Transcript Wednesday: Lecture 1

Molecular Dynamics Studies of
the Gating Mechanism of a
Mechanosensitive Ion Channel
Molecular Dynamics Simulation
of MscL in POPC Membrane
Overview
• What are mechanosensitive (MS) channels,
where are they found, and why are they
interesting?
• What has been learned about MS channels
already?
• How can molecular dynamics help us
understand MS channels?
MS channels are ubiquitous
• Eukaryotes: Mid1 gene in yeast (Kanzaki et
al, Science (1999), v285, 882-886.
• Mammals: TRAAK (Maingret, JBC 274,
1999.
• Haloferax volcanii, a halophilic archaeon.
• Prokaryotes: MscL in E. coli,
Mycobacterium tuberculosis, many others.
Biological Roles of MS Channels
hearing
balance
touch
gravity
cardiovascular regulation
Membrane tension and osmotic
pressure
Osmotic
downshock
H20
Membrane
tension
increases
H20
K+, osmoprotectants
excreted
Physical Properties of Lipid Bilayers
• Diverse lipid
composition –
differences in
lateral tension
profile
• Elastic modulus
much higher than
either shear or
bending modulus
Figure from Voet & Voet
How Cells Sense Pressure
• Most eukaryotic MS channels require
coupling to the cytoskeleton and/or the
extracellular matrix (Sachs and Morris,
1998).
• Bacterial MscL is functional in
reconstituted lipid bilayers (Sukharev et al.,
1994).
Discovery and Isolation of MscL
• MS channels discovered in chick skeletal
muscle cells (Guharay and Sachs, 1984)
• Patch-clamp studies of E. coli revealed
three MS activities: MscL, MscS, and
MscM
• MscL identified as a 17-kD protein, and the
corresponding mscl gene cloned (Sukharev
et al., 1994)
Physiological role of MscL
• MscL is an emergency safety valve for bacteria –
the line of last defense against cell lysis.
• MscL gates at tensions approaching the membrane
rupture tension.
• Of the three E. coli Msc channels, MscL has the
highest conductance and allows the largest solutes
to pass through, without resulting in lysis of the
cell.
Prokaryotic MscL Homologs
• High degree of
conservation in
primary sequence,
especially in
transmembrane
helix regions.
Chang et al., Science, v282, 1998, pp 2220-2226.
Pentameric Structure of MscL
o
85 A
o
35 A
o
50 A
K31 Mutations in E.coli
• K31D and K31E slow the growth of E. coli
• The effect is partially mollified by highosmolarity solvent.
• These mutants retain less K+, and mutant
channels open at lower tension.
• Comparison of whole-cell and patch-clamp
results indicates role of membrane potential.
Gain-of-function Mutations
Ou et al, PNAS v95, pp. 11471-11475, 1998.
Helix Mutations
Mapping of mutations
onto MscL structure
Yellow: Very severe
Ala20, Val21, Gly24,
Thr28.
Green: Severe
Leu17.
Hydrophobicity of a Key Residue
Controls Gating
Gly22 was mutated to all other 19 amino acids The hydrophobicity of
the mutant residue modulated the gating threshold, which affected the
growth rate.
Yoshimura et al., Biophys. J. 77, 1998.
Patch-clamp calibration of MscL
• Measure conductance vs. pressure of MscL in lipid patch
• Relate pressure to tension via T = p X r/2
Free Energy Changes in Gating
Patch-clamp
experiments relate
membrane tension
T to channel open
probability P0:
P0 = 1/[1+exp(DETDA)/kBT].
But discrepancies
with kinetic data
indicate that this is
not a two-state
system!
Single channel current measurements
Multiple Conductance States in MscL
• Model single-channel kinetics
using linear sequential model:
C1-S2-S3-S4-O5
• Measure rate as a function of
membrane tension: k = k0
exp(aT), where a is the tension
sensitivity.
Summary of calibration results
• Rate limiting step is k12, for which the energy
barrier is 38 kBT.
• All states > S1 have about the same energy and are
insensitive to tension.
• Calculated cross-sectional area (based on
conductance measurements and assuming a 4 nm
channel thickness) gives a channel cross-section of
2.7-3.6 nm, hence an outer diameter of 5.5-6 nm.
Molecular Dynamics Simulation
of MscL in POPC Membrane
Side View
Red: TM1
Blue: TM2
Gray: N-terminus
Pink: C-terminus
Top View
o
85 A
System Setup
• Protein structure from PDB (entry 1msl).
• Residues 1-9 were disordered in the crystal
structure; these were not modeled.
• POPC membrane from previous (constant volume)
simulation by Heller (1993); membrane was
‘squared off’ and the protein was inserted in the
middle.
• Equilibrated water box added for solvation.
• Total of 52,473 atoms.
Simulation Protocol
• Molecular dynamics carried using the program NAMD2.
• Force field: CHARMM26, TIP3P water, periodic boundary
conditions, full electrostatics using Particle Mesh Ewald
(PME) summation.
• Dynamics: NpT ensemble using Langevin dynamics /
Langevin piston. Langevin decay time 10 ps-1. Isotropic
pressure of 1 atm in a flexible rectangular cell.
• Equilibration for 1 ns at 310 K with velocity reassignment,
followed by 2 ns of Langevin dynamics.
Unit Cell Dimensions
The periodic cell
flattened and widened
while maintaining a
nearly constant
volume. Mainly due
to electrostatics.
RMSD from initial structure
All Ca
Ca of TM helices
RMS fluctuations
for Ca atoms
• Removed center-ofmass translation
from helix Ca.
• Largest fluctuations
in loop regions.
RMS fluctuations for TM helices
RMS fluctuations for helices taken separately
TM1 (residues 15 to 43
TM2 (residues 69 to 89)
Future Simulation Work
• Model the N-terminus residues – amphipathic
helix?
• Analyze lipid-protein interactions
• Analyze correlated equilibrium motions of the
protein (normal mode analysis, principle
component analysis, singular value
decomposition)
• Measure water accessibility of helix residues
Modeling Challenges
• Large-scale conformational changes are too
slow to observe in normal MD simulations.
• Multi-scale analysis: implicit water, implicit
lipid bilayer.
• Role of the membrane potential
Acknowledgements
• Justin Gullingsrud and Dorina Kosztin
• Computational resources: National Center
for Supercomputing Applications,
Pittsburgh Supercomputing Center
• Figures created using VMD and Tachyon