1. p7 folding and its interaction with amantadine Chee Foong Chew Structural Bioinformatics & Computational Biochemistry Unit Department of Biochemistry, University of Oxford

2. The HCV life cycle Adapted from www.tibotec.com

3. The HCV life cycle p7 monomer TM1 TM2 cytosol lumen Hexamer? Heptamer?

4. What is Molecular Dynamics? Describe the forces on all atoms: –bonded (bonds, angles, dihedrals) –non-bonded (van der Waals, electrostatics) Describe the initial atom positions: Integrate: F = ma (a few million times…) Result: positions and energies of all atoms during a few microseconds

5. Bond & Sansom, J. Am. Chem. Soc. (2006) in press. Main chain particle (1 per residue) Protein-optimised bond lengths & angles, particle type defined by H- bonds - N0/Nd/Nda Weak harmonic distance restraints between H-bonded atoms (0.4-0.6 nm for  -strand, 0.55-0.65 nm for  -helix) Bond & Sansom, J. Am. Chem. Soc. (2006). Side chain particle (1-2 per residue) Summary: ►Ala/Leu/Ile/Val/Pro=C; Ser/Thr=P ► Cys/Met=N0, Asn/Gln=Nda ► Asp/Glu=Qa, Lys/Arg=C+Qd ► Phe=C+C, Tyr=C+Nd, Trp=Nd+C, His=C+Nda water +ve ion -ve ion Adapting Lipid CG Model for Proteins Marrink et al, J. Phys. Chem. B (2004) 108, 750. ~4:1 heavy atom:particle mapping, 4 particle types – polar (P), mixed (N), hydrophobic (C) & charged (Q). Weak bonds/angles. Shifted Coulomb potential, dielectric (  =20) for Q. Particles interact via shifted LJ potential (5 levels), tuning for H-bonds lipid P Q+ Q- C N Q

6. PC-rich lipid mixture PE-rich lipid mixture 0 ns 2 ns 10 ns Results

7. Prediction via CG Simulations: p7 CG simulations of folding & insertion of 2 helix model into a bilayer All simulations 5 x 2 µs 2 TM helix hairpin in POPE rich bilayers & in DPC micelles Incomplete insertion of 2 nd helix in DOPC rich bilayers P7 in POPE:POPC 4:1bilayer p7 in POPE:DOPC ( 4:1 ) bilayerp7 in POPE:DOPC ( 1: 4 ) bilayer

8. PE-rich bilayer Changing the lipid environment alters the electrophysiological behaviour of p7 PC-rich bilayer

9. PE rich vs PC rich environments: Synchrotron CD p7 in TFE: 82%  -helical, 3% strand, 7% turns, 6% unordered p7 in POPE:DOPC (4:1): 76%  -helical, 2% strand, 11% turns, 11% unordered p7 in DOPC:POPE (4:1): Similarity to NB-influenza

10. p7 (a) PC rich PE rich Griffin et al p7 (b) Simulation studies p7 folding model N N N N N C C C C C

11. X 6 pore X 6 pore (a) (b) p7 oligomer ??? N C N C

12. Amantadine oldest drug on the market to treat flu “Channel blocker” for M2, p7, NMDA receptors anticholinergic (side effects – anxiety, insomnia, difficulties in concentrating) on-going clinical trials on Hepatits C virus (Triple cocktail- interferon,ribivirin,amantadine)

13. Amantadine and lipids Neutron and X-ray scattering suggest interfacial location EPR data (but spin label almost the same size as amantadine itself) NMR (using fast-tumbling bicelles) –But quantified distribution of amantadine in the bilayer difficult Uneven distribution of lipid protons Overlapping methylene resonances Use of diffusion coefficients to determine Kp

14. How does amantadine block channel? Cork in the bottle From the interface Does it interact with the individual monomer?

15. PMF and Umbrella Sampling U i = internal energy k B = Boltzmann constant T = temperature PMF utilize the partition coefficient relationship to obtain the Gibbs free energy:- Thus sampling of high energy states is important – but this is difficult in normal simulations so we add a restraint which is corrected post-simulation. Method originally proposed by Torrie and Valleau (1977):-

16. How does amantadine interact with the lipid? 1.How and where does amantadine interact with the bilayer? 2.How does amantadine permeate through the lipid bilayer? POPC bilayer Restrain at 0.05Å windows – needed to ensure adequate sampling of high energy positions (states) Equilibrate for 200 ps at each step Production of 1 ns per step Use WHAM to move back to Boltzmann Calculate the potential of mean force (PMF) to obtain  G as function of a reaction coordinate (in our case the bilayer normal) using umbrella sampling

17. Permeation of amantadine Yellow=phosphate Green=headgroup carbon Red=oxygen Blue=nitrogen Grey=lipid tails Cyan=amantadine carbon White=hydrogen

18. Interaction with the membrane

19.  G of amantadine in POPC bilayer

20. Agreement with NMR data… z Data from interfacially located windows (ie where  G is at its minimum) Amantadine preferentially occupies the interfacial region Is there a preferred orientation? Recent NMR value is quoted at 30  (Tim Cross’s group)

21. Water Permeation Passage of amantadine accompanied by water wires Waters quickly return back to interface after wire breaks.

22. Amantadine-Lipid Interactions How does the ammonium group interact with the lipid headgroups? Lipid coreinterface

23. Lipid Packing

24. Protonation State of Amantadine Does it cost more free energy to deprotonate and transport that across the membrane than the energy required for the charged species? Experimental pKa is 10.68 =  G of +15 kcal/mol. Barrier at centre of bilayer is removed Recall that protonated amantadine has barrier of 16.05 kcal/mol. Ie. Thermodynamically comparable. Deprotonated amantadine at 310K

25. Change in pKa Can use difference in PMFs (protonated versus deprotonated) to work out the change in pKa.

26. Summary –Channel blocker or channel structure modifier? –Water plays a significant role in the local microstructure around amantadine. –Transport across the membrane would involve large free energies comparable to that required for deprotonation. Large deformation of the lipid membrane possible though.

27. Improvements… Application of transmembrane potential Coarse-grained PMF of amantadine More than one amantadine molecule – are the effects additive? Effect of size of bilayer on PMF Polarizable force-fields

28. Residues critical for infectivity

29. Establishment of a bilayer membrane Incorporation of synthetic p7 peptide ( J. Scheinost, J. Offer, P. Wentworth) Application of a command voltage Electrophysiology experiments

30. N CC N Coming soon….. Which and if any of these give rise to ion channel recordings?

31. Acknowledgements All members of the Structural Bioinformatics Computational Biochemistry Unit, University of Oxford especially Prof Mark Sansom, Dr Philip Biggin,Dr Pete Bond, Chze Ling Wee, Ranjit Vijayan, Dr Kia Balali Mood. Dr Nicole Zitzmann, Thomas Whitfield from Antiviral Drug Discovery Unit, University of Oxford. The Wellcome Trust

32. Email: chee.chew@chch.ox.ac.uk