Methods
Homology Models
Hv1 shares 11%, 15.2% and 19.7% sequence identity with the Kv1.2 VSD (PDB ID
2A791), KvAP VSD (PDB ID 1ORS2), and Kv1.2-2.1 chimera VSD (PDB ID 2R9R3)
for which x-ray crystal structures are available at resolution 1.9, 2.9 and 2.4 Å
respectively. We used these as templates for building models of the open-state
structures of Hv1. Each x-ray structure was aligned against sequences of closely
related homologues, and this was used to calculate a structural profile (i.e. a position
specific substitution matrix – PSSM). A second sequence-only profile was obtained
for the Hv1 target sequence, also based on an alignment containing close sequence
homologues. Sequence homologues were obtained from the non-redundant protein
sequence database UniRef1004 (http://www.ebi.uniprot.org). Non-iterative BLAST
was run with 10-5 e-value threshold, excluding significantly truncated hits. The query
was re-aligned against the obtained sequences using MAFFT alignment program5 and
BLOSUM62 as a substitution matrix6 (see supplementary Fig. S3 and S4). The
iterative option was used to ensure the best alignment quality.
Using the profile-profile alignment method of FUGUE7, which incorporates
environment specific substitution matrices, a structure-sequence alignment was
generated. Manual adjustments were introduced in a small number of ambiguous
positions ensuring key regions are well conserved.
The adjusted FUGUE alignment was used to build 3D models with MODELLER,
version 8.08. Ten models were generated satisfying maximum restraints. These were
evaluated on the basis of the energy and violation values calculated via MODELLER,
in addition to the sequence-structure compatibility scores of pG9 and those computed
from PROSA200310 and VERIFY3D11. Regions in the models found to be unreliable
and associated with ambiguous regions in the alignments were improved by altering
the alignments manually using ViTO12. Finally, the best model was selected.
A homology model of the open-state structure for the tetrameric bacterial voltagegated sodium channel NachBac13 were built following a similar procedure.
Coarse-grain simulations
Coarse-grain (CG) molecular dynamics (MD) simulations were performed using
GROMACS 3.3.314, implementing the GROMOS96 force field15, as described in
previous studies16. CG parameters for lipid molecules (Dioleoyl-phosphocholine,
DOPC and Palmitoy-oleoyl-phosphocholine, POPC), Na+, Cl-, K+ ions and water are
as in reference17 and for amino acids as in reference18. A CG protein model was
generated from the corresponding atomistic structure, and contains a chain of
backbone particles with side chain particles attached to it. Details of protein bond and
angle potential can be found in ref.18. Lennard–Jones interactions were shifted to zero
between 9 and 12 Å, and electrostatics were shifted to zero between 0 and 12 Å, with
a relative dielectric constant of 20. The non-bonded neighbour list was updated every
10 steps. Simulations were performed at constant temperature, pressure, and number
of particles. The temperature of the protein, lipid, and solvent were each coupled
separately using the Berendsen algorithm19 at 310 K, with a coupling constant T = 40
ps. The system pressure was anisotropically coupled using the Berendsen algorithm at
1 bar with a coupling constant P = 40 ps and compressibility of 5 x 10-6 bar-1. The
time step for integration was 40 fs.
1
CG models were generated for the Hv1 and NachBac homology models and for the xray structures with the PDB entries: 1ORS (KvAP VS); 2A79 (Kv1.2); 2R9R (Kv1.22.1, chimera); and 3BEH (Mlotik1).
The tertiary structure for each protein was maintained by using an elastic network
model20. Harmonic restraints were applied between all backbone particles within 7 Å
of one another, each with a force constant of 10 kJ mol-1Å-2 and an equilibrium bond
length equal to that in the starting structure.
Hv1 and KvAP VS were each placed in a box of 13 x 13 x 13 Å; chimera, Kv1.2,
NachBac and MlotiK1 were each placed in a box of 15 x 15 x 13 Å.
Each CG model was energy minimized using <1000 steps of the steepest decent method,
to relax any steric conflicts within the protein. Subsequently, each system was combined
with randomly positioned CG lipids (Table S1). Each system was solvated with CG water
particles, and sodium or chloride counter-charge ions were added to keep overall
electrical neutrality. Each system was energy-minimized further for <1000 steps, to relax
any steric conflicts between protein, lipid, and solvent. Production simulations were then
run on Linux machines.
Full-atom simulation setup
Full-atom MD simulations were prepared as described in previous studies21. Sidechain ionization states were determined based on pKA calculations performed using
PROPKA22. All of the ionizable residues were predicted to be in their default states
(i.e. the ionization states that were expected at pH 7 based on the standard pKA values
of the residues).
Full-atom (AT) lipid bilayers were reconstituted from the final configuration of the
CG simulations by modeling AT fragments based on the CG particles and building
them into entire lipid molecules, which were then energy-minimized (Stansfield et al.
unpublished) (Table S2). The AT protein was superposed on the CG protein by leastsquare fit of the coordinates of the C atoms and particles. The resultant AT proteinbilayer systems were energy minimized, and solvated with SPC water molecules
followed by removal of any water molecules too close to either protein or lipid
molecules. The systems were then subjected to further energy minimization. Countercharge ions were added to both systems by replacing water molecules with ions at the
most favorable electrostatic potential positions.
Prior to the production run a 1 ns equilibration run was performed during which all of
the heavy (i.e. not-H) protein atoms were harmonically restrained with an isotropic
force constant of 1000 kJ mol-1 nm-1. Restrained MD runs were performed at 300 K
for each protein-bilayer system. Finally all positional restraints were removed and 20
ns duration production run simulations were performed for each system.
Simulations of KvAP VS were taken from a previous work21.
Full-atom simulation details
MD simulations were performed using GROMACS 3.314, implementing the
GROMOS96 force field15. The lipid parameters were based on GROMOS96,
supplemented with additional bond, angle and dihedral terms23. All energy
minimization procedures used < 1000 steeps of steepest descent method, in order to
relax any steric conflicts generated during system setup. Long-range electrostatic
interactions were calculated using the particle mesh Ewald (PME) method, with a 12
Å cutoff for the real space calculation24. A cut-off of 12 Å was used for the van der
2
Waals interactions. The simulations were performed at constant temperature, pressure
and number of particles. The temperature of the protein, lipid and solvent (waters and
ions) were separately coupled using the Nose-Hoover thermostat25 at 310 K, with a
coupling constant T = 0.1 ps. System pressures were semi-isotropically coupled using
the Parrinello-Rahman barostat26 at 1 bar with a coupling constant P = 1 ps and
compressibility = 4.5 x 10-5 bar-1. The LINCS algorithm27 was used throughout to
constrain bond lengths. The time step for integration in both simulations was 2 fs.
All analyses used GROMACS tools and locally written code. Molecular graphics
images were generated using VMD28.
.
3
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