plosPLoS Comput BiolploscompPLoS Computational Biology1553-734X1553-7358Public Library of ScienceSan Francisco, USA08-PLCB-RA-0522R310.1371/journal.pcbi.1000289Research ArticleBiophysics/Macromolecular Assemblies and MachinesBiophysics/Membrane Proteins and Energy TransductionBiophysics/Protein FoldingBiophysics/Theory and SimulationCell Biology/Neuronal Signaling MechanismsConformational Changes and Slow Dynamics through Microsecond
Polarized Atomistic Molecular Simulation of an Integral Kv1.2 Ion ChannelMicrosecond Kv1.2 SimulationBjelkmarPär1NiemeläPerttu S.2VattulainenIlpo345LindahlErik1*Center for Biomembrane Research & Stockholm Bioinformatics
Center, Department of Biochemistry & Biophysics, Stockholm University,
Stockholm, SwedenVTT Technical Research Center of Finland, Espoo, FinlandDepartment of Applied Physics, Helsinki University of Technology,
Helsinki, FinlandMemphys—Center for Biomembrane Physics, Physics Department,
University of Southern Denmark, Odense, DenmarkDepartment of Physics, Tampere University of Technology, Tampere,
FinlandRouxBenoitEditorWeill Medical College of Cornell University, United States of America* E-mail: lindahl@cbr.su.se
Conceived and designed the experiments: IV EL. Performed the experiments: PB
PSN. Analyzed the data: PB PSN. Wrote the paper: PB PSN IV EL.
The authors have declared that no competing interests exist.
22009202200952e100028930620086120092009Bjelkmar et alThis is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
Structure and dynamics of voltage-gated ion channels, in particular the motion of
the S4 helix, is a highly interesting and hotly debated topic in current
membrane protein research. It has critical implications for insertion and
stabilization of membrane proteins as well as for finding how transitions occur
in membrane proteins—not to mention numerous applications in drug
design. Here, we present a full 1 µs atomic-detail molecular dynamics
simulation of an integral Kv1.2 ion channel, comprising 120,000 atoms. By
applying 0.052 V/nm of hyperpolarization, we observe structural rearrangements,
including up to 120° rotation of the S4 segment, changes in
hydrogen-bonding patterns, but only low amounts of translation. A smaller
rotation (∼35°) of the extracellular end of all S4 segments is
present also in a reference 0.5 µs simulation without applied field,
which indicates that the crystal structure might be slightly different from the
natural state of the voltage sensor. The conformation change upon
hyperpolarization is closely coupled to an increase in 310 helix
contents in S4, starting from the intracellular side. This could support a model
for transition from the crystal structure where the hyperpolarization
destabilizes S4–lipid hydrogen bonds, which leads to the helix
rotating to keep the arginine side chains away from the hydrophobic phase, and
the driving force for final relaxation by downward translation is partly
entropic, which would explain the slow process. The coordinates of the
transmembrane part of the simulated channel actually stay closer to the recently
determined higher-resolution Kv1.2 chimera channel than the starting structure
for the entire second half of the simulation (0.5–1 µs).
Together with lipids binding in matching positions and significant thinning of
the membrane also observed in experiments, this provides additional support for
the predictive power of microsecond-scale membrane protein simulations.
Author Summary
Proteins that transport ions across the cellular membrane are essential for
cellular life. The proteins conducting positively charged potassium ions are key
players in heart beat and nerve impulse generation because they are regulating
the electrical excitability of the cell (together with proteins transporting
other ions). These particular ion channels open and close in response to voltage
changes across cellular membranes, but the details of this process are still not
fully understood. It is, however, known that the main protein element
responsible is a helical section containing several charges. Through new
computer simulation methods, we have been able to run unprecedentedly long
atomic simulations of an entire potassium channel embedded within a patch of
membrane to help to shed new light on this gating process. Upon changing the
voltage across the membrane, we observe a change in structure of this helical
protein segment that appears to be an early sign of transition from the open to
the closed state of the channel. This has also been previously proposed to be
critical for the gating process. Understanding these structural changes on an
atomic level is essential for both advancing basic science and enabling drug
design targeting of voltage-regulated ion channels.
Supercomputing resources were provided by The Finnish IT Center for Scientific
Computing (CSC) and National Science Foundation award CNS-0619926 to Stanford
University. This work was supported by grants from the Swedish Foundation for
Strategic Research (EL), Swedish Research Council (EL), and the Academy of
Finland (PN, IV).Introduction
Potassium channels are the single most common type of ion channels in nature. The
subclass of voltage-dependent potassium channels enable controlled ion transport
over the cell membrane and are hence pivotal for a wide range of functions such as
nerve impulse action potentials, our heart beats, insulin secretion upon low ATP,
and many diseases [1]. The first X-ray structure to be determined was the
bacterial tetrameric KcsA K+ channel [2], which is pH-modulated
with an opening mechanism likely controlled by protonation and salt bridges/hydrogen
bonds [3]. Recently determined structures of voltage-modulated
potassium channels such as KvAP [4],[5] and Kv1.2 [6],[7] in the open state share
the same central pore domain (PD), but for these the actual voltage-sensitivity is
introduced by adjacent 4-helix voltage sensor domains (VSDs) in each monomer. The PD
is formed by eight transmembrane helices (and their connecting loops), two from each
subunit, and it contains an ion-conducting channel connecting the inside and the
outside of the cell. This water-filled pore consists of a rather large intracellular
cavity leading into a narrow ion-selectivity filter at the extracellular end.
The access to the intracellular cavity, and hence to the ion-conducting pore, is
controlled by a gate that is opened in response to the membrane potential. It is
well established that the gating within the PD is regulated by conformational
changes in the VSDs in response to the varying membrane potential. In particular,
the gating is caused by the voltage sensor S4 helix of the VSD which contains
several positively charged amino acids [8]–[10]. When the
charged S4 helix moves it creates an experimentally measurable current called the
gating current, which is distinct from the so called
α-current caused by ions passing over the membrane through
the protein channel. The actual opening of the channel pore domain and initiation of
the α-current has experimentally been shown to occur in the
millisecond range [11], but Sigg et al. have reported an early component
of the gating current with a time constant as short as 12 µs [12].
When the cell is at rest, the membrane is hyperpolarized (negative potential on
intracellular side) and the gates of the voltage-sensitive potassium channels are
closed, their S4 helices located near the intracellular membrane border, referred to
as the down or “resting” (R) state in Figure 1. Upon depolarization of the membrane,
channel opening is thought to occur in a stepwise fashion where each voltage-sensing
domain first activates independently, by transferring some of the positive charges
within the voltage sensor helix from the intracellular side in the down state to the
extracellular side of the membrane, the up state. In this transient activated state
of the protein, the ion-conducting pathway is still obstructed by the gate formed by
the intracellular ends of the four pore-lining S6 helices of the PD. Subsequently,
the channel gate is opened by an additional cooperative motion [13], yielding the
open-activated state (A) in Figure
1. For long periods of depolarization, the channel is believed to undergo
slow inactivation caused by conformational changes around the selectivity filter
[14]–[17], making it
impermeable for ions even though the gate is open (the open-inactivated state, AL).
Going from R over A to AL, i.e., opening and eventually inactivating the protein
after long depolarization, is referred to the forward direction in literature.
However, since the experimental structure corresponds to the AL (or possibly A)
state, the current simulations aim to assess the conformational changes in the
backward direction, both due to hyperpolarization and equilibration in a more fluid
bilayer.
10.1371/journal.pcbi.1000289.g001
Model of voltage gating.
Based on Villalba-Galea et al. [27], not
including the RL state. At hyperpolarization the VSD is in resting (R) state
and protein is not conducting. Upon depolarization it transfers to an
activated (A) state, likely due to S4 voltage-dependent motion and coupled
to the channel opening. This state is transient and converts into a more
stable relaxed state (AL) at prolonged depolarization (non-conducting).
Crystal structures likely correspond to this open-inactivated state.
Several experimental studies have shown that the gating involves displacement of just
over three charges per subunit across the membrane [9],[10],[18],[19]. Although the exact
nature of the gating process is not yet fully understood, several ideas have been
proposed, for instance S4 rotation combined with crevice reshaping to effectively
move charges between inside/outside (transporter model [20]), larger rotation
and translation of S4 inside a rigid environment (helical screw [21],[22]) or S3b/S4 helices moving as a hairpin (paddle
model [4]).
These models have mostly converged at smaller necessary S4 displacements combined
with rotation/tilt, but there are still important differences, for instance whether
S4 moves independently of S3b or not, whether the charged arginine residues at some
point are facing the membrane or not, and in which order the motion events occur
[23].
Note that small S4 displacements still could explain the effective charge
displacement due to significant water depressions in the VSDs that cause local
focusing of the electric field [24]–[26].
Additional interesting conformational changes upon gating have recently been
proposed. In the new Kv1.2 crystal structure [7], the intracellular end of
the S4 helix adopts a 310 conformation leading the authors to propose
that this secondary structure alteration is relevant for the gating. This is also
supported by recent data from Villalba-Galea et al. [27] suggesting that
the R to A transition of the voltage sensor takes place as 310 helix
whereas the A to AL transition changes the voltage sensor conformation to
α-helix.
The experimental structures in combination with theoretical models of the closed
state [28] has led to considerable interest in understanding
these structural transitions. Simulations of isolated voltage sensors have confirmed
water-filled VSD crevices (on both intracellular and extracellular sides), S4
stabilization and field focusing [29],[30]. Treptow et al. used short (9 ns) simulations of
an integral Kv1.2 channel to show how the S4 gating charges can be stabilized [31], and
Jogini/Roux have reported on structural flexibility and arginine side chain dynamics
in the voltage sensors from 20 ns-simulations [26],[32]. Coarse-grained
representations of Kv1.2 systems [33] have been used to reach 350 ns of simulation,
although the pore in this case collapsed to an apparently closed state even without
any applied field. Recently, Nishizawa et al. reported on S4 motion in an isolated
VSD when running at elevated temperatures applying strong fields of 0.15 V/nm and
position-restraining adjacent helices. While interesting, it is unclear how
realistic this is since the position restraints will prevent side chains in adjacent
helices from stabilizing S4, and near-instantaneous membrane rupture was observed at
0.2 V/nm (in our longer simulations, fields exceeding 0.1 V/nm lead to electrostatic
breakdown in 100 ns) and no significant S4 motion occurred in 30–40 ns
simulations at lower voltages [34]. While all these studies provide new insights
into Kv1.2 dynamics, they are all partly limited, either by short timescales or
other approximations; VSDs might for instance behave differently than integral ion
channels, and any charged elements will move if the voltage applied is high enough
and the rest of the structure is restrained. Ideally, one would want to simulate an
entire ion channel system for long times at low field strengths, which previously
has not been possible.
Here, we present a full atomic detail microsecond simulation of an integral Kv1.2
system comprising 120,000 atoms, with an applied field of 0.052 V/nm, corresponding
to approximately 500 mV of potential drop over the membrane (see methods and Figure S1). The
seed for this project was our recent development of new parallelization techniques
in the GROMACS molecular simulation code [35] that have enabled us to
reach a microsecond with high-accuracy simulation settings in just a month of runs
(roughly 125,000 CPU-hours). Analysis of the S4 helix structure, dynamics and
interactions in applied fields is of course of particular interest. Another
challenge is to compare and contrast results to the new high-resolution chimera
Kv1.2/Kv2.1 structure [7] that appeared during the project, and where the
intracellular part of S4 assumes 310 helix conformation. The primary
questions we wanted to address are whether previously observed motions in short ion
channel and VSD simulations do represent the early stages of transition towards the
resting state or merely short-time fluctuations? To what extent can molecular
simulations started from lower-resolution structures predict native conformations
and/or lipid-protein interactions, and can we use it to better understand channel
opening/closing?
MethodsSystem Assembly
The initial ion channel conformation used in this study was constructed from the
X-ray structure of the Kv1.2 channel [6], PDB accession ID
2A79. The monomer coordinates were assembled into a tetramer channel which
kindly was provided directly from MacKinnon's laboratory. To reduce the
system size the T1 domain was excluded. The parts used were chains B, from
residue T219 (inclusive), C from UNK33 corresponding to A162 in Kv1.2, and chain
D. Since parts of the transmembrane helices were of limited X-ray resolution it
was necessary to complement it with side chain and loop modeling to arrive at a
model suitable for simulations. The voltage sensor domains together with the
pore domain helices from the separate chains were structurally aligned to the
corresponding part in the ROSETTA model of the open state of the Kv1.2 channel
[28], using the program STRUCTAL [36].
Subsequently, the missing parts of the crystal structure (i.e., residues denoted
UNK, missing side chains and loops) were copied from the ROSETTA model
structure. This essentially resulted in a model using the experimental data for
the backbone and most side chains, with the remaining side chains and missing
loops from the ROSETTA model. The ion channel structure was immersed in a lipid
membrane by using the Membrane package of VMD [37]. Starting from a
pure POPC bilayer, the head group of every fourth lipid was randomly (in each
monolayer) exchanged for the negatively charged PG head group. The ion channel
was subsequently inserted and overlapping lipids deleted, after which 313 POPC
and 111 POPG lipids remained in the membrane. Although somewhat similar to a
prokaryotic membrane, this mixture was used to better mimic conditions used in
the crystallization protocols [6],[7] and it has previously also been used in VSD
simulations [38]. The negative head groups in particular could
have important interactions with arginine, and PG force field parameters are
better tested than PS. The bilayer was first melted with MD at 300 K, while the
protein was kept frozen and the z-coordinates of the lipid chain methyl groups
position restrained
(Fc = 1000
kJ/mol/nm2) to keep the membrane intact while it packed around
the protein for 1 ns. The system was subsequently solvated with 26,632 SPC
waters [39], of which 127 were replaced with potassium
ions to neutralize the net system charge, followed by another 1 ns of
equilibration with only the protein frozen. The final assembly reached 119,913
atoms, and an approximate system size of 12*12*10 nm3
(Figure 2).
10.1371/journal.pcbi.1000289.g002
Kv1.2 immersed in a membrane.
The protein is colored by subunits (blue, yellow, purple, green) and
lipids chains are drawn in gray. Water molecules were left out for
clarity. The full system consists of roughly 120,000 atoms. The lower
panel shows the same system without lipids.
Molecular Simulations
The protein part of the system was described with the OPLS-AA/L force field [40],
and the lipids with the Berger force-field [41]. The reason for
this choice is simply that the Berger force field has been shown to accurately
reproduce experimental results [42],[43] combined with a low
computation cost due to the united CH2 atoms in the lipid chains, and
then it is natural to combine it with OPLS from which the Berger FF was derived.
The OPLS-AA combination rules and 1,4 scaling factors (0.5) were used when
mixing united and all-atom models. Atomic names of the VMD lipids were converted
with an in-house script (available upon request).
All simulations were performed with a development version of GROMACS 4.0 [35],[44] that enabled efficient scaling and high
performance (50–60 ns/day) using 170 cores on a Cray XT4
supercomputer. Bond lengths were constrained with the LINCS algorithm [45] while
SETTLE [46] was used for water molecules. A newly developed
non-iterative parallel constraints algorithm (P-LINCS) [47] enabled us to
introduce virtual interaction sites to remove all internal vibrational degrees
of freedom of hydrogens even when using domain decomposition, which in turn made
it possible to extend time steps to 5 fs while maintaining energy conservation
[35]. (All software is freely available through
http://www.gromacs.org.) Electrostatic interactions were
calculated every step with the Particle-Mesh Ewald algorithm [48].
Due to the slightly worse scaling properties of PME (3D Fourier transforms) it
proved efficient to move interactions to direct space by using longer cutoffs of
12 Å both for PME and van der Waals interactions, so the PME grid cell
dimensions could be reduced to 88×88×68 (for technical
details, see Ref. [35]). Neighbor lists were saved and reused for 5
steps. All simulations were performed at constant temperature and with
semi-isotropic pressure scaling. The temperature of the system was coupled to
300 K using the weak coupling algorithm with a time constant of
τT = 0.1
ps [49]. The X+Y (isotropic) and Z box
dimensions were coupled independently to reference pressures of 1 bar with
Berendsen weak coupling, a
τP = 10.0
ps time constant, dispersion corrections to pressure, and a system
compressibility of 4.5·10−5 bar−1[49].
The assembled system was equilibrated in three steps with gradually weaker
position restraints on the protein (1000 kJ/mol/nm2 for 1 ns, 100
kJ/mol/nm2 for 10 ns, 10 kJ/mol/nm2 for another 10
ns). Side chains were only restrained in the first of these runs. Production
runs were performed without restraints; an initial 50 ns without applied
potential followed by a microsecond simulation with an applied electrical field
of 0.052 V/nm along the z system axis, with lower potential on the intracellular
side. With a box length of ca 10 nm in the z direction, this corresponds to a
potential drop of ∼500 mV, which is due to depolarization at water/lipid
interface and will occur mostly over the membrane part (Figure S1).
The treatment of external potentials in membrane systems has recently been
covered in detail [50], and it has also been reported that the
periodicity effects of the PME algorithm artificially can increase polarization,
corresponding to almost 50% higher field in pure water [51].
As additional tests, the non-polarized simulation was extended to 0.5
µs, and to assess the effect of higher temperature/potential a
separate third 0.5 µs simulation was started from the conformation
after 0.8 µs of lower applied field, but increasing temperature to 343
K and field strength to 0.1 V/nm. Finally, a 50 ns test simulation (starting
from 1 µs) was performed where the interactions between R300-E183 and
R303-E226 were turned off using energy exclusion groups in GROMACS.
ResultsMicrosecond-Scale Dynamics of the Kv1.2 Channel
In terms of structural stability there are two obvious factors that could affect
the integrity of the ion channel negatively in extended simulations; the
significant number of rebuilt side chains and the complete removal of the
non-membrane-spanning T1 domain believed to be partly responsible for
tetramerization/stabilization of the Kv1.2 chains [6]. Microsecond scales
are still quite hard to reach even for small globular proteins without the
complex long-range electrostatics critical in membrane and ion channel systems,
and to our best knowledge it has not previously been performed for systems of
this size. This is particularly critical since the slow lipid diffusion and
reorientation slows down all motion in the membrane, so full stabilization (or
unfolding) of a structure should be expected to take at least hundreds of
nanoseconds for a membrane protein.
We directly observe distortion of the membrane close to the protein with
significant local bending, lipids pointing in toward polar groups and deep water
depressions (though not full transmembrane pores) forming in all voltage sensor
domains, just as previously observed in shorter simulations both of isolated
voltage sensor domains [29],[38] and complete ion
channels [26],[31],[52].
The Cα coordinate root mean square displacement (RMSD) of the transmembrane
helices relative to the (2.9 Å resolution) crystal structure rapidly
increases during the initial 50 ns of free equilibration, but levels off around
2 Å. When the hyperpolarization field is applied it very slowly (0.25
µs) continues to grow to 2.6 Å, where it is stable for the
remainder of the simulation (Figure
3A). The central pore-lining helices are even more rigid, although
they are actually part of four different chains. For the first 0.2 µs
they are roughly within 1 Å of the crystal structure, after which it
increases just slightly to 1.2 Å as a consequence of the other
structural changes in the rest of the structure. Since this was achieved
without the tetramerization domain attached this suggests
the extra domain might actually not be critical to maintain the Kv1.2 structure,
at least on microsecond scales. The structural flexibility of the voltage
sensors has previously been reported by Jogini [26] and is quite
striking; one possible explanation is that it could be necessary to enable the
structural transitions required for the channel function.
10.1371/journal.pcbi.1000289.g003
Protein flexibility.
(A) Root-mean square displacement (RMSD), smoothed by a 1 ns running
average, of the transmembrane Cα atoms in the entire protein
(black), and for the pore domain (red), compared to initial simulation
conformation and to the new chimera crystal structure of Kv1.2 (PDB ID
2R9R) (green). (B) Root-mean square fluctuation (RMSF) of Cα,
with each subunit displayed separately. The secondary structure regions
of the protein are identified for clarity.
Since the starting model was derived from the Kv1.2 crystal structure [6] in
combination with the ROSETTA open model [28] it is quite
illustrating to compare the RMSD of corresponding parts of the transmembrane
(TM) helices to the newer 2.4 Å chimera structure, PDB accession ID
2R9R [7]. As the simulation proceeds beyond 0.3
µs the RMSD of the transmembrane part shows that the protein becomes
less similar to the starting model, leveling out at about 2.6 Å,
compared to the new crystal structure to which the RMSD is 2.2–2.3
Å. Thus, even when starting from imperfect coordinates, the simulation
ensemble is closer to the new high-resolution chimera structure. However, as
recently addressed [27],[53], it is not trivial
to interpret the crystal data: the current structures of Kv1.2 might not have
captured the protein in its open-activated state but rather in the
open-inactivated state due to prolonged depolarization.
To separate inherent flexibility from simple drift/unfolding of the different
Kv1.2 domains the Cα root mean square fluctuations (RMSF) around the average structure of
each chain from the 1 µs-simulation were calculated (Figure 3B). All four subunits
exhibit virtually identical patterns, with the two largest peaks in the curve
correspond to the large flexible extracellular loops. Experiments have shown
that these two loops can be omitted and the resulting protein remains functional
[7].
At least the endpoints of the S3&S4 helices are more mobile than the
S1&S2 helices, and the pore helices are quite rigid. Interestingly, the
protein regions located “behind” the selectivity filter in
the three-dimensional structure (the C-terminal part of the S5 helix and the
P-loop in residues 361–373, together with the selectivity filter
itself in residues 374–378) proved remarkably stable throughout the
simulation. This is consistent with experimental data; residues in the P-loop in
the highly homologous pore domain of the KcsA potassium channel have been shown
to be involved in filter-stabilizing interactions [17]. Two
potassium ions were located within the selectivity filter throughout the
simulations and no events of ion transfer through the filter were recorded.
While stable, the ion channel is still quite a sensitive system, which became
obvious for the high-temperature/field (343 K&0.1 V/nm) simulation. The
membrane stayed more or less intact over 500 ns, but the RMSD of the TM part
gradually increased to 5 Å (data not shown). This was
coupled with very large distortions of the VSDs and even partial unfolding of
alpha helices. In our opinion, the system is simply unfolding under these
conditions. Fields higher than 0.1 V/nm consistently lead to electrostatic
breakdown on scales of 100 ns in test simulations. As recently reported by
Böckmann et al. there appears to be an exponential dependence of pore
formation time vs. applied field [51], and we therefore
decided to limit the analysis to the simulations closer to long-time stable
conditions.
Perhaps surprisingly, we only observed a couple of Ångström of
S4 helix translation along the bilayer normal towards the intracellular side and
no effect on the pore radius was observed. Since the transition between open and
closed conformations of the channel is a process of one or a few milliseconds
[11], it is not expected to occur completely in
µs-scale simulation. There is however a small but significant
component of the gating movement, the fast gating component, which appears to
occur on µs timescales [12] and it is consequently feasible that initial
stages of channel structural transitions could be captured by the current
simulation. The fact that the crystal structure might not be in the
open-activated state (A) but rather the open-inactivated state (AL) is also
important since even when hyperpolarization is applied the structure might have
to go through the A state before being able to access resting conformations, a
process not expected to be coupled to any significant charge transfer [27].
S4 Motion and Stabilization
To characterize the behavior of the key charged residues in the S4 helix, the
movement along the z-axis (parallel to the membrane normal) was tracked. This is
non-trivial since the protein position fluctuates a lot over a microsecond due
to membrane undulations. It was thus calculated as the difference in the
z-coordinate of the Cα atoms of these residues after removing the rigid body motion of the
non-S4 helices of each VSD by fitting each frame of the simulation trajectory to
the corresponding Cα atom of the starting structure of the protein. The hydrogen-bonding
pattern of these cationic side chains were also analyzed since conformational
changes often are associated with alterations in the hydrogen-bonding.
The relative displacement of the Cα atoms along the z-axis (after removal of overall VSD movement) is
shown in Figures 4 (A
& C subunits), S2 (B & D subunits), and S3 (A & C
subunits, depolarized simulation).
10.1371/journal.pcbi.1000289.g004
Dynamics of charged amino acids in the S4 helix of subunits A and C.
Panels A & F show the relative Cα translation along the
membrane normal, with positive direction towards the extracellular side.
Note the difference in scales on the y-axis. Panels B/G & C/H
indicate rotation of Cα and the outermost heavy atom around the
local helix axis, respectively (clockwise rotation being positive when
viewed from the N-terminal). Panels D/I & E/J display the number
of hydrogen bonds formed with the rest of the protein and lipids,
respectively (acceptor-donor distance<3.5 Å,
angle<30 degrees). A 1 ns running average is used in all
panels.
In general, fluctuations of 5–10 Å were seen over hundreds of
nanoseconds, suggesting that it is quite difficult or impossible to draw
conclusions from shorter simulations. However, it is quite striking that there
is really only one case—subunit C—where we see significant
changes due to the applied field (for reference: simulations had identical
starting conditions). The net movement towards the intracellular side is quite
low in all cases (with and without applied field), up to ∼3 Å
for R300 in subunit C, which really is the same order of magnitude as the
fluctuations on microsecond scale. R303 and K306 also moved marginally in the
same direction while R294 adopted a more extracellular position hence extending
the N-terminal part of the helix. A visually more appealing way to represent
this is depicted in Figure
5. It shows that the S4 helix in this subunit is less bent due to
straightening of the extracellular portion of the helix (see next section). It
has the effect that the mass center of the S4 helix backbone is shifted towards
the intracellular side, especially the N-terminal half, explaining the relative
downward movement of R300 but also the lack of corresponding movement in R294
and R297 which both end up on a more extracellular phase of the helix (due to
rotation) compared to the reference structure.
10.1371/journal.pcbi.1000289.g005
Sideview of subunit C, helix S1 to S4–S5 linker helix.
For a clearer view of the S4 helix, the protein is visualized as viewed
from the pore domain. The extracellular side is “up”
in the figure. The starting structure (gray) and the last frame of the
simulation (cyan) are shown. They were structurally aligned using the
Cα coordinates of the helices S1 to S3. In both structures, the
Cα atoms of the four top arginine residues (R294, R297, R300 and
R303) in S4 are shown as blue spheres for comparison. Simulation box is
visualized in inset (same orientation).
Hydrogen-bonding patterns of the key cationic side chains are
also displayed in Figures 4,
S2,
and S3.
Hydrogen bonds formed with other protein residues and lipids are shown, not with
water, but in practice all hydrogen bonding donors and acceptors in these
residues are matched. Consequently, the total number of hydrogen bonds formed by
an arginine is 5 (3 for lysine), one for each side chain hydrogen connected to
an electronegative atom (nitrogen). R294 and R297 formed strong hydrogen bonds
with oxygens in the lipid head groups in the extracellular leaflet and water
molecules in the extracellular VSD crevice but they also interacted transiently
with residues in the neighboring S5 helix of the pore domain (D352 and E353).
R300 and R303 on the other hand, mostly formed stable salt-bridges with
negatively charged residues within the VSD, in particular E183 in S1 and E226 in
S2, something that also has been shown in the crystal structures of Kv1.2 and in
other modeling/simulation attempts of the open Shaker channel
[26],[28],[54], and
with water (in the extracellular crevice). For subunit C, where we did see clear
differences with/without hyperpolarization, the formation of new hydrogen bonds
with the protein and water in the extracellular VSD crevice instead of
interaction with lipids (for example in R297 in subunit A and similar events in
the other subunits, B and D) was highly correlated with a large rotation around
the helical axis (see next section). As the rotation in the extracellular half
of the S4 helix took place in subunit C under hyperpolarization, the total
number of hydrogen bonds formed between lipids and R294 and R297 decreased as
these side chains gradually reduced their exposure to the lipid environment. The
lysine residue, K306, formed hydrogen bonds with protein, first with E236 in S2
(except in subunit C) and later with D259 in S3 (interactions confirmed by
experimental findings in Shaker [55]), and water
molecules in the intracellular VSD crevice. Interestingly, the formation of the
hydrogen bond between K306 and D259 in subunit C at 0.3 µs is highly
correlated to onset of 310 helix growth in S4 of subunit C.
The charge movement in S4 towards the intracellular side upon channel
open-to-closed gating is thought to be associated with hydrogen-bonding pattern
changes. The two most extracellular arginine residues should, as they move down
in response to the hyperpolarization, start competing for the hydrogen bonds
formed between the following two arginine residues (R300 and R303) and the
anionic residues in the VSD center (E183 and E226). As they do, these latter two
arginine residues become accessible to the water in the intracellular crevice
and/or other polar/charged residues residing there [56]. It is likely that
these changes in hydrogen-bonding within the VSD contribute to the free energy
barrier between the intermediate states of gating. Curiously, no systematic
change in this hydrogen-bonding pattern was observed, with the exception of R300
in subunit C under hyperpolarization, where we see a complete loss of its
hydrogen bonds to protein.
This could of course be due to the fact that this process is much slower compared
to the simulation timescale, as might be possible if this really is the main
free energy barrier of structural transformations between the open and closed
states. To investigate this further we attempted to selectively exclude the
interactions between the residues participating in these hydrogen bonds in the
open state of the channel. After 50 ns of such a simulation (continuing from the
last frame of the normal hyperpolarization simulation at 1 µs) we
still did not see any of these structural and binding rearrangements. This
indicates that the free energy barrier could rather be attributed to the
hydrophobic area surrounding S4 in the central part of the VSD, formed by
isoleucine residues in S1 and S2 (corresponding to the Kv1.2 residues I177 and
I230 respectively) as proposed by Campos [57] and/or by
phenylalanine (F233) as proposed by Long [7], separating the
extracellular and intracellular water crevices. To fully close the channel, the
S4 helix must be translated towards the intracellular side according to all
current models. It will force its arginine residues to cross this hydrophobic
area devoid of possible hydrogen-bond acceptors, and hence the hydrogen bonds
between the arginine residues and the anionic residues located extracellularly
of the hydrophobic area (E183 and E226 in particular) are almost certain to
break as the former move across the barrier without stabilizing hydrogen bonds,
which would lead to a kinetic free energy barrier for this transition. Once the
side chains have crossed the hydrophobic area, they can again form hydrogen
bonds with other polar residues of the protein or with water in the
intracellular crevice of the VSD.
Secondary Structure Alteration Might Be Necessary for S4 Translation
Rotation of the S4 helices was observed and quantified by measuring the local
rotation for all residues within the helix in each subunit with respect to the
position of the corresponding residue in the starting conformation of the
protein, defined from a local helical axis. For the key positively charged
residues in S4—the four most extracellular arginine residues (R294,
R297, R300 and R303) and the downstream lysine residue (K306)—both the
rotation of Cα and the outermost heavy atoms were analyzed (Figure 4 for subunits A & C, Figure S2
for subunits B & D).
Interestingly, S4 helices in all subunits exhibit a limited rotation in
all simulations, even in the depolarized state. Significant
rotations were always counterclockwise around S4, as seen from the extracellular
side, turning the two most N-terminal arginine residues slightly toward the VSD
interior (see Figure 6). All
four subunits had a similar initial behavior with respect to S4 rotation (Figures 4 and S2, S3 in
depolarized state); a rotation of around −30 to −40°
for the Cα atoms of R294 and R297 while their side chains underwent a somewhat
smaller rotation at the beginning due to favorable hydrogen bonds to lipid head
groups. Some of these side chains were subject to additional rotations, highly
correlated with changes in their hydrogen-bonding, as is apparent for R297 in
subunit A at ∼370 ns (and similar events for R294 and R297 side chains
in subunits B and D, Figure S2), where the creation of two
hydrogen bonds with protein caused the side chain to rotate to the level of the Cα atom. The more intracellularly located residues in the helix, e.g.,
R300/R303/K306, underwent marginal rotation, if any.
10.1371/journal.pcbi.1000289.g006
Rotation of the S4 helix in subunit C.
An extracellular view of the S4 helix at the beginning (gray) and end
(cyan) of the simulation. The top three arginines in each conformation
are shown in stick representation and the large counter-clockwise
rotations of R294 and R297 indicated by arrows. The end-conformation was
been aligned to the initial using the Cα atoms in helices S1 to
S3 after which the S4 helices were aligned to each other in order to
compare their relative rotation.
In addition to the small but clear rotation of the S4 helix present in all
chains, the extracellular half of the helix in subunit C also exhibited a
significantly larger but slow rotation in the 0.3–0.7 µs
timeframe when the hyperpolarization field was applied. R294 and R297 rotated
about −80 to −120° (peak values as much as
−150°), whereas the minimal rotation of R300 and R303 was
kept. This had the consequence that the helix became longer as the pitch angle
between helix turns increased and the positively charged residues in the helix
aligned on the same phase, all pointing toward the VSD interior, forming a
significant stretch of 310 helix. The Cα rotation is again closely correlated with side chains (the backbone
might even precede the sidechains). Together with the rearrangement taking 0.4
µs to complete, it indicates a predominantly entropy-limited process.
To double-check that the α helix vs. 310
helix states are correctly modeled, the energies for an ACE-Ala20-NME
peptide in the two secondary structures were calculated, confirming that the
α helix enthalpy is lower by roughly 5 kJ/mol per
residue. This agrees well with earlier studies using both OPLS [58] and other parameters [59] that also calculated
an entropic contribution to oppose roughly 1/3 of this. The final free energy is
thus roughly 3–4 kJ/mol lower per Alanine residue for an alpha helix
in solvent, with a low barrier around 1 kJ/mol (from 310 to
α helix) in earlier OPLS studies [58]. In addition, the force field employed here has
also been succesful at reproducing the free energy landscape for the
310 helix-containing Trp-cage protein to within 1.5 Å [60], so
we do not expect the transition to be an artifact from parameters.
The larger rotation was a progressive event, not associated with single
hydrogen-bonding formation/breakage as in the abrupt R297 side chain rotation in
subunit A. Note that no corresponding rotation occurred in any of the subunits
in the simulation without external field. The 310 helix extension
(which even starts from the intracellular side) was associated with formation of
a new hydrogen bond between K306 and D259. S3b motion was clearly coupled with
S4 in fluctuation motions and initially even rotates slightly counter-clockwise
around S4 to maintain interaction surfaces. However, at least in the present
simulations, these interactions were broken when S4 rotated >70°
degrees, and the extracellular part of S4 turned almost a third of a turn
relative to S3b. Hydrogen-bonding to lipids decreased in R294 and R297 after
approximately 470 ns (in favor of hydrogen bonds to water and to some extent
protein residues in the case of R297), but most of the rotation had already
taken place at that point. Consequently, the lipids involved in hydrogen-bonding
with these two arginine residues were dragged in towards the VSD/PD interface as
the field-driven subunit C rotation took place.
Secondary structure plots from the hyperpolarization simulation are presented in
Figures 7 (subunits A,C)
and S4
(subunits B,D), while Figure S5 shows the secondary structure in
subunits A,C in the non-polarized simulation. They provide additional evidence
of protein stability; the S1–S6 transmembrane helices keep their core
α-helical structure while the helix ends and the
connecting loops are somewhat more variable in their secondary structure as
expected. The region around the selectivity filter (residues 274–278)
also shows a high structural stability throughout the simulation.
10.1371/journal.pcbi.1000289.g007
The secondary structure of subunits A and C as calculated by DSSP.
Note the significant growth of the 310 helix in subunit C that
correlates with S4 rotation.
The rotation of the extracellular half of the S4 helix in subunit C caused this
part of the helix to be more tightly wound and somewhat elongated because the
pitch angle between following helix turns increased. The secondary structure in
this region accordingly underwent a change from α-helix
to 310-helix. In fact, the handful most C-terminal residues of the S4
helix in all subunits adopt 310 secondary structure already early in
the simulation. However, the significant 310-helix growth in S4 of
subunit C starts after some 200 ns and continued for 250–300 ns,
perfectly correlated with the rotation of the extracellular end of this helix.
After the rotation, the 310-helix consistently encompassed a full 17
residues in S4 (residues 293–309), including the two most
extracellular arginine residues R294 and R297. No corresponding
310-helix growth was seen in any of the subunits in the simulation
without the applied electric field.
Our model was derived from the original crystal structure of the Kv1.2 channel
[6]
but it has very interesting similarities to the new fully-resolved Kv1.2 chimera
crystal structure [7]. In particular, the new structure features
310-helices C-terminally of the second arginine in S4 (R297). One
idea put forward by MacKinnon and coworkers is that the 310 portion
of the secondary structure of the S4 helix could be important for the function
of the protein by turning the arginine residues away from the lipid membrane as
they start the motion towards the intracellular side in response to the
hyperpolarization of the membrane. The easy solution would be if the side chains
are simply pulled down by the hyperpolarizing field, essentially dragging the
helix into 310 helix. However, this would not explain why the
transition in vivo can take up to milliseconds, or why there
are separate open-active and open-inactive states. In the hyperpolarization
simulations, the backbone and side chain rotation appears to be quite correlated
and slow, and the 310 helix is even growing from the intracellular
side. As mentioned above, this rather points to an entropic effect of packing S4
to the remaining VSD helices, somewhat akin to finding a keyhole in the
dark—it takes time, and pushing harder will not help. It also supports
an overall ‘screwing’ motion of S4, but in particular a
model where S4 first might have to transition to 310 helix (AL to A)
for the actual closing motion (A to R) to be possible, as recently also
suggested by Villalba-Galea et al. [27].
Protein-Membrane Interactions
The lipid bilayer thickness was studied by calculating time-averaged bilayer
thickness between sn-2 carbons in the glycerol group (the carbon at the
branching point between the two acyl chains and the head group) on a grid in the
plane of the membrane (the xy-plane). The result is shown in Figure 8. Membrane proteins
have been shown to induce large distortions in the surrounding bilayer in some
cases and that the lipid membrane can modulate protein function [61].
Clearly, the transmembrane protein has a large effect on the bilayer thickness;
in general, the thickness is obviously reduced close to the protein and in
particular in the grooves between neighboring VSDs (down to ∼25
Å) compared to the bulk thickness (40–50 Å).
10.1371/journal.pcbi.1000289.g008
Lipid bilayer thickness.
Distance between sn2 carbons (second carbon of the glycerol backbone) on
opposite sides of the bilayer, calculated on a grid in the xy-plane
(viewed from the intracellular side) and averaged over 100 ns in the
beginning, in the middle and in the end of the simulation. White areas
are representing grid-points where the lipid densities are low in both
leaflets, i.e., protein regions.
The Kv1.2 chimera structure [7] has highly resolved lipid molecules in the
crystal forming a bilayer-like structure, especially in the grooves between
laterally protruding neighboring VSDs, indicating that these lipids are an
integral part of the protein structure and probably contribute to its stability.
This suggest strong lipid-protein interactions in those regions which in turn
support the data that shows that these parts of the lipid bilayer are very
important for lipid and lipid-soluble channel-regulators, such as drugs and
toxins [62]–[64]. Even though the
number of resolved lipid molecules is rather low in the crystal structure, a
direct comparison to the simulations is tempting. In addition to several lipid
acyl chain fragments, three polar lipid head groups are resolved in the unit
cell (containing a single subunit); one in contact with the C-terminal part of
S6, the second wedged between the VSD and the S4–S5 linker, both part
of the intracellular leaflet, whereas the last is located close to the P-loop on
the extracellular side. This latter region had already been proposed to be a
lipid-interaction site due to lipid head group electron density in the KcsA
crystal structure [65]. The lipid close to the S4–S5
linker in the intracellular leaflet was noted to have a considerable different
level of burial in the bilayer. By mapping snapshots of the simulation onto the
crystal structure we estimate a thinning specifically on the
intracellular side of at least 8 Å in this region. These
two lipid molecules are shown in Figure 9 (lipid close to the S4–S5 linker on the right),
together with two POPC lipids from the simulation at very similar
positions—note that this packing was not assigned in the starting
conformation. The simulation suggests a thinning, not only of the same
magnitude, but also in the same region of the bilayer.
10.1371/journal.pcbi.1000289.g009
Comparison of lipid structure between a simulation snapshot and the
new Kv1.2 chimera crystal structure.
A sideview of the S4–S5 linker to S6 region of the crystal
structure (gray) superpositioned on a snapshot of the simulation (cyan).
Lipid molecules of the crystal structure are shown as van der Waals
spheres. Two lipids molecules from the simulation at corresponding
positions are shown in stick representation.
The lipid-exposed positively charged arginine residues in S4 perturbed the
membrane bilayer in their neighborhood because of their hydrogen-bonding to
lipid head groups. However, they never interacted directly with
the hydrophobic phase. On average, lipids were dragged down to be able to form
these interactions and the bilayer hence became thinner in this region which can
be seen by the mostly blue-colored regions of the membrane in Figure 8, close to the PD at
the right edges of the VSDs as viewed radially from the protein pore (region at
x,y-coordinates ∼(4,4.5) nm in plot, for subunit A). Such interactions
between the positively charged arginine residues of S4 and the negatively
charged lipid phosphodiester groups are crucial for the protein function, as
they are stabilizing the open conformation [66]. Moreover, this
significant thinning of the bilayer in the proximity of the voltage-sensing
arginine residues further helps the electric field focusing [25],[67].
Other simulations have also reported on the thinning of the membrane with
immersed voltage-gated ion channels or VSDs. In simulations of the VSD in
POPC/POPG bilayer mixtures at 3∶1 molecular ratio, Sands et al. found
a thinning of the bilayer of ∼7 Å close to the voltage sensor
compared to bulk (measuring the P-P distance of opposing phospholipids) [38]. In
simulations of an isolated S4 helix (from KvAP) in a POPC bilayer, Freites et
al. [68] found such a deformation of the membrane in the
vicinity of the peptide where the hydrophobic core was reduced to a mere 10
Å. In particular, they noted that one lipid in a simulation snapshot
spanned the entire membrane in a configuration resembling that of a monolayer.
Roux and coworkers [26] also reported significant membrane thinning
at the intracellular side in their simulation of Kv1.2 due to increased
interactions between basic residues and (DPPC) lipid head groups. Although these
studies are hard to compare due to differences in methodology, lipid and protein
compositions, thinning of the bilayer around the VSD is present in all cases and
the phenomenon most likely has functional importance because it both stabilizes
the open state and focuses the electric field around the voltage-sensing
arginine residues of the S4 helix.
To characterize the nature of the lipid-protein interactions, the number of POPC
and POPG lipids within 9 Å of the protein surface (measured from the
phosphor atom in the lipid head group) was analyzed and compared between the
first (random) and last (equilibrated) frames of the simulation, see Figure 10. The POPC/POPG ratio
at the start of the simulation is 2.61 while it decreases to 2.28 at the end,
compared to the overall ratio of 2.82 (313/111), clearly indicating an
enrichment of negatively charged POPG lipids close to the protein. A closer look
at the data reveals that the contribution to the POPG enrichment is mainly due
to increased interactions between POPG and positively charged residues on the
intracellular side of the protein, far away from the voltage-sensing arginine
residues in the open state. This might not be critical for the protein function,
but experimental studies have shown that the presence of the phosphodiester
moiety of the lipid head group is important for the stabilization of the open
state of the voltage-dependent K+ channels [66].
Rather, this enrichment might simply be explained by electrostatics; the charge
distribution of the protein differs significantly between the intracellular and
extracellular sides of the membrane with a clear overweight of positively
charged residues at the intracellular side, which hence attracts negatively
charged POPG lipid molecules. Biological relevance of specific interaction sites
of anionic lipids can not be ruled out, however, since they have been detected
in KcsA [69] and they are thought to modulate the selectivity
filter stability [70]. In the same spirit, cardiolipin binding
sites have been reported for a number of membrane proteins such as the ADP/ATP
carrier, and a cardiolipin molecule has also been found in the structure of the
cytochrome bc1 complex [71]. Finally, they could be important for
accessibility of lipid-soluble protein functional regulators binding at or in
close proximity to these sites.
10.1371/journal.pcbi.1000289.g010
Change in number of contacts between the protein and lipids.
A contact is defined between the protein and a lipid if at least one
protein atom is located within 9 Å from the phosphorous atom
in the lipid head group. The absolute initial number of contacts are 102
and 39 for protein-POPC and protein-POPG, respectively.
Discussion
The environment of the charged arginine residues in S4 after equilibration confirms
previous reports that the two most extracellular arginine residues are only partly
exposed to the lipid environment where they hydrogen bond to lipid head groups, in
addition to transient interactions with water molecules in the extracellular VSD
crevice and protein residues of the neighboring pore domain. The downstream two
arginines were found to participate in stable salt-bridges with negatively charged
residues from the neighboring S1 and S2 helices, forming a structural barrier
separating the extracellular and intracellular water cavities within the
voltage-sensing domains.
The initial 30–40° rotation of the upper part of S4 in all subunits
(both with and without hyperpolarization applied) indicates that the equilibrium
state in a bilayer is likely somewhat different from the crystal structures. The
rotation partly moves the two arginine residues within this segment away from the
membrane-exposed surface of the VSD, towards its interior. Recently, Lewis et al.
[53]
presented a study that supports the notion that the voltage sensor in the crystal
structure might not have the exact orientation expected in the open-activated state,
based on experimentally derived distance-constraints between the most N-terminal
arginine in S4 (R294) and residues in the S5 helix of the pore domain, but they
should be rotated by ∼37°. Our simulations agree virtually perfectly
with this, and enabled interactions (although transient) between R294 and relevant
S5 residues.
However, since this rotation occurs also in the simulation without external applied
field (which should probably correspond to the AL state, if that is indeed the state
of the crystal structure), this motion might not be representative of the AL to A
transition, but rather an equilibration of the X-ray structure due to different
environment (e.g., ion concentrations) in the crystal vs. the simulation. It is not
trivial to say which one of these is closer to the native state. To focus on
possible simulation shortcomings, one could, e.g., argue that the rotation might be
be due to incomplete initial lipid packing around the protein, which is important
for the stability of the S4 helices. While theoretically possible, we find this
somewhat unlikely since the ion channel coordinates were restrained for the first 20
ns of simulation to pack lipids efficiently around the protein, and the rotation
subsequently took another ∼50 ns to complete. Furthermore, the membrane used
for solvation did not have the four-fold symmetry of the ion channel, so it is
improbable that incomplete local solvation would cause quantitatively similar
rotation in all four VSDs.
The slower, large-magnitude, rotation of S4 observed in the VSD of subunit C (roughly
120°) is quite striking, and most likely caused by the hyperpolarization of
the membrane since no corresponding rotation is observed in the simulation without
the applied electrical field. Equally important—this rotation is closely
coupled to most of S4 forming 310 helix (17 residues), which we hence can
attribute to the hyperpolarization with fair confidence. Together, this has the
effect that the positively charged residues in S4 align on the same phase of the
helix, all pointing to the VSD interior. While some caution is advised considering
the limited amount of data, this does look like an early stage of the voltage sensor
transitioning. This is supported by considerable axial rotation of the S4 helix (as
much as ∼180° in recent disulfide and metal bridge experiments [57]) and a
change of the tilt angle of the helix [72].
A shorter stretch (two turns) of 310 helix is also present in the new
chimera Kv1.2/Kv2.1 structure [7], and the authors have suggested it could be
important for gating. In a new experimental paper by Villalba-Galea et al. [27]
this idea has taken further and they propose a mechanism of gating where the S4
helix totally adopts a 310 conformation as it moves between the resting
and activated states (R and A). Moreover, their data suggest that the slow
inactivation of the protein (the A to AL transition) takes the S4 helix from the
310-conformation to α-helical in the
open-inactivated state. Interestingly, since the crystal structures of the
Kv-channels are thought to be in the open-inactivated state, this seems to support
the transition observed, which in that case would correspond to the backward
direction from open-inactivated AL state towards the activated A state (S4 forming
310 helix), which later would enable the A to R transition since a
tighter helix with aligned charges could be easier to translate vertically.
Since stepwise activation of the VSDs has been observed experimentally [13],[73], we
expect similar transitions to later occur in the remaining three subunits too.
However, since even the transition motion takes close to half a microsecond to
complete, even these fairly long simulations are at the very lower boundary of the
timescales required to overcome the free energy barriers involved.
Since gating is a reversible process, the hyperpolarization should return the protein
to the closed resting state. However, it is not obvious that this mechanism of
charge transfer is strictly the reverse of activation, in particular not when
starting from the open-inactivated state. It is for instance likely that the closing
process too is initiated in the VSD, and conformational changes propagated to the
channel such that the entrance to the ion-conducting pore closes. In this case, it
is also quite possible that S4 first might have to rotate away from the lipids when
destabilized by the hyperpolarization, and later translate down due to entropic
packing (whether this corresponds exactly to the AL-A-R transition or not is a
separate question). While high potential/temperature or coarse-grained models are
extremely useful from an understanding point of view, they can also easily alter
this order of events or produce artificial motion. In fact, based on recent
experiments [27] it is doubtful that a VSD should be able to
transition (in particular not directly from AL to R states) on 10–100
nanosecond timescales while not changing secondary structure!
So, which is the chicken and which is the egg? Does the 310 helix
formation drive the rotation or vice versa? Unfortunately this is not easy to
resolve even from simulations since the two appear to be extremely
coupled—the 310 helix of subunit C extended from the C-terminal
side of the helix with a duration directly correlated with the large rotation after
hyperpolarization. The seed for this alteration in structure seems to have been a
hydrogen bond forming abruptly between K306 in S4 and D259 in S3, putting a local
strain on the S4 helical backbone which subsequently was mediated
‘upwards’ in the S4 helix. Prior to this, the VSD helices became
less tightly packed transiently as reflected by a 10% increase in the
radius of gyration around the z-axis, possibly pushing the structural flexibility
needed to initiate the 310 formation in this otherwise tightly packed
central part of the VSD. On the other hand, the first part of the rotation at the
N-terminal end of S4 began directly as the positional restraints were released and
cannot be attributed to the change in secondary structure which started after about
0.2 µs in subunit C. Further, if the subsequent large rotation in subunit
C was caused solely by the 310 formation, then one would expect that the
more C-terminally located R297 would start to rotate before R294.
In practice, we believe it is a collective entropic process of re-packing S4 against
the adjacent helices when the field changes, which would explain both the slow
transition and why S4 can be transiently stable as 310 helix in the
open-active state.
The interactions between lipids and the membrane protein constitute an entire chapter
by itself. Generally speaking, the coupling between membrane protein activation and
membrane composition has been reported previously [74], illustrating the
importance of specific lipid-protein interactions as well as the effects of membrane
elastic properties on protein structure and dynamics. The thinning of the bilayer in
the grooves between neighboring VSDs is evident, and this is actually also the
region with resolved lipids in the new crystal structure of the chimera Kv1.2
protein [7],
indicating favorable interactions with the protein. There are several experimental
reports of functionally important interactions between the voltage sensor and
lipids, both for stabilizing the open state of the voltage-dependent
K+ channels [66] and for the focusing
of the electric field around the voltage sensor [25],[67], that are mapped to this
area of the bilayer. Additionally, interaction sites with protein function
regulators, such as drugs and toxins, have been shown to be located on the protein
surface facing these lipid regions [62]–[64]. Hence, the bilayer
behavior within these grooves seems to be of a very different nature compared to the
bulk lipid bilayer and other parts of the membrane close to the protein. Quantifying
these differences more thoroughly is something we will focus on in a future
project.
Supporting Information
Average actual electric field and potential in the 1 microsecond simulation.
Shown as a function of z-position with and without the applied field. The
field and potential is calculated by single and double intergration,
respectively, of the charge density according to the Poisson equation. Due
to depolarization at the water/lipid interface, virtually the entire
potential shift occurs over the membrane part of the system.
(0.71 MB TIF)
Charged residue dynamics in S4 for subunits B and D. Panels A&F show
the relative Cα translation along the membrane normal, with positive
direction towards the extracellular side. Note the difference in scales on
the y-axis. Panels B/G&C/H indicate rotation of Cα and the
outermost heavy atom around the local helix axis, respectively (clockwise
rotation being positive when viewed from the N-terminal end of the S4
helix). Panels D/I&E/J display the number of hydrogen bonds formed
with the rest of the protein and lipids, respectively.
(2.75 MB TIF)
Dynamics of charged amino acids in subunits A and C without external field
(Compare Figures
4&S2). Panels A&F show the relative
Cα translation along the membrane normal, panels B/G&C/H
rotation of Cα and the outermost heavy atom around the local helix
axis, and Panels D/I&E/J the number of hydrogen bonds formed with
the rest of the protein and lipids. Interestingly, all subunits show limited
rotation (30–40 degrees) even without applied field, indicating
the membrane environment could be slightly different from the crystal
structure.
(1.92 MB TIF)
The secondary structure of subunits B and D as calculated by DSSP. Note that
no 310 formation in S4 is present (compare Figure 7).
(2.97 MB TIF)
DSSP secondary structure of subunit A and C without external electric field.
There is no significant growth of 310 helix contents in S4.
(0.93 MB TIF)
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