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Interactions of Angiotensin II with water and water/ethanol mixtures

studied with Molecular Dynamics Simulations.

Hari Leontiadou1, Ioannis Samios2*, Zoe Cournia1*

Biomedical Research Foundation, Academy of Athens, 4 Soranou Ephessiou, 11527, Athens, Greece

Department of Chemistry, Faculty of Natural Sciences, National University of Athens, Panepistimiopolis-

Zografou, 11521, Athens, Greece

ABSTRACT: (Word Style BD_Abstract). All manuscripts must be accompanied by an abstract. The abstract should briefly state the problem or purpose of the research, indicate the theoretical or experimental plan used, summarize the principal findings, and point out the major conclusions. Abstract length is one paragraph.

The octapeptide angiotensin-II (H-AspArgValTyr
IleHisProPhe-OH, AII) is one of the oldest peptide
hormones, known for its multiplicity of biological actions
related to endocrine or connected to the central and peripheral nervous system1. It is produced by the conversion
of angiotensin-I (H-AspArgValTyrIleHisProPhe
HisLeu-OH, AI) to AII by the action of the angiotensin-I
converting enzyme (ACE) of the vascular endothelium.
AII is a potent pressor agent, which has a vital role in the
regulation of blood pressure, in the conservation of total
blood volume and salt homeostasis. AII binds to the AII
receptor AT1, which is a transmembrane protein. As the
binding site of AT1 receptor is exposed to the lipid membrane, AII is directly exposed to the lipid bilayer. An X-ray
structure of Angiotensin II-Fab complex shows that the
hormone peptide adopts a compact U-shaped structure
when it is bound to the receptor2. However, photolabeling
studies of [Bpa3]AngII with the AT1 receptor indicate a
model where the peptide in its bound form adopts a rather extended -strand structure3. Furthermore, several
NMR studies of AII in aqueous and organic solvents indicate the existence of a mixture of unfolded and folded
conformations ranging from extended -structures to the
more compact U-shaped ones4-8. Organic solvents such as
TFE and heating have been reported to favor the fold of
the angiotensin amide in a rather compact structure determined through circular dichroism measurements9.
However, recent NMR and Molecular dynamics studies of
[Val5] AngII analogue in a water/ethanol (35%v/v) binary
solvent at low temperatures, suggest that the peptide is
primarily in an extended -structure and that most regions of the peptide are preferentially solvated by ethanol

molecules10-11. Despite the inherent flexibility of the octapeptide, another possible reason for the divergent structural models proposed over the years could be the use of
different solvents and experimental conditions.
Mixtures of ethanol and water are solvents that can influence stability and conformational properties of biological molecules. Use of such solvents in experiments with
biomolecules that usually operate in environments that
are only partly aqueous may produce results that help in
understanding the role(s) of water in maintaining macromolecule structure and activity. In other arenas, mixtures of water and ethanol provide reaction media that
enable enzymes to act on substrates that are not soluble
in water, although substrate specificity, reaction rates and
protein stability may be altered by the solvent mixture. In
all of these systems, the effects observed may be the result
of direct interactions of solvent alcohol molecules with a
protein. In general, preferential solvation of proteins by
organic co-solvents and clustering of organic molecules in
the binary solution are considered possible mechanisms
that could either allow or block proteins from jumping
between different conformational states12-14.
Moreover, ethanol/water mixtures may be considered
as a bilayer mimic considering the amphiphilic nature of
In this paper, we provide an atomic-level picture of the
intermolecular interactions governing AngII-water and
AngII-water-ethanol mixtures using MD simulations.
Structural and dynamical properties of these systems are
outlined in the context of identifying the solvent microenvironment around the AngII peptide and in describing
the representative conformations of AngII in these two

media. Our results are in excellent agreement with relevant experimental data and provide insights into the bioactive conformation of this hormone.
System Preparation
Human Angiotensin II (AngII) was chosen -Asp-ArgVal-Tyr-Ile-His-Pro-Phe-for the conformational study of
the peptide in water and ethanol/water-35% (v/v) solvent
at different temperatures. The initial structure of the octapeptide was taken from the Protein Data Bank,
1N9V.pdb7. The apparent pH was chosen to be 4 in order
to match most of the experiments. So, in the model peptide the terminals are ionized. Asp is not protonated and
charged (-1), Arg is protonated and charged (+1) and His is
protonated and charged (+1). The system was solvated in
pure water or 35% ethanol-1,1-d2-water (v/v) and neutralized with 1 Cl- ion.

ically, the distance between the center of mass of the Cand N-terminal residues (ASP-PHE) reaches the lowest
values ( ~1.4-1.1nm ) in water and (~1.4-1.0nm) in water/ethanol the same time periods that the radius of gyration adopts also its minimum values (Fig.1 iii, iv). NMR
experiments have revealed that the C- and N- terminal
residues distance of AngII in water is 0.72nm8 while in
water/trifluoroethanol and in dimethylsulfoxide (Spyroulias, 2003) is around 1.78 nm. In our simulations we observe that the folding/unfolding events of AngII at elevated temperatures (310K, 323K) seem to be significantly
more frequent in the binary solvent (water/ethanol) than
in water.

Molecular Dynamics (MD) simulations

MD simulations were performed using gromacs 4.6.1
double precision15 running on BA and CURIE supercomputing centers [?]. The AMBER99SB-ILDN force field was
chosen for the peptide and tip4p was used for the water
model. The model for the ethanol molecule was the one
previously studied by Gerrig, [2013]. A 2fs time step is
used together with PME for the calculation of the longrange electrostatics. A cut-off of 1.4 is used for both the
electrostatics and the van der Waals interactions. The
non-bonded neighbor list is updated every 5 steps. LINKS
is used as a constraint algorithm and the Berendsen coupling is used for the pressure and temperature. Table S1
summarizes the simulated systems. The analysis of the
trajectories has been performed by using the gromacs
4.6.1 tools. The first 30ns from the trajectories were considered as relaxation time and were discarded.
Results and Discussion
AngII structure and dynamics
The root mean square deviation (rmsd) from the initial
structure has been calculated for all different systems
(Fig. S1). The average rmsd for both systems in water and
water/ethanol solvent remains the same. However, it is
observed that for elevated temperatures (310K, 323K) the
rmsd fluctuations increase indicating a folding/unfolding
pattern of the peptide. The folding/unfolding behavior of
the peptide is noticeable also in the plots of the radius of
gyration of AngII in all different systems (Fig.1 i,ii). Although, the average value of the radius of gyration of AngII is around 0.75-0.8nm there are instances where it decreases significantly (~0.6-0.65nm). Arrows in Figure 1 are
indicating the folding events of AngII in water (i) and
water/ethanol (ii). Interestingly, the distance of the terminal residues of the peptide is following the same pattern of fluctuations as the radius of gyration. More specif-

Figure 1. Running averages measured every 1000ps of the

radius of gyration for the AngII peptide in water (i) and
water/ethanol (ii) solvent at various temperatures. Arrows
indicate folding events. Running averages measured every
1000ps of the distance between the center of mass of the
termini residues of AngII (ASP_PHE) in water (iii) and
water/ethanol (iv).
Representative conformations of AngII observed in
Water and in Water/Ethanol
A cluster analysis was performed for the peptide in order
to find out the representative conformations of AngII in
pure water and in water/ethanol during the simulation at
different temperatures. The conformations were clustered
by comparing the rmsd of the Calpha atoms of the peptide according to

Arg2 hydrogen bond of about 1-4% in pure water and 312% in water/ethanol (see Table S2).

Figure 2. The first cluster representative conformations

of AngII (i) in water and in (iv) water/ethanol are shown
at various temperatures. U-shaped conformations are less
populated in water at (ii) 310K and (iii) 323K and in water/ethanol at (v) 310K and (vi) 323K. Peptides are represented in cartoon while some key residues are shown in
sticks. Structures at 278K, 298K, 310K and 323K are colored in yellow, magenta, cyan and blue respectively.

the gromos method implemented in Gromacs, using a

cutoff of 0.15nm. The representative conformations from
the most populated clusters of each system are shown in
Fig.2. We observe that the dominant conformation of
AngII in water and water/ethanol is mainly an extended,
coil structure (Fig.2 i, iv). Note that at the lowest simulated temperature 278K the AngII peptide is mainly in its
open extended conformation that remains stable
throughout the simulation. However, at elevated temperatures there is a small population of folded conformations
that resemble the U-shaped molecule found in other
studies8. More specifically, the percentage of the Ushaped structure in water is 7% at 310K (Fig. 2 ii) and 6%
at 323K (Fig. 2 iii). In water/ethanol the same percentage
is 6% at 310K (Fig. 2 v) and 17% at 323K (Fig. 2 vi).
Intra-Peptide Hydrogen Bonds
The formation of intra-peptide hydrogen bonds in pure
water and in water/ethanol is presented in Table S2. As
mentioned before, during all different simulations the
peptide is mainly in the open, extended conformation
where the formation of intra-peptide hydrogen bonds
does not seem to be favoured. However, it is important to
mention the occurrence of some intra-peptide hydrogen
bonds that are characteristic of the AngII octapeptide and
compare these with known experimental models. Therefore, common features between our model and the X-ray
structure of AII bound to mAb Fab13116 as well as the NMR
structure of AII in aqueous solution8 are a) the formation
of a hydrogen bond between Asp1-side-OD1 and Arg2main-NH. In fact in our simulations we additionally observe Asp1-Arg2 side chain-side chain hydrogen bonds
resulting in a total percentage of occurrence of the Asp1-

b) the formation of a His6-side-ND1 and Pro7-main-O

hydrogen bond with a percentage of occurrence of 6-13%
in water and 2-8% in water/ethanol. Interestingly, the
Asp1-Arg2 and His6-Pro7 hydrogen bonds discussed
above are common for all simulated systems, sampling
both extended and U-shaped conformations, although the
former is marginally present in pure water. A hydrogen
bond that appears only in the water/ethanol system at
elevated temperatures (310K, 323K) is the one formed between Arg2 and Phe8 residues with an occurrence of 38%. This hydrogen bond could bring the two peptide
termini in a close distance and stabilize a compact Ushape structure (Fig. 2 v).
Clustering of the three aromatic rings
Based on previous studies5, the model of a clustering of
the aromatic rings of AngII Tyr4-His6-Phe8, as a charge
relay system has been proposed. However, in our simulations analysis of the distances between the center of mass
of the aromatic rings excludes the presence of such an
aromatic ring clustering (Fig. S2).
Peptide Dynamics
The root mean square fluctuation per residue is presented
in Figure S3. The increase of the temperature enhances
the flexibility of the residues in both water and water/ethanol systems. However, a pronounced flexibility
increase appears for Ile 5 and Phe 8 of the peptide in water/ethanol at 310K and 323K.
Diffusion of AngII peptide in water and in water/ethanol
Table 1 and 2 present the calculated translational diffusion coefficients for AngII octapeptide in both systems
(water and water/ethanol) and for ethanol molecules,
respectively. Comparison with previous available experimental and simulation data shows a very good agreement
which allows us to explore solvent structural properties in
a more detail.
Preferential binding of solvent molecules at the peptide surface
It is well known that preferential interactions between the
peptide residues and the different solvent components are
important for peptide/protein structure and dynamics10, 13,
. Therefore, the radial distribution functions of important residues/atoms of the system have been calculated for all systems at all temperatures. The radial distribution function (RDF) between the center of mass of each
residue in the peptide and the atoms of the solvent and
co-solvent has been

Table 1 Diffusion of AngII Dpeptide 10-5 cm2s-1


In Water

In Water/Ethanol

Gerigs sim.



0,1846( 0,0458)

0,0579 ( 0,0025)

0,053 ( 0,007)



0,6045 ( 0,0245)

0,3497 ( 0,0899)

0,11 ( 0.02)



0,9412 ( 0,3155)

0,1563( 0,0141)


2,7192 ( 0,6804)

0,4776 ( 0,1569)

*Simulations and experiments for AngII in ethanol/water-35% (v/v), reported in Gerig, 2013.

Table 2 Diffusion of Ethanol molecules Dethanol 10-5 cm2s-1


In Water/Ethanol

Gerigs sim.



0,7344( 0,0034)

0,342( 0,013)



1,2809 ( 0,0105)

0,839 ( 0.022)



1,7711 ( 0,0645)


2,3184 ( 0,0908)

*Simulations and experiments for AngII in ethanol/water-35% (v/v), reported in Gerig, 2013.

calculated. The temperature dependence of the radial

distribution function is shown in Fig.S4 for two terminal
residues. According to the results, the temperature increase does not affect the positions of the peaks and the
valleys of the RDFs but only slightly their heights and
depths. Furthermore, comparison of the RDFs of the residues center of mass (COM) with the oxygen atom of water and ethanol molecules at the same temperature clearly
indicates differences at the heights but not the positions
of the first peaks (Fig. S5). A slight preference for water
molecules is observed for Phe8 where the RDF for the
oxygen of water molecules starts at a distance of 0.3 nm
indicating probably the formation of a hydrogen bond
(Fig. S5, last figure). In addition, comparison of the RDFs
of peptide residues COM with water molecules in pure
water and water molecules in the binary water/ethanol
solvent does not indicate any change in the positions of
the peaks and valleys that could be due to the presence of
the ethanol (data not shown). In simulations of
[val5]AngII in water/ethanol (35% v/v) Gerig [2013] has
reported preferential solvation of peptide hydrogens by
ethanol molecules. More specifically, he reports that hydrogen atoms of valine and tyrosine side chains preferen-

tially interact with ethanol molecules while hydrogens of

Arg1, Arg2, Phe8 and His6 side chains are in a solvent
environment rich in water molecules. In our system
which is similar to the one of Gerig [2013] except for the
fact that we have used the human [Ile5]AngII analogue,
we have assessed the preferential solvation of the peptide
in a different way, by calculating the RDF of the center of
mass of each peptide residue with the Owater and Oethanol of
the solvent. Integration of the RDFs plots up to the first
valleys gives an indication of the coordination numbers
for the first solvation shell around each residue (Figure
S6). As seen in Table 3 most of the peptide residues interact mainly with water molecules except for the apolar
residues of Val3 and Ile5. The strong interaction of the
peptide with water is seen also in the number of hydrogen
bonds formed between the peptide and the solvent molecules (Fig. S7). There are on average around 15 hydrogen
bonds formed between the peptide and water and only
around 2 hydrogen bonds between the peptide and ethanol molecules.

Table 3. Coordination numbers of each peptide residue with O ethanol and Owater in the first solvation shell for the system of AngII
in water/ethanol.


Res. 1

Res. 2

Res. 3

Solvent Structural properties

The hydrophobic clustering of ethanol molecules in bulk
aqueous solutions has been experimentally investigated1821
showing that ethanol is primarily monomeric for mole
fractions of eth<0.07 while it self-associates when
0.07<eth<0.74. Mass spectrometric analysis indicates that
the ethanol rich clusters consist of less than 8 ethanol
molecules20. In our system, where eth is around 0.14, we
do observe some minor ethanol aggregation. The percentage of ethanol molecules that self-associate is around
14% and the average size of the clusters formed is 2 ethanol molecules independent of the temperature. Larger
aggregates of ethanol are also observed comprising 3-4
molecules but with a low percentage 1-3% of occurrence.
A detailed analysis of the polar interactions between water and ethanol molecules has been performed for an area
with r=0.4nm around the peptide. Figure S8 presents the
number of hydrogen bonds formed between water-water,
ethanol-ethanol and water-ethanol molecules in the proximity of the peptide. There are on average two hydrogen
bonds formed between ethanol molecules around the
peptide while the ethanol-water hydrogen bonds are outnumbered indicating a clear preference of ethanol to associate with water molecules.

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Res. 4

Res. 5

Res. 6

Res. 7

Res. 8


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CCR2, CC chemokine receptor 2; CCL2, CC chemokine ligand
2; CCR5, CC chemokine receptor 5; TLC, thin layer chromatography.

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