Download multiscale modeling of the organizations of receptor transmembrane

MULTISCALE MODELING OF THE ORGANIZATIONS
OF RECEPTOR TRANSMEMBRANE DOMAINS
IN LIPID MEMBRANES
Wei Dai, Manolis Doxastakis
Dept. of Chemical & Biomolecular Engineering
University of Houston
ECD
TMD
ICD
Jura et al., Mol. Cell
(2011)
• Membrane proteins 25% but few structures
characterized
• Signal transduction pathways; Key cellular
process
• Increasing evidence that TMD provide critical interactions
Finger et al., Sci. Signal (2010); Moore et al., Structure, (2008)
• Hypothesis: multiple states that are separated by weak free energy barriers
• Active role of TMD supports that membrane composition is also a factor
Pike., Biochemistry (2002)
• Wealth of indirect experimental evidence as FRET, crosslinking, mutations,
but indirect.
Polyansky et al., J.Am.Chem.Soc.
(2012)
• Recent simulation studies support TMD interactions
Janosi et al., Biophys. J. (2010); Prakash et al.,
Biophys. J. (2010)(2011)
• Amino acid sequence – membrane
composition
• Spatial and temporal limitations
rotations at (O)(10-6) -- (O)(10-4) s
Rigby et al., Biochemistry (1996);
Sharpe et al., Biochemistry (2002)
de Meyer et al., Biophys. J. (2010)
Glycophorin A (GPA)
• Mesoscopic protein models in complex
environments
• Implicit membrane models
• Coarse-Grained models with a level of
amino-acid specificity
Marrink et al., J. Phys. Chem. (2007)
MacKenzie et al., Science (1997)
• Parallel Monte Carlo simulations with
hundreds of unconstraint pairs
algorithm
Janosi et al., J. Chem. Phys. (2009);
Janosi et al., Biophys. J. (2010)
• GpA in single component bilayer
 Free energy of dimerization
-7 to -9 kcal/mol
 Lipid induced interactions significant at
short separations
 Prediction of TMD dimer in excellent
agreement to experimental data
• ErbB family TMDs
• ErbB2 in DPPC + Cholesterol
 Cholesterol alters association pathway
Prakash et al., Biophys. J. (2010) (2011)
DPPC
• Cholesterol promotes dimerization
Anbazhagan and Schneider,
Bioch. Biophys. Acta, (2010)
• Decreased association affinity in
mammalian membranes (~30%
cholesterol) , -3.9 kcal/mol
Single
component
+30% chol
Chen et al., J. Am. Chem. Soc., (2010)
• Dramatic destabilization in certain
lipid mixtures
Single
component
Hong and Bowie., J Am Chem Soc. (2012)
Anbazhagan and Schneider, Bioch. Biophys. Acta, (2010)
E70PEITLIIFGVMAGVIGTILLISYGIR96
• Single GpA helix atomistic molecular
dynamic simulations
(Gromacs, Forcefield Gromos 53a6, 100ns)
• Extract secondary structure which is a
required input in the MARTINI model
 No significant change in helicity
• CG simulations of a single helix:




Monotonic change of thickness
Tilt angle decreases with cholesterol
Significant differences with ErbB2
Minimal response to the change of
thickness
 At low tilt angles protein precession
entropy is significant
Lee and Im, Phys. Rev. Lett. (2008)
• DPPC molecules maintain a similar arrangement
• Cholesterol preferentially locates in proximity
• Less change of lipids distribution around protein, less tilt
angle change
Pure DPPC
10% CHOL
• Cholesterol promotes dimerization of GpA, for 10% and 20% CHOL
• Increase of repulsive barrier of lipids drives rearrangement so that direct
protein-protein increase
• 10% CHOL, contact at PHE78 and ALA82
• Preliminary results support that for 30%, unfavorably lipid-induced
interactions destabilize significantly the dimer
• Reconstruction algorithm: simulate annealing, CG and atomistic structures
are coupled via restraints Rzepiela et al., J.Comput. Chem. (2010)
• Added restraints for hydrogren bonds
Without
Restraints
Restraints
E260EDFYFPWLLIIIFGIFGLTVMLFVFLFSKQQR292
• The growth hormone receptor
(GHR) – class Ⅰ cytokine
receptor
• Important biological functions:
cell division, regeneration and
growth
• Present model: Preformed inactive
dimers. GH binding induces
realignment associated with
increased separation of ICDs &
asymmetric ECD domain
Brooks and Waters, Nature. (2010)
Pang et al., PLoS.Comput.Biol. (2012)
• MC free energy calculations
reveal two different states in
proximity
• Left-handed dimer: increased Cterminal separation asymmetric
tilt angles
• Long AA MD simulations (1 microsecond) in DPPC bilayer
• Cysteine mutations per each amino acid to compare to experimental
crosslinking data
• Crosslinking
parallel interface
inactive
• Single point-mutations
LH
active
• Linking left-handed dimer to
GH:ECD receptor (PDBid:3HHR)
• Generate missing residues by
Modeller
• Supplement our predictions with
homology modeling to build the
complete receptor
Brooks et al, submitted
• Stable in explicit bilayers for
>0.5μs
• Cholesterol promotes dimerization of GpA up to 20%
• New Mechanism for GHR activation : Binding of GH to ECDs induces a
transition from inactive parallel dimer to left-handed crossover dimer.
• Current work:
– General framework for activation of receptors (EPOR)
– Multiscale methodology combines the efficiency of CG model and the
accuracy of AA model
ACKNOWLEDGMENTS
• Prof. M.Waters (Univ. of Queensland).
• National Science Foundation, CBET
• UH Research Computing Center & High Performance Computing.
ACKNOWLEDGEMENTS
• Prof. M.Waters (Univ. of Queensland).
• National Science Foundation, CBET
NSF/CBET 1067356.
• UH Research Computing Center & High Performance
Computing.
• IBM BlueBioU and Rice.
RESEARCH AIM
1. Extends studies in multicomponent membranes
• Recent published experimental data support the active role of
the membrane environment for GpA
2. Multiscale Modeling of TMD association
3. Linking TMD arrangement to function
CONTACT INTERFACE
• In pure DPPC, contact interface GxxxG motif
• 10% CHOL, contact at PHE78 and ALA82
• PHE interaction increases contact interface area, stronger proteinprotein interaction
Contact interface in pure DPPC
GLY79
GLY83
GLY86
Contact interface in 10% CHOL
PHE78
ALA82
EXPERIMENTAL RESULTS
• Asymmetric ECD structure with GH binding (active configuration)
• Proximity of upper TMD leads to activation
Crossing linking before 260EED (CEED) proximity of upper TMD
•
Mutations EED by alanines, activation concomitant to increased separation
of the intracellular sequences
• Crosslinking experiment without GH binding, inactive TMD dimer
interface: with Phe 276 and 283, without GxxG motif
• 150ps umbrella COM pulling for
each configuration – average
force
• 32 configurations for each separation,
moving average for all configuations,
decreased standard deviation
• CG model softer lipids, AA more
repulsive forces
-41kJ/mol/nm
-187kJ/mol/nm