Supplementary informations: Towards new
insights in the sterol/amphotericin nanochannels
formation: a molecular dynamic simulation study
Khaoula Boukaria, Sébastien Balmeb, Jean-Marc Janotb, and Fabien Picauda,*
a
Laboratoire de Nanomédecine, Imagerie et Thérapeutique, Université Franche-Comté,
Centre Hospitalier Universitaire de Besançon, UFR ST, 16 route de Gray, 25030 Besançon
cedex, France
b
Institut Européen des Membranes, UMR5635 CNRS-UM2-ENSCM, Place Eugène
Bataillon, 34095 Montpellier cedex 5, France
*corresponding author:
fabien.picaud@univ-fcomte.fr
S1.Force Field Parameters
To determine the force field parameters we first optimized the initial geometry molecule
(Fig SI1, left) inside water solvent with DFT method using the B3LYP hybrid functional
at the 6-31++G medium-sized basis set. Then, partial charges and Hessian matrix were
extracted according to the Force Field Toolkit Procedure (Fig SI1, middle). The stability
of the molecule and the structure was then performed in water using molecular dynamic
simulation (Fig SI1, right).
Fig SI 1 Comparison between the initial AmB structure (left panel), the optimized structure
using DFT calculations in water solvent (middle panel) and the structure obtained after 2 ns of
molecular dynamic simulations in water (right panel).
The large set of parameters obtained for this molecule is difficult to summarize precisely.
To obtain the details of it, please feel free to contact the corresponding author that will
send it you back. All atoms were considered as different in the force field building. This
leads to the parametrization of 140 bonds, 252 angles, and 389 dihedrals for the bonded
interaction while 138 charges and van der Waals values were attributed for the nonbonded interaction.
S2.Comparative study at short MD time (t=250 ps)
Baginski et al.(Baginski, Resatb & Borowski, 2002) studied the AmB-ERG and AmB-CHL
complexes stabilities embedded inside DMPC membrane using short MD simulations of
duration equal to 250 ps. To compare we analyze our simulations at short MD time (t=250 ps)
and before letting all the systems evolve until stabilization.
Fig. SI 2 shows the geometry adopted by the different couples inside the membrane at
t=250ps. We can already observe in these snapshots that the three couples of molecules do not
behave identically during this small MD duration while the initial geometries of the
simulation were about the same.
Fig SI 2 Snapshots of the simulation at t=250ps. a) For AmB-ERG, b) For AmB-CHL, c) For
AmB-LAN. Yellow, cyan, green and red van der Waals spheres are for amphotericin,
ergosterol, cholesterol and lanosterol molecules respectively. Water, ions and lipid molecules
are not shown for clarity.
According to literature(Baginski et al., 2002), both intramolecular and intermolecular
properties play an important role for these two systems. We have calculated the number of
intramolecular hydrogen bonds formed inside each AmB molecules for all the systems (Fig.
SI 3a). To carry out this calculation, we have considered the distance between donor and
acceptor not longer than 3.3 Å and the planar acceptor-hydrogen-donor angle not bigger than
90°. The distribution of the total number of AmB intramolecular hydrogen bonds showed that
16 hydrogen bonds were formed while in our calculations, two peaks appear centered on 12
and 20 hydrogen bonds for the AmB-ERG complex. In the case of AmB-CHL complex, these
intramolecular hydrogen bonds presented a similar distribution compared to the AmB-ERG
channel (centered on 16 hydrogen bonds) in the literature. Here we found a distribution of
hydrogen bonds with a double peak conformation whose average was around 24 hydrogen
bonds. Therefore, we found in these simulations that AmB-CHL channel presented more
intramolecular hydrogen bonds compared to AmB-ERG one. The small differences observed
with literature could be due to the initial starting point of the geometrical molecular
arrangement that could lead to a deformation of the AmB molecule in the presence of
cholesterol compared to ergosterol. For AmB-LAN complex, a well peaked distribution was
obtained centered on the same number of hydrogen bonds than that observed in the AmBERG (18 measured hydrogen bonds) (see Fig. S2a).
Fig. SI 3 a) Total number of AmB-AmB intramolecular hydrogen bonds for AmB-ERG,
AmB-CHL and AmB-LAN channels. b) Total number of AmB-Sterol intermolecular
hydrogen bonds for AmB-ERG, AmB-CHL and AmB-LAN channels
Then, the intermolecular properties of AmB-ERG, AmB-CHL channels and AmB-LAN
were investigated. The number of hydrogen bonds formed between molecules were calculated
and compared to literature. In this study, we found 9 hydrogen bonds for AmB-ERG complex
compared to 10 in reference(Baginski et al., 2002). If we look at the AmB-CHL complex, our
results show that AmB molecules formed 12 hydrogen bonds compared to 14 hydrogen
bonds in reference(Baginski et al., 2002). For AmB-LAN, only 7 hydrogen bonds were
obtained. While small differences were observed between these two studies, we can argue that
statistically, MD simulations gave the same behavior. Indeed, the strong instability of the
system in terms of energy and structure at this short time (during equilibration phase) can
modify rapidly the properties of the system from one simulation to another. Moreover, since
the inside of these complexes is hydrophilic, this will allow water molecules and ions to pass
through it (especially for AmB-ERG complex). We have calculated the evolution of the
formation of hydrogen bonds between water molecules and AmB molecules inside the pore.
In the case of AmB-ERG complex, we found 27 formed hydrogen bonds (compared to 23 in
reference(Baginski et al., 2002)). For the AmB-CHL complex, the water molecules form 24
hydrogen bonds with the AmB ones (22 hydrogen bonds in Ref.(Baginski et al., 2002)). We
have finally evaluated the number of water molecules confined inside the nanopore at 250 ps.
For AmB-ERG complex, 364 water molecules were found while in the case of AmB-CHL
system, 322 water molecules were localized inside the nanopore. The number of water
molecules and hydrogen bonds are more important for the AmB-ERG channel due to its wider
pore diameter. As awaiting the nanopore offering the most number of water molecules
allowed the formation of the most important number of hydrogen bonds between them and
AmB molecules. This reasoning is verified since, for AmB-LAN complex, we found around
30 hydrogen bonds between the 432 water molecules in the pore and the polyenes (see Fig. SI
4).
24
21
frame (%)
18
15
AmB-ERG
12
AmB-CHL
9
AmB-LAN
6
3
14
19
24
29
Number of hydrogen bonds
34
39
Fig SI 4. Total number of AmB-water hydrogen bonds for AmB-ERG , AmB-CHL and
AmB-LAN channels
To understand where these small differences came from, we investigated the size of the
pore diameter. At 250 ps, we found a mean diameter for the AmB-ERG pore equal to
13.0±0.6 Å (value comparable with reference(Baginski et al., 2002), i.e. around 14 Å). For the
AmB-CHL system, we found a pore diameter equal to 12.0 ±0.5 Å in agreement with the
calculations of Baginski et al., who obtained a pore diameter ranging between 12 Å and 13 Å.
For AmB-LAN, this distance is around 15±2 Å, showing an important increase of the pore
diameter depending on the sterols incorporated inside the membrane (see Fig. SI 5). The
AmB-ERG pore diameter channel is slightly larger than the AmB-CHL as demonstrated in
reference(Baginski et al., 2002). This may explain the difference in the number of hydrogen
bonds formed in each channel. The distance between AmB molecules should be shorter in the
case of AmB-CHL complex which allow them to form more hydrogen bonds between each
other's than in the case of AmB-ERG channel. However, as shown in Fig. SI 5, the short MD
durations cannot give any information on the dynamical evolution of the pore since the
diameter of each remains constant during 250 ps. It cannot be concluded on the stability of
these structures and the necessity to increase the MD duration is here evident.
17
O-O distance (Å)
16
15
ERG-AmB
14
CHL-AmB
13
AmB-LAN
12
11
0
50
100
150
200
250
Time (ps)
Fig SI 5. AmB pore diameter for AmB-ERG, AmB-CHL and AmB-LAN channels
Another crucial property should be studied to verify the stability of the systems. The root
mean square deviation denoted RMSD, was calculated for the eight AmB molecules and
sterols separately as a function of the time simulation (Fig. SI 6). A close inspection of the
RMSD reveals the presence of some small increasing fluctuations during 250 ps which
indicate that the AmB-ERG couples are moving with each other.
2.2
2.6
a)
b)
1.8
AMB
1.4
ERG
RMSD (Å)
RMSD (Å)
2.2
1.8
AmB
CHL
1.4
1
1
0
50
100
150
Time (ps)
200
250
0
50
100
150
Time (ps)
200
250
c)
3
RMSD (Å)
2.6
2.2
AmB
1.8
LAN
1.4
1
0
50
100
150
200
250
Time (ps)
Fig SI 6. a) RMSD for AmB (blue curve) and ERG (green curve). b) RMSD for AmB (blue
curve) and CHL (green curve). c) RMSD for AmB (blue curve) and LAN (green curve).
The RMSD for the three complexes showed the same behavior with a concomitant
evolution of the polyene geometry and the sterol one. The RMSD fluctuations were however
less important for AmB-CHL, which indicated stronger interaction between AmB molecules
and this sterol.
Our results show that the type of sterol plays a major role in the formation of the nanopore,
especially in the size of the pore diameter. At 250 ps in the case of AmB-CHL channel, pore
diameter is smaller than the AmB-ERG one and water forms less hydrogen bonds with AmB
molecules (in agreement with Ref.(Baginski et al., 2002)). Therefore, having a larger pore
diameter promotes the leakage of water molecules through the nanopore. Moreover, the
RMSD calculations show that the three systems have not converged in terms of geometrical
stability and further MD simulations are necessary to improve these preliminary conclusions.
S2.Stability at the end of the simulations
We plotted in Figs. SI 7 and SI 8 the RMSD and total energy as a function of simulation
time for the whole simulation. As clearly shown, it is necessary to stand for 70 ns to reach
structural and energetic stabilities of the system. The constant evolution of the structure
during simulations indicates that the sterol plays a major role in the AmB behavior. We stop
the simulations for the AmB-LAN system at t=50ns since it behaves comparatively to AmBERG one.
20
RMSD (Å)
16
12
ERG-AmB
8
CHL-AmB
LAN-AmB
4
0
0
20
40
60
Time (ns)
Figure SI 7: RMSD of AmB-ERG (blue curve), AmB-CHL( red curve) and AmB-LAN
(green curve).
-2,40e+5
-2,45e+5
E (kcal/mol)
-2,50e+5
-2,55e+5
-2,92e+5
-2,93e+5
-2,94e+5
-2,95e+5
-2,96e+5
0
20
40
t (ns)
60
Figure SI 8: Total energy of AmB-ERG (blue curve), AmB-CHL( red curve) and AmBLAN systems (green curve)
The averages performed between 35/50ns and 55/70ns are a bit different between the two
ranges of time especially for the AmB-ERG interactions but the standard deviations increase
also. However, considering two equivalent time ranges, LAN and ERG present the same
value for this kind of contribution. We could thus hypothesize on the same behavior for the
two systems containing different sterols. Concerning the inter polyene interaction, the AmBERG system presents a small increase of its energy while AmB-CHL has an inverted behavior
between table SI1 and table SI2. This corroborates the expansion of the AmB-ERG system
compared to the cohesion of the AmB-CHL one. The system built with LAN presents a higher
energy (less stable) than ERG and CHL. This would thus lead to the expansion of the system
as for ERG.
System
AmB-ERG
AmB-CHL
-100±5
-188±8
VdW energies AmBsterol (Kcal/mol)
-330±20
-250±10
VdW energies AmBDMPC (Kcal/mol)
-53±9
-85±9
VdW energies AmBAmB (Kcal/mol)
Table SI 1 : VdW energies estimated as an average between t=35 and t=50ns
AmB-LAN
-102±9
System
AmB-ERG
AmB-CHL
-88±9
-190±9
VdW energies AmBsterol (Kcal/mol)
-320±20
-260±10
VdW energies AmBDMPC (Kcal/mol)
-49±6
-90±9
VdW energies AmBAmB (Kcal/mol)
Table SI 2 : VdW energies estimated as an average between t=55 and t=70ns
AmB-LAN
-290±20
-35±7
Baginski, M., Resatb, H., Borowski, E. 2002. Comparative Molecular Dynamics Simulations of
Amphotericin B–Cholesterol/Ergosterol Membrane Channels, . Biochimi. Biophys. Acta. 1567
63–78.