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

touch

gravity

balance

cardiovascular regulation

Membrane tension and osmotic pressure

Osmotic downshock

H20 H20

Membrane tension

increases

K+, osmo- protectants

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

85 Ao

35 Ao

50 Ao

K31 Mutations in E.coli

• K31D and K31E slow the growth of E. coli

• The effect is partially mollified by high-osmolarity 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.

Mapping of mutations onto MscL structure

Yellow: Very severe Ala20, Val21, Gly24, Thr28.

Green: Severe Leu17.

Helix Mutations

Hydrophobicity of a Key Residue Controls Gating

Yoshimura et al., Biophys. J. 77, 1998.

Gly22 was mutated to all other 19 amino acids The hydrophobicity of the mutant residue modulated the gating threshold, which affected the growth rate.

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 GatingPatch-clamp experiments relate membrane tension T to channel open probability P0:

P0 = 1/[1+exp(E-TA)/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(T), 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

Red: TM1 Blue: TM2 Gray: N-terminus Pink: C-terminus

Side View

Top View

85 Ao

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 DimensionsThe periodic cell flattened and widened while maintaining a nearly constant volume. Mainly due to electrostatics.

RMSD from initial structure

All C

C of TM helices

RMS fluctuations for C atoms

• Removed center-of-mass translation from helix C

• 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