Keeping Aquaporin Channels Constitutively Open
for Biotechnology Applications and more
MEMPHYS, Center for Bio-Membrane Physics,Center of Excellence funded by
The Danish National Research Foundation
Southern Denmark University, Odense, Denmark
Aquaporin: the water pore
Peter Agre, JHU2003 Nobel Prize in Chemistry 50%
Periplasm
Cytoplasm
Transmembrane water transporters
Aquaporin
• 109 water molecules per second– Fast
• Very water selective– Pure
• Bidirectional, passive
• Mechanically/Osmotically driven transport
• Robust protein
Can be used to filter water in industrial applications?
The Aquaporin (MEMBAQ) project
The goal of the project is to explore the possibilities to incorporate recombinant aquaporin molecules in different
types of industrial membranes for water filtration.
Produce recombinant aquaporin
Construct stable membranefilm
Built the membrane film into a composite membrane
Test the membrane system for real applications
simulation simulation
Laboratory testing Laboratory testing
Porous hydrophilic support of lipid bilayer, like mica, cellulose
PorousTeflon film or other hydrophobic material
Planar lipid bilayer membrane with incorporated aquaporins.
Aquaporin molecule
Phospholipid molecule or other amphiphilic lipid molecule
Concept
ΔP
Courtesy: PH Jensen, Aquaporin
MEMBAQ: 9 partners
Role of Simulations in MEMBAQ
At MEMPHYS, the objective is to implement computer simulations of aquaporins (AQPs) embedded in different
nanotechnological membrane materials, and to use the data from computer simulations in the design of better
membrane materials.
Recombinant aquaporin
Stable membrane film
Testing in real applications
Simulations: Optimize design of nanotech membrane materials
Simulations: Aid design ofbetter aquaporins
.. More technical details ..
SoPIP2;1: Spinach Leaf Aquaporinwill be used in MEMBAQ
• Most aquaporins’ channels are always (constitutively) open
• Unlike most mammalian AQPs, SoPIP2;1 is a gated channel– Sometimes open,
sometimes closed– The gating is controlled by
several mechanisms
Crystal Structure of SoPIP2;1
• OPEN and CLOSED conformations were trapped and crystallized
• OPEN and CLOSED conformations differ in certain respects
Tornroth-Horsefield et al. Nature, 2006, 439, 688-694
Open and Closed States of SoPIP2;1
Closed
Open
Closed
Open
SoPIP2;1 is a GATED water channel
OpenClosed SoPIP2;1 Courtesy: Urban Johanson, Lund. U
OPENCLOSED D-LOOP BLOCKING THE PORE BY LARGE MOVEMENT
Courtesy: Urban Johanson, Lund. U
What drives the gating ?
• Phosphorylation at Ser274 and Ser115 opens the channel
• Calcium is required for keeping it closed
• Protonation of His193 closes the channel
All these can independently alter the conformation of the D-loop
Loop D N-terminus
• The D-loop links to the N-terminus via a network of H-bonds mediated by R190, D191, R118
• The network of H-bonds is broken by phosphorylation of Ser-115
What drives the gating ?
Tornroth-Horsefield et al. Nature, 2006, 439, 688-694
Gating by Ser115
Tornroth-Horsefield et al. Nature, 2006, 439, 688-694
Overall Objectives Molecular Dynamics Simulations
• Quantitative estimation of the water conduction rates through SoPIP2;1– No experimental measurements yet
• Enhance water permeation rate of SoPIP2;1– Drive it towards a constitutively open
conformation
Simulation Methods and Setups
• Trajectories of molecular systems in time using Newton’s equation of motion
• Time and length scales of nanoseconds and nanometers are accessible
• Thermodynamic properties can be calculated
(Why) Molecular Dynamics Simulations
Molecular Dynamics Simulations
• Each atom represented by point mass and point charge
• Interactions between atoms described by springs, electrostatics, and so on
• Evolution of a molecular trajectory
• Based on Newton’s classical equations of motion
• Macroscopic thermodynamic properties can be calculated using the principles of statistical mechanics
• No black magic !
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dE m
dt
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( )bond stretch bpairs
E K b b
i kelectrostatic
nonbondedpairs
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Simulation Setup
Tetrameric model of SoPIP2;1 embedded in a fully hydrated (POPE) and/or phosphatidylcholine (POPC) lipid bilayer
Molecular Dynamics of SoPIP2;1 in Membranes
• NPT ensemble
• Temperature: 310 K
• Pressure: 1 atm.
• N ~ 110000 atoms– 270 lipids
– Protein
– ~ 17000 water
– ions
• 105 x 105 x 80 Å
• Time step: 1 x 10-15 s
• CHARMM force field
• Ser115 and Ser274 not phosphorylated
18 Å
Simulations Implemented
• CLOSED and OPEN conformations– In POPC or POPE lipid membranes
• CLOSED mutants to improve Permeability– With POPC membranes
Simulations Completed~ 0.3 μs, 100,000 cpu hours
Number Conformation ofSoPIP2;1
Lipid Type Simulation Time (ns)
Wild-type
1 CLOSED POPC 41.20
2 CLOSED POPE 39.20
3 OPEN POPC 33.10
4 OPEN POPE 36.10
Mutants
1 R190A-D191A POPC 54.55
2 R190A-D191A(2) POPC 41.4
3 TRUNC POPC 42.6
Simulations run on DCSC
ResultsSingle Channel Permeability
Single Channel Permeability
• In principle, experimental measurements are possible
• Estimation of single channel osmotic permeability is possible from equilibrium MD simulations
• Estimates from simulations are usually within an order of magnitude of experimental measurements
• However, RELATIVE estimates (permeability of one channel versus another) are reliable
jW = pf ΔCS
Jensen & Mouritsen (2006) Biophys. J., 90, 2270-84
Single-channel Permeability Constants
Osmotic Permeability (pf)
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Zhu, et al. (2004) Phys. Rev. Lett., 93(22), 224501
www.ks.uiuc.edu
Single-channel Permeability Constants
owd kvp
Diffusive Permeability (pd)
k0 = # water molecules that traverse the channel per unit time
vw = Molar volume of water
Zhu, et al. Phys. Rev. Lett., 93(22), 224501
www.ks.uiuc.edu
Single Channel Permeability
Simulation pf (cm3/s) x 10-14 <L> (Å)
CLOSED-POPC 0.33 22.25
CLOSED-POPE 0.41 22.72
OPEN-POPC 0.68 22.06
OPEN-POPE 0.73 22.29
Khandelia and Mouritsen, unpublished data
Single Channel Osmotic Permeability pf
AQP1: 6 x 10-14 cm3/s
Khandelia and Mouritsen, unpublished data
Low pf of SoPIP2;1
• Simulations predict an absolute pf one order of magnitude lower than AQP1, two to threefold lower than GlpF.
• However, no experimental data is available for SoPIP2;1 to compare absolute values with
• Ratio of pf(closed)/pf(open) is similar to experimentally measured values
Suga and Maeshima (2004): Plant Cell Physiol, 45(7), 823-30
ResultsInfluence of Lipid Type
Influence of Lipid Type
The type of lipid (POPC vs. POPE) should not influence permeability
Results
Enhancing Permeability of SoPIP2;1Shifting Conformational Equilibrium towards the OPEN state
In collaboration withProf. Per Kjellbom & Dr. Urban Johanson
(Lund University, Sweden)
Gating of SoPIP2;1How to improve water conductivity ?
• Unlike most mammalian AQPs, SoPIP2;1 and other plant aquaporins are gated
• SoPIP2;1 can switch between CLOSED and OPEN states
• From the MEMBAQ perspective, it is important that the conformational equilibrium of SoPIP2;1 is driven towards the OPEN state for maximal filtration efficiency
– How ?
Enhancing Water Conductivity 1. Role of R190 and D191 in Gating
Arg190 and Asp191 on the gating loop anchor the loop to the Calcium ion
Tornroth-Horsefield et al (2006). Nature, 439, 688-694
It has been shown that homologous mutants in Arabidopsis thaliana PIP2;2 could not be closed
C Tournaire-Roux, et al. (2003) Nature, 425(6956), 393-7
Loop D
N-terminus
Hedfalk et al. (2006) Curr. Opin. Struct. Biol, 16, 447–456
Enhancing Water Conductivity2. Longer D-loop in SoPIP2;1
Two More (Successful) Strategies Tested in Simulations
• R190A-D191A mutation: Might disrupt the H-bonded network, and release the D-loop from the N-terminus
• Truncation of the D-loop: Deletion of the extra residues 193 through 196 should remove steric hindrance to water transport
(Both mutants of the CLOSED conformation)
Simulations Completed
Number Conformation ofSoPIP2;1
Lipid Type Simulation Time (ns)
Mutants
1 R190A-D191A POPC 54.55
2 R190A-D191A(2) POPC 41.4
3 TRUNC POPC 42.6
Both Mutants Increase Permeability
Khandelia and Mouritsen, unpublished data
Molecular Basis for Increased Permeability
Mutant R190A-D191AWild Type
Molecular Basis for Increased Permeability: Role of Ser36
Only one monomer is shown, ~ 40 ns
Ser36 is conserved in PIPs
R190-D191A
Wild TypeInitial Final
Molecular Basis for Increased Permeability: Role of Ser36
Khandelia and Mouritsen, unpublished data
OPENCLOSED D-LOOP BLOCKING THE PORE BY LARGE MOVEMENT
Courtesy: Urban Johanson, Lund. U
Summary
• Single-channel osmotic permeability constants computed
• Mutants with higher water conductivity were designed, which may be more suitable for future prototypes in MEMBAQ
• New fundamental insights into the molecular basis for gating of SoPIP2;1: Ser36 involved in gating ?
• Mutants are being tested in the lab.
Future Research• Mechanical properties of free standing and solid-
supported lipid bilayers (with and without protein)
• Effect of a pressure gradient on permeation dynamics, and on supported bilayer properties
ΔP
Courtesy: Peter Holme Jensen, Aquaporin
Acknowledgements
• Ole G. Mouritsen, the director of MEMPHYS
• MEMBAQ, for the funding
• DCSC, for the supercomputing resources.
• Urban Johanson and Per Kjellbom (collaborators at LUND, Sweden)
Molecular Basis for Increased Permeability: Role of L263, V264
Molecular Basis for Increased Permeability: Summary
E.coli AqpZ 2.5 A,Savage et. al, PloS Biology, 2003
E.coli GlpF, 2.2 A,Fu et. al, Science, 2000
Half-membrane spanning repeats
Selectivity Filter:
W48, G191, F200, R206, (GlpF)
F43, H174, T184, R189, (AqpZ)
Conserved NPA motifs
Aquaporin Architecture
Water in single file~20 A constriction
Only water transport Glycerol too
Single Channel Permeability
Simulation pf (cm3/s) x 10-14 pd (cm3/s) x 10-14 <L> (Å)
CLOSED-POPC 0.33 0.07 22.25
CLOSED-POPE 0.41 0.08 22.72
OPEN-POPC 0.68 0.171 22.06
OPEN-POPE 0.73 0.26 22.29
Unpublished data