p7 folding and its interaction with amantadine

Chee Foong Chew

Structural Bioinformatics & Computational Biochemistry Unit

Department of Biochemistry, University of Oxford

The HCV life cycle

Adapted from www.tibotec.com

The HCV life cycle

p7 monomer

TM1 TM2

cytosol

lumen

Hexamer?

Heptamer?

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

• 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

PC-rich lipid mixture

PE-rich lipid mixture

0 ns

0 ns

2 ns

2 ns

10 ns

10 ns

Results

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 2nd helix in DOPC rich bilayers

P7 in POPE:POPC 4:1bilayerp7 in POPE:DOPC ( 4:1 ) bilayer p7 in POPE:DOPC ( 1: 4 ) bilayer

PE-rich bilayer

Changing the lipid environment alters the electrophysiological behaviour of p7

PC-rich bilayer

PE rich vs PC rich environments: Synchrotron CD

p7 in different lipid environments

-40

-20

0

20

40

60

80

100

190 200 210 220 230 240 250 260

wavelength (nm)

MR

E

• 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

p7(a)

PC rich

PE rich

Griffin et al

p7(b)

Simulation studies

p7 folding model

NN N

N N

C

C

C

C

C

X 6

pore

X 6

pore

(a)

(b)

p7 oligomer ???

N C

N

C

AmantadineNH3+

• 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)

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

How does amantadine block channel?

• Cork in the bottle

• From the interface

• Does it interact with the individual monomer?

PMF and Umbrella Sampling

levelsall

i B

i

Tk

UQ

_

1

exp

Ui = internal energykB = Boltzmann constantT = temperature

PVQTkG B ln

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):-

WBN

wBNN

TkrW

TkrWrAA

/exp

/exp

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

Permeation of amantadine

Yellow=phosphateGreen=headgroup carbonRed=oxygenBlue=nitrogenGrey=lipid tailsCyan=amantadine carbonWhite=hydrogen

Interaction with the membrane

G of amantadine in POPC bilayer

Agreement with NMR data…

zData 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)

Water Permeation

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

Amantadine-Lipid Interactions• How does the ammonium group interact with the lipid headgroups?

Lipid coreinterface

Lipid Packing

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

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

change in pKa.

-2

-2

-1

-1

0

1

1

2

2

-4 -3 -2 -1 0 1 2 3 4

Z coordinate /nm

Ch

ang

e in

pH

un

its

Summary

• 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.

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

Residues critical for infectivity

V command voltage

current measurement

cis trans

• Establishment of a bilayer membrane

• Incorporation of synthetic p7 peptide (J. Scheinost, J. Offer, P. Wentworth)

• Application of a command voltage

Electrophysiology experiments

N

C C

N

Coming soon…..

Which and if any of these give rise to ion channel recordings?

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

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