1. 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

2. Aquaporin: the water pore Peter Agre, JHU 2003 Nobel Prize in Chemistry 50% Periplasm Cytoplasm Transmembrane water transporters

3. Aquaporin 10 9 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?

4. 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 membrane film Built the membrane film into a composite membrane Test the membrane system for real applications simulation Laboratory testing

5. 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 ΔPΔP Courtesy: PH Jensen, Aquaporin

6. MEMBAQ: 9 partners

7. 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 of better aquaporins

8. .. More technical details..

9. SoPIP2;1: Spinach Leaf Aquaporin will 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

10. 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

11. Open and Closed States of SoPIP2;1 Closed Open Closed Open SoPIP2;1 is a GATED water channel

12. Open Closed SoPIP2;1 Courtesy: Urban Johanson, Lund. U

13. OPEN CLOSED D-LOOP BLOCKING THE PORE BY LARGE MOVEMENT Courtesy: Urban Johanson, Lund. U

14. 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

15. Loop DN-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

16. Gating by Ser115 Tornroth-Horsefield et al. Nature, 2006, 439, 688-694

17. 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

18. Simulation Methods and Setups

19. 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

20. 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 ! + -

21. Simulation Setup Tetrameric model of SoPIP2;1 embedded in a fully hydrated (POPE) and/or phosphatidylcholine (POPC) lipid bilayer

22. 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

23. Simulations Implemented CLOSED and OPEN conformations –In POPC or POPE lipid membranes CLOSED mutants to improve Permeability –With POPC membranes

24. Simulations Completed ~ 0.3 μs, 100,000 cpu hours NumberConformation of SoPIP2;1 Lipid TypeSimulation Time (ns) Wild-type 1CLOSEDPOPC41.20 2CLOSEDPOPE39.20 3OPENPOPC33.10 4OPENPOPE36.10 Mutants 1R190A-D191APOPC54.55 2R190A-D191A(2)POPC41.4 3TRUNCPOPC42.6 Simulations run on DCSC

25. Results Single Channel Permeability

26. 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 j W = p f ΔC S Jensen & Mouritsen (2006) Biophys. J., 90, 2270-84

27. Single-channel Permeability Constants Osmotic Permeability (p f ) \]’ Zhu, et al. (2004) Phys. Rev. Lett., 93(22), 224501 www.ks.uiuc.edu

28. Single-channel Permeability Constants Diffusive Permeability (p d ) k 0 = # water molecules that traverse the channel per unit time v w = Molar volume of water Zhu, et al. Phys. Rev. Lett., 93(22), 224501 www.ks.uiuc.edu

29. Single Channel Permeability Simulationp f (cm 3 /s) x 10 -14 (Å) CLOSED-POPC0.3322.25 CLOSED-POPE0.4122.72 OPEN-POPC0.6822.06 OPEN-POPE0.7322.29 Khandelia and Mouritsen, unpublished data

30. Single Channel Osmotic Permeability p f AQP1: 6 x 10 -14 cm 3 /s Khandelia and Mouritsen, unpublished data

31. Low p f of SoPIP2;1 Simulations predict an absolute p f 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 p f (closed)/p f (open) is similar to experimentally measured values Suga and Maeshima (2004): Plant Cell Physiol, 45(7), 823-30

32. Results Influence of Lipid Type

33. Influence of Lipid Type The type of lipid (POPC vs. POPE) should not influence permeability

34. Results Enhancing Permeability of SoPIP2;1 Shifting Conformational Equilibrium towards the OPEN state In collaboration with Prof. Per Kjellbom & Dr. Urban Johanson (Lund University, Sweden)

35. Gating of SoPIP2;1 How 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 ?

36. 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

37. Hedfalk et al. (2006) Curr. Opin. Struct. Biol, 16, 447–456 Enhancing Water Conductivity 2. Longer D-loop in SoPIP2;1

38. 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)

39. Simulations Completed NumberConformation of SoPIP2;1 Lipid TypeSimulation Time (ns) Mutants 1R190A-D191APOPC54.55 2R190A-D191A(2)POPC41.4 3TRUNCPOPC42.6

40. Both Mutants Increase Permeability Khandelia and Mouritsen, unpublished data

41. Molecular Basis for Increased Permeability

42. Mutant R190A-D191AWild Type Molecular Basis for Increased Permeability: Role of Ser36 Only one monomer is shown, ~ 40 ns Ser36 is conserved in PIPs

43. R190-D191A Wild Type Initial Final

44. Molecular Basis for Increased Permeability: Role of Ser36 Khandelia and Mouritsen, unpublished data

45. OPEN CLOSED D-LOOP BLOCKING THE PORE BY LARGE MOVEMENT Courtesy: Urban Johanson, Lund. U

46. 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.

47. 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 ΔPΔP Courtesy: Peter Holme Jensen, Aquaporin

48. 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)

49. Molecular Basis for Increased Permeability: Role of L263, V264

50. Molecular Basis for Increased Permeability: Summary

51. 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

52. Single Channel Permeability Simulationp f (cm 3 /s) x 10 -14 p d (cm 3 /s) x 10 -14 (Å) CLOSED-POPC0.330.0722.25 CLOSED-POPE0.410.0822.72 OPEN-POPC0.680.17122.06 OPEN-POPE0.730.2622.29 Unpublished data