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28 Frishman, D. and Mewes, H. W. (1997) Nat.
Struct. Biol. 4, 626-628
29 Arkin, I. T., Brunger, A. T. and Engelman, D. M.
(1997) Proteins 28, 465-466
30 Jones, D. T. (1998) FEBS Lett. 423,281-285
31 Deber, C. M. and Li, S. C. (1995) Biopolymers 37,
295-318
32 Whitley, P., Saaf, A., Gafvelin, G., Johansson, M.,
Wallin, E. and von Heijne, G. (1995) Biochem. SOC.
Trans. 23,965-967
33 Orengo, C. A., Jones, D. T. and Thornton, J. M.
(1994) Nature (London) 372,63 1-634
34 Orengo, C. A., Michie, A. D., Jones, S., Jones,
D. T., Swindells, M. B. and Thornton, J. M. (1997)
Structure 5, 1093-1 108
35 Baldwin, J. M., Schertler, G. F. X. and Unger,
V. M. (1997) J. Mol. Biol. 272, 144-164
Received 2 April 1998
Molecular dynamics simulations of membranes with embedded proteins and
peptides: porin, alamethicin and influenza virus M2
M. S. P. Sansorn*', D. P. Tielernant, L. R. Forrest* and H. J. C. Berendsent
*Laboratory of Molecular Biophysics, University of Oxford, The Rex Richards Building, South Parks Road,
Oxford 0x1 3QU, U.K., and tBlOSON Research Institute and Department of Biophysical Chemistry,
University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
Introduction
Computer simulations of the dynamics of atoms
within large molecular systems are now possible
for systems comprising tens of thousands of
atoms and are feasible for time spans of several
nanoseconds. Such molecular dynamics (MD)
simulations give a great deal of insight into the
details of molecular motion in biological systems
as related to function, but are limited to processes that can be sampled on a nanosecond
time scale. Bilayer membranes are good
examples of biological systems with essentially
nanosecond dynamics; even slow processes such
as the transport of small molecules through the
membrane can be quantitatively studied. While
interfacial structure and mobility can be adequately simulated, it is not (yet) possible to
follow long-time-scale events such as phase
separation within a bilayer, aggregation phenomena of embedded peptides or proteins, or the
full process of insertion of proteins into a bilayer.
T h e method of MD simply solves Newton's
equations of motion in small time steps (in practice a time step of 2 f s is allowed when bond
lengths are constrained). T h e quality of the
results depends on the quality of the force field
Abbreviations used: MD, molecular dynamics; Alm,
alamethicin; Aib, a-amino isobutyric acid; Phol,
phenylalaninol; TM, transmembrane; DPPC, dipalmitoyl-phosphatidylcholine; POP(C/E), palmitoyl-oleoylphosphatidyl(choline/ethanolamine); RMSD, root
mean square deviation.
'To whom correspondence should be addressed.
Volume 26
used, which describes the forces on each atom
given the positions of all atoms. Popular force
fields are adequate for practical purposes, but
still lack accurate evaluation of long-range interactions and treat non-pair-additive contributions
in an average way only. Methods are available to
evaluate thermodynamic quantities such as
entropy and free energy, and consequently
properties such as binding constants, partition
coefficients, equilibrium constants, etc., but they
rely in general on the construction of a reversible
path requiring a large number of independent
simulations.
MD simulations have been reviewed recently
[l]. T h e overall picture that emerges from simulation is that of a strongly fluctuating fluid bilayer
in which specific molecular groups (such as the
headgroup atoms) are spread over a distance of
about 1 nm perpendicular to the membrane
plane. This picture is consistent with experimental data from X-ray diffraction, neutron scattering, and deuteron magnetic resonance. One
can distinguish four regions in a typical membrane [Z]: (i) the aqueous region; (ii) the headgroup region; (iii) the relatively well-ordered
lipid chain region right behind the head groups;
(iv) the less ordered hydrocarbon region in the
middle of the membrane. A detailed study of the
permeability of water through a dipalmitoylphosphatidylcholine (DPPC) bilayer [31 has
shown that the resistance to permeation is
located primarily in region (iii) where the solubility of water (determined by its free energy) is
low due to the hydrophobic nature of the
Modelling and Membrane Proteins
environment, and the diffusion constant is also
low because of the rather dense packing in that
region.
Dieckmann and M. S. P. Sansom, unpublished
work). The sequences of Alm and of the transmembrane (TM) domain of M2 are:
Porin
Alm: Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln7-Aib-Val-
It is relatively straightforward to incorporate a
protein with known structure, such as porin
(which is a trimeric protein of the outer bacterial
membrane with a molecular mass of approx.
130 kDa allowing small polar molecules to permeate), in a membrane. However, if all atoms of
the protein, lipids and water are incorporated,
the system to be studied comprises some 70000
atoms, and simulating nanoseconds becomes a
major undertaking. Such a simulation was
carried out recently [4], showing that water in
the aqueous channels has special properties: in
certain regions in the channel it is so strongly
oriented by local electric fields that it must lose
its normal 'bulk' dielectric properties. The channel width, and therefore its permeability, fluctuates in time. These detailed simulations
suggest that one must be very careful with
reduced system computations in which both lipid
and water molecules are omitted and the protein
is restrained so as not to deviate significantly
from its X-ray structure.
a-Helix bundles
Simple pore-forming peptides (or other amphipathic molecules) are thought to form pores by
aggregation. They represent an important class
of compounds with antimicrobial or antifungal
activity, with potential applications in the food
and pharmaceutical industries. In addition they
are model compounds for more complex and
specialized channel molecules that regulate the
specific permeation of ions and small molecules
in biological cells. The best studied examples of
pores formed by aggregation are those formed by
bundles of amphipathic a-helices [5].
Two examples of ion channels formed by
a-helix bundles have been studied by MD simulations in a phospholipid bilayer with an explicit
aqueous environment: alamethicin (Alm) and the
M2 protein from influenza virus type A. Alm is a
fungal peptide that forms ion channels in bilayer
membranes, and has been much studied as a
simple model of an ion channel [6]. M2 is a
small (97 residue) protein that forms lowpH-activated proton-selective channels in the
membrane of the influenza A virion, and is the
target for the anti-influenza drug amantadine
([7-91; L. R. Forrest, W. F. DeGrado, G. R.
Aib-Gly-Le~-Aib-Pro'~-Val-Aib-Aib-Glu'~Gln-Phol
M2:
A~-Leu~~-Val-Ile-Ala-Ala-Ser~'-Ile-IleGl$4-Ile-Leu-His37-P he-Ile-Leu-Trp-IleL ~ u ~ H2
~-N
The sequence of Alm contains a preponderance
of the helicogenic amino acid a-amino isobutyric
acid (Aib). There is a central proline at position
14, and a C-terminal phenylalaninol residue
(Phol). The T M sequence of M2 (as predicted
using MEMSAT [ 111) is close to the N-terminus
of the protein, is 18 residues long and is largely
hydrophobic with the exception of Ser-31 and
His-37. Both sequences adopt an a-helical conformation when in a membrane or membranemimetic environment. Alm channels are formed
by voltage-induced self-assembly of parallel
a-helix bundles within a lipid bilayer. Such
bundles may contain between approx. n = 5 and
n = 12 helices, with the size of the central pore
(and hence the single-channel conductance)
increasing as n increases. In the current study we
have modelled the n = 6 helix bundle as the
simplest representative of the properties of most
of the conductance levels. The helices are
believed to be oriented such that their hydrophilic surface (defined by the Gln-7 sidechains)
is directed towards the centre of the pore. The
M2 protein is a tetramer, which forms protonselective channels in bilayer membranes. A wide
range of mutagenesis studies have identified the
Ser-31, Gly-34 and His-37 residues as forming
part of the lining of the channel [7,12].
Both channels have been modelled using
restrained M D and a simulated annealing protocol to explore helix-packing modes [9,13,14]. For
Alm, the resultant models have been shown to
provide an explanation of the effects of modification of the Gln-7 sidechain on channel stability
[15], and good agreement between predicted and
observed single-channel conductances [ 161 and
current-voltage relationships [ 171. For M2, a
model of the n = 4 helix bundle generated
by restrained M D ([9]; L. R. Forrest, W. F.
DeGrado, G. R. Dieckmann and M. S. P.
Sansom, unpublished work) agrees remarkably
well with an independently derived model based
on analysis of cysteine-scanning mutagenesis
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Biochemical Society Transactions
440
bilayer. T h e Alm model was inserted into the
cavity thus created. This system (Alm n = 6
bundle plus POPC) was solvated with SPC [20]
water (approx. 30 water molecules per lipid
molecule), giving a final system of 6 Alm helices,
104 POPC molecules and 3528 water molecules,
a total of 17000 atoms. This system (Figure k4)
was then energy minimized before use in MD.
T h e set-up used for the M2 helix bundle
was essentially the same as that for Alm, except
that a smaller hole was used in the POPC
bilayer. T h e His-37 residues were assumed to be
in their neutral (unprotonated) form, as we
wished to examine the high-pH closed form of
the channel [8,21]. Thus the system (Figure 1B)
consisted of 4 M2 helices, 110 POPC molecules
and 4860 water molecules, yielding a total of
21 016 atoms.
All MD simulations of the helix bundles in a
lipid bilayer were run using GROMACS [22].
T h e lipid parameters were as in a previous MD
study of lipid bilayers [23], and simulation details
were based on those in [4]. Both the Alm and
M2 simulations were of 2 ns duration.
data [12] and with data from solid-state NMR
experiments [18]. Thus, in both cases there is
good reason to believe that the models derived by
in vucuo restrained MD simulations are substantially correct and merit more detailed simulation
studies. In particular, we were anxious to use
unrestrained MD simulations in a lipid bilayer to
assess the stability of the two helix bundle
models.
T h e simulation system used for Alm will be
described first. Note that the Glu-18 sidechains
were assumed to be in their protonated state as
electrostatics calculations [ 191 suggest that the
pKAs of these sidechains would be raised substantially by their proximity to one another and
by their location at the C-terminus of an a-helix.
An equilibrated palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayer with 128 lipid molecules
was used. A cylindrical hole was made in the
centre of this by removing lipids whose P atoms
fell within 1.55 nm of the central axis of the
cylinder. A short MD simulation with a radially
acting repulsive force was used to drive any
remaining atoms out of the cylinder into the
Figure I
Molecular graphics images of the two simulations systems
(A) Hexameric Alm r-helix bundle (ribbon format, in white) inserted in a POPC bilayer (light grey) with water (dark grey) on either side
of the membrane and within the central pore ( 6 ) Tetrameric influenza A M2 transmembrane r-helix bundle (spacefilling format, white)
inserted in a POPC bilayer (with the carbonyl oxygens of the fatty acyl chains shown as light grey van der Waals spheres) with water
(darker grey) on either face of the membrane The approximate locations of the water (w), interfacial (I) and hydrophobic core (h) regions
are indicated
A
B
w
i
h
i
L
t
Volume 26
w
Modelling and Membrane Proteins
Alamethicin
T h e behaviour of the Alm simulation was found
to depend on the protonation state of the ring
of Glu-18 residues that contribute to the
C-terminal mouth of the pore. If these were all
in their ionized (deprotonated) state, then the
helix bundle did not remain intact over the duration of the simulation, but instead suffered relatively large movements of its constituent helices
relative to one another as a consequence of
electrostatic repulsions. However, as discussed
above, it is more likely that the Glu-18 residues
will be all or mostly protonated with the Alm
helix bundle. Over the course of the 2 ns MD
simulation with fully protonated Glu-18 side
chains, the helix bundle remained quite stable.
As shown in Figure 2, if one superimposes the
Ca traces for the Alm bundle at t = 0, 1 and 2 ns,
it can be seen that the basic structure of the
bundle is maintained. This is particularly so for
the N-terminal helical segments (residues 1-1 l ) ,
which are more closely packed together within
the bundle. There is greater evidence of some
flexibility in the C-terminal helical segments.
Examination of the Ca root mean square deviation (RMSD) versus time (Figure 3 ) shows that
this rises over approximately the first 300 ps
until it reaches about 2.3w, and then fluctuates
around this value for the remainder of the simulation. This is comparable with the behaviour of
the C a RMSD for the OmpF porin trimer when
simulated in a palmitoyl-oleoyl-phosphatidylethanolamine (POPE) bilayer [4]. It is encouraging that the drift of the model structure for the
Alm helix bundle is about the same as that of the
porin simulation, which starts with an X-ray
structure. More detailed analysis of the Alm MD
simulation confirms the visual impression that
the largest fluctuations occur in the C-terminal
halves of the Alm molecules. Thus analysis of
secondary structure versus time shows more frequent deviations from a-helicity in the
C-terminal segments. To some extent this may
be viewed as a hinge-bending motion about the
central kink introduced into the Alm molcule by
the Gly-Xaa-Xaa-Pro motif. Both NMR/amide
exchange [24] and MD (D. P. Tieleman, M. S. P.
Sansom and H. J. C. Berendsen, unpublished
work) studies of isolated Alm molecules in solution suggest that such motion occurs. T h e extent
of secondary structure fluctuations in the
C-terminal helices of the Alm helix bundle is,
greater than for isolated Alm helices in methanol
or inserted into a POPC bilayer, but less than
those of isolated Alm helices in water. Despite
the relative flexibility of the C-terminal helical
segments, the overall shape of the pore formed
by the Alm helix bundle is retained, with the
narrowest region of the pore formed by the ring
of Gln-7 sidechains. As has been seen in MD
simulations without a lipid bilayer [25], water
molecules within the Alm-bundle pore show
reduced mobility and their dipoles are oriented
anti-parallel to the Alm cr-helix dipoles. T h e
water orientation is the result of a large effective
electric field (approx. 10"V.mp') within the
channel, caused by the dipole moments of the
helices, which are all parallel [26]. T h e field in
Figure 2
Ca traces of the Alrn a-helix bundle
Superimposed Ca traces of the hexamertc Alm a-helix bundle at t
perpendicular to ( A ) and down (6) the pore (z) axis.
A
=
0, I and 2 ns, viewed
B
C
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Biochemical Society Transactions
the channel is likely to increase the hydrophilicity of the channel itself, while the water dipoles
oppose the helix dipoles and thus reduce the
unfavourable mutual repulsion of the parallel
helices [%I.
spond to the closed form of the M2 channel [9].
This is supported by the simulation, as a continuous pore does not form through the centre of
the helix bundle. Instead, water molecules are
accommodated within a pocket inside the helix
bundle, close to the rings of Ser-31 and Gly-34
residues.
Thus, these simulations suggest that the M2
helix bundle is stable when simulated in a lipid
bilayer. However, the problem remains of the
extent of the T M helices in the intact M2 protein. MD simulations of single extended (26-mer
and 34-mer) M2 helices in-a POPC bilayer (L.
R. Forrest and M. S. P. Sansom, unpublished
work) have been used to help define the optimum length of the T M helix. Results from such
studies suggest that a 22-mer helix is most
likely; n = 4 bundle models corresponding to this
22-mer a-helix have been constructed by
restrained MD simulations in oucuo and will be
used as the basis of further multi-nanosecond
MD simulations of the M2 helix bundle in a
POPC bilayer.
Influenza virus M2
Conclusions
The M2 system is more complicated than Alm,
in that it represents the T M domain excised
from the larger M2 protein. One difficulty is that
of exact definition of the extent of the T M
a-helices. These can be predicted from the M2
sequences using a number of different methods
[27]. Although these methods all agree that the
M2 subunit sequence contains a single T M helix,
and agree on which residues form the hydrophobic core of that helix, they differ as to the
exact start and end residues. For our initial
model we have taken a relatively conservative
definition of 18 residues for the T M helix core,
as predicted by MEMSAT [ 111. As can be seen
from Figure 1(B), a tetrameric bundle model
formed from this 18-mer helix is just big enough
to span the hydrophobic core region of the POPC
bilayer. It was therefore of considerable interest
to see that the n = 4 M2 a-helix bundle model
retained most of its structure over the course of
the 2 ns MD simulation. From Figure 3 it can be
seen that at the end of the simulation, the Ca
RMSD was just under 2 % i.e. a little less than
that for the Alm helix bundle. The M2 helix
bundle model exhibits a marked left-handed
supercoil structure that is maintained throughout
the simulation. The state of the system simulated, with the four His-37 residues in their
neutral (deprotonated) state, is believed to corre-
Overall, the present study shows the feasibility of
multi-nanosecond MD simulations of models of
ion channels formed by a-helix bundles
embedded in an atomistic model of a phospholipid bilayer. The accessible time scale suffices to
study the dynamic behaviour of both water and
lipid molecules and indicates the stability of particular helical aggregates. This MD approach can
be used to help refine models of such channels,
both for channel-forming peptides such as Alm,
and for bundles formed by T M helices from
more complex channel proteins. In the present
study, the emphasis has been on demonstrating
the stability of the models during such simulations. Having established this, it will now be possible to exploit such simulations to explore the
properties of channels in more detail. For Alm,
the simulations will be extended to channel
models with numbers of helices per bundle ranging from e.g. n = 5 to n = 8 [14], and to analysis
of the energetics and dynamics of ion permeation
through such channels. For M2, MD simulations
in lipid bilayers will be used to refine the basic
model, and then to explore possible mechanisms
of proton permeation.
We conclude that the technique of detailed
MD simulation is a valuable tool with which to
investigate details of atomic behaviour, and
especially of the properties of water in aqueous
Fimre
- 3
RMSD of Alm and influenza virus M2
442
Ca RMSDs versus time for the Aim (grey, thick line) and influenza
virus M2 (black, thin line) simulations.
M2
Volume 26
1
Modelling and Membrane Proteins
channels. It is also powerful in evaluating the
stability of proposed aggregation states of poreforming peptides. What MD cannot do in its
present form is predict such aggregation states
or give reliable thermodynamic quantities that
relate to the stability of different aggregation
states. The study of the thermodynamics and
kinetics of aggregate with atomic detail is beyond
MD and requires reduced system descriptions on
mesoscopic time and length scales. The same is
likely to apply to the details of the insertion
mechanism by which peptides enter a lipid
bilayer from the aqueous phase. MD studies of
the latter process are at present in progress in a
joint project of our laboratories. For such processes future systematic reductions of system
complexity are necessary, such that events on
much longer time scales can be approached.
Careful evaluation of the reductions by comparing reduced with detailed simulations is likely to
lead to reliable simulation tools by which slow
aggregation phenomena in lipid bilayers can be
studied as well.
Work in M.S.P.S.’s laboratory is supported by The
Wellcome Trust. L.R.F. is an MRC research student.
D.P.T. was supported in part by the European Union
under contract CT94-0124.
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