures
investigated in this study supports the growing pore pathway.
A nuance to this pathway is that noninserted arcs can
oligomerize on the membrane surface (prepore pathway) and
can stochastically insert to form transmembrane arcs.15
Finally, we point out that the process of unregulated pore
formation on a membrane surface accompanied by lipid
displacement from the pore lumens could drive membrane
buckling and tension generation. The number of ClyA arcs and
pores required to lyse erythrocytes31 has been shown to be
∼105 at the onset of lysis, thus leading to a maximum estimated
coverage of 7% of the erythrocyte cell surface. Note that in our
simulations the protein to lipid ratio is significantly higher.
Because the actual scenario is an extremely dilute regime, the
manner in which membrane mechanical properties are
modulated by ClyA pores is still an open question. The
situation is further complicated by the multicomponent cell
membrane and the presence of an actin cytoskeleton,
embedded sugars, and integral membrane proteins.
In summary, the combined observations provide strong in
silico evidence at molecular resolution for simultaneous lipid
evacuation from the central pore lumen as oligomerization
proceeds and the ability of all membrane-inserted n-mers
including the single transmembrane protomer to spontaneously
form stable water channels capable of leakage. We speculate
that modulating ClyA arc distributions by operating in the
appropriate concentration regimes in suitably packed mem-
branes may prove useful for selective membrane permeabiliza-
tion and size-based analyte separations using ClyA nanopores
and arcs.89
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.lang-
muir.7b02277.
All-atom simulations, spring constants in MARTINI
sampling, protein confirmational changes, and simula-
tions set up using CHARMM-GUI protocols (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: ayappa@chemeng.iisc.ernet.in. Phone: +91 (80) 2293
2769. Fax: +91 (80) 2360 8121.
ORCID
Rajat Desikan: 0000-0002-0785-8187
Prabal K. Maiti: 0000-0002-9956-1136
K. Ganapathy Ayappa: 0000-0001-7599-794X
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors thank the Department of Science and Technology
(DST), Government of India, for a grant under which this work
was carried out. The authors thank Sandhya S. Visweswariah, J.
K. Basu, Narendra M. Dixit, Rahul Roy, and Pranesh
Padmanabhan for extensive discussions.
DOI: 10.1021/acs.langmuir.7b02277
Langmuir 2017, 33, 11496−11510
Langmuir
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11510 DOI: 10.1021/acs.langmuir.7b02277

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