A computational model to investigate
the aquaporin mechanism of action
Nicole Hasler and Léon Othenin-Girard
Laboratory for Biomolecular Modelling, EPFL
Prof. Matteo Dal Peraro
Supervisor: Alessio Prunotto
1. Introduction
Aquaporins are integral membrane proteins that form pores and transport water
molecules across membranes without letting other substances, such as ions or
various solutes, flow into the cell1. This feature allows for faster transport than
simple diffusion through the phospholipid bilayer. All aquaporins are impermeable
to charged molecules, which prevents fast diffusion of protons, and therefore are
essential in the conservation of the electrochemical gradient between the
cytoplasm and the extracellular space1.
Two areas of the proteins are especially important as regards water filtration. The
NPA motif, which consists of the amino acids aspargine, proline and alanine,
constitutes the first constriction site of aquaporin2. The second, the ar/R selectivity
filter, is a tetrad that is formed from residues on helices B, and E and two residues
from loop E. It is often the narrowest section of the pore, where water molecules
can be selectively bound to amino acids and allowed passage through the protein3.
Due to their essential role in water transport, aquaporins are vital for the proper
function of cells. Therefore, they are important for understanding normal
physiology as well as pathophysiology of clinical disorders. Molecular dynamics
can be used to study the structural properties of the protein and to investigate its
action mechanism. The method has limitations, as not all factors that influence a
system in vivo can be incorporated into a simulation, let alone all relevant chemical
laws. This project will attempt to produce a realistic model of an aquaporin
embedded in a small membrane patch and evaluate the extent to which an
accurate simulation is possible.
2. Methods
Using VMD
VMD is a program in which one can visualize the models created and output
trajectories. First, the different functions of VMD were explored. Various molecules
were displayed and moved in 3D space to gain a working knowledge of the
program. Then different styles of graphical representation and coloring methods
were investigated. Sections of a protein were colored, using different setting to
color parts of a molecule with certain characteristics and properties.
Another important aspect was learning to adapt the trajectories and use the
animation tools provided by the program to see how the protein changes over
time, especially when exposed to compression and pulling forces. This included
1
Tajkhorshid, Emad. "Structure, Dynamics and Function of Aquaporins." 15 10 2006. Theoretical and
Computational Biophysics Group. NIH Center for Macromolecular Modeling and Bioinformatics. 14.
3. 2016
2
Beckman Institue. "Introduction to Aquaporin Structure." NIH Institute for Macromolecular
Modelling and Bioinformatics. 17. 3. 2016
3
Jung et al. "Molecular structure of the water channel through Aquaporin CHIP: The hourglass
model." Journal of Biological Chemistry and Molecular Biology 269.May 20. (1994).
<http://www.ks.uiuc.edu/Training/Tutorials/science/aquaporin/tutorial_aqp-html/node3.html>.
1
learning to display multiple frames to better compare the development over a
certain period of time.
After this introduction to the program, the same commands were exercised using
TKconsole and text-based commands. Aligning molecules to better compare their
structures was also a vital part of this first process, as it is helpful for comparing
different types of the same protein, for example human aquaporin and E.coli
aquaporin.
Another possibility given by VMD is the option to label atoms and calculate
distances between them and even to create a graph displaying change in distance
over a certain time period during which the protein is stretched or compressed.
The root mean square deviation, a measure for how much each atom moves on
average, is also computable at the click of a button.
Creating the membrane
After successfully familiarizing oneself with the function and setup of VMD, one can
proceed to trying to gain a better understanding of phospholipid bilayers. To
create a model of a biological membrane, it is advisable to use a program like
LipidBuilder, an application developed by the EPFL itself.
Phosphatidylethanolamine (PE) was selected as the phosphate head of the
phospholipid, and the meaning of phospholipid nomenclature was researched to
allow determination of the hydrocarbon tail length and structure. PE(16:0-18:1
(9Z)) provided the formula for a membrane component with a PE head and two
hydrocarbon tails, one with 16 carbons in the backbone and one with 18 carbons
and one double bond. Then, the tool was used to build a membrane of the size 120
x 120 Angstrom and care was taken to pack it as densely as possible.
Creating the protein
Following this step, the KcsA membrane protein became the subject of
investigation. The goal was to embed KcsA in a fully hydrated membrane. The
channel is, like aquaporin, a tetramer composed of four identical subunits, and also
features a selectivity filter that only conducts potassium ions across the membrane
at a high rate4.
The PDB file of a KcsA monomer, which contains the coordinates of each atom in a
molecule, was downloaded from the protein database. This monomer was
replicated three times and transformed in a matrix to fit the monomers together.
Then, the individual files were merged in Terminal to create the tetrameric
molecule. This same process was applied to aquaporin.
Next, AutoPSF was used to create corresponding PSF for the PDB of the protein
created. The resulting PSF file contains information on the bonds between the
4
Alek Aksimentiev, Marcos Sotomayor, David Wells. "Tutorials." 2 2012. Theoretical and
Computational Biophysics Group. NIH Center for Macromolecular Modeling and Bioinformatics. 14 3
2016 <http://www.ks.uiuc.edu/Training/Tutorials/science/membrane/mem-tutorial.pdf>.
2
individual atoms in the molecule, and is therefore important for running a
molecular dynamics simulation.
Solvating the protein was another step. The water layer was made 3 Angstrom
thick, and the resulting PDB file was combined with the rest of the system.
Unwanted water molecules that overlapped with the protein were removed by
selecting molecules near the protein residues.
Embedding the protein in the membrane
A different membrane was built using the MembraneBuilder tool provided by
VMD. This membrane was also made using PE heads, forming a POPE membrane.
Next, the protein was inserted into the membrane by moving it to the center of
mass of the membrane. The hydrophobic regions of the protein were colored to
ensure that these were fully surrounded by the phospholipid bilayer and thus not
in contact with the surrounding water. The lipids as well as the water molecules
solvating the phosphate heads that overlapped with the protein were removed.
Problems arose when attempting to solvate the system, as a glitch in the created
file prevented the “solvate” function of VMD from solvating the entire system with
water. Therefore, it was decided to continue with the molecular dynamics
simulation without solvating the membrane patch with its contained protein, as
there were already some water molecules present on either side.
Running the simulation
The molecular dynamics simulation was run after modifying a file containing
instructions for this process to better suit our protein-membrane complex’
parameters. The size of the membrane patch was adjusted, and the sections
pertaining to the solvation of the membrane were removed, as this step had been
left out. This simulation took some time to run, as the created system contained a
very large amount of atoms (over 60’000). Because it was taking too long, it was
decided to run it on the clusters of the EPFL. However, sadly, the cluster crashed in
the middle of the night, disrupting the simulation. Due to a lack of time, it was not
possible to run it a second time. Thanks to the fact that so far, no previous group
had even been able to reach this stage of the project, the Laboratory for
Biomolecular Modeling had a prepared simulation that was available to be
evaluated.
3. Results
The simulation was run and evaluated by analyzing the RMSD. After the one
hundredth frame, an equilibrium was reached, as the RMSD oscillated between
two points from this time on. This can be seen in figure 1. The model needed a
certain time of equilibration to reach the physiological state that is common for in
vivo processes.
3
Figure 1 RMSD graph for all atoms
Another representation method used to analyze simulations is the Ramachandran
plot. This graphically represents the bond angles that occur in amino acids in
nature and compares these acceptable angles with the ones present in the
simulation. We found that the bond angles in the simulation were concentrated
around the areas of naturally occurring bond angles, as seen in figure 2.
Figure 2 Ramachandran plot for all amino acids
Next, the RMSD plots of the important functional groups that affect pore function
were analyzed. To make this possible, the residues facing each other on the interior
surface of each aquaporin channel were selected (residues 79 to 88 and 196 to
204). Then, the reentrant loops next to the NPA motif were selected (residues 74 to
78 and 191 to 195). The NPA motif itself (residues 78 to 80 and 194 to 196), an
important arginine residue (no. 197) which stabilizes the pore and binds water
molecules, and glutamates, which stabilize the reentrant loops, were also colored.
Analysis of the RMSD graphs of these residues showed that they reach a state of
equilibrium very soon, after the fiftieth frame.
Additionally, the simulation showed that the water molecules pass through the
pores in single file, as they are supposed to. They also switch orientation within the
pore, flipping so that the oxygen molecule faces the top. Both of these aspects
mimic behavior of the oxygen molecules passing through an aquaporin in nature.
4
Figure 3 Water molecules flip inside pore
4. Discussion
The regular oscillations of the RMSD indicate that the system simulated did reach
equilibrium. The representations in the Ramachandran plot also indicate that the
model does not differ very strongly from the bond angles that occur in nature.
These aspects, combined with the fact that the water molecules behave in a
realistic manner as well, lead us to conclude that the molecular dynamics
simulation constitutes a relatively good representation of aquaporin function in
vivo. Of course, many aspects cannot be accurately reproduced in models, as the
laws of chemistry are far too numerous and complex to incorporate in a simulation,
but the system produced reaches and acceptable level of realistic aquaporin
function.
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5. Sources
1. Agre et al. "Aquaporin water channels - from atomic structure to clinical
medicine." The Journal of Physiology 542.1 (2002).
2. Alek Aksimentiev, Marcos Sotomayor, David Wells. "Tutorials." 2 2012.
Theoretical and Computational Biophysics Group. NIH Center for Macromolecular
Modeling and Bioinformatics. 14 3 2016
<http://www.ks.uiuc.edu/Training/Tutorials/science/membrane/memtutorial.pdf>.
3. Beckman Institue. "Introduction to Aquaporin Structure." NIH Institute for
Macromolecular Modelling and Bioinformatics. 17. 3. 2016
<http://www.ks.uiuc.edu/Training/Tutorials/science/aquaporin/tutorial_aqphtml/node3.html>.
4. Jung et al. "Molecular structure of the water channel through Aquaporin CHIP:
The hourglass model." Journal of Biological Chemistry and Molecular Biology
269.May 20. (1994).
5. Tajkhorshid, Emad. "Structure, Dynamics and Function of Aquaporins." 15 10
2006. Theoretical and Computational Biophysics Group. NIH Center for
Macromolecular Modeling and Bioinformatics. 14 3 2016
<http://www.ks.uiuc.edu/Research/aquaporins/ >.
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