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: [email protected]
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