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 !

2

2i

i i

dE m

dt

r2

01,2

( )bond stretch bpairs

E K b b

i kelectrostatic

nonbondedpairs

q qE

D

ikr

+ -

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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)()(

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tCRi

ii

t

ttztztL

dttn

ttnDn 2/)(2

nwf Dvp

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