Ion Transit Pathways and Gating in ClC 1H 2Cl Antiporters

1
Ion Transit Pathways and Gating in ClC 1H/2Cl-
Antiporters
Thomas L. Beck Department of Chemistry University
of Cincinnati thomas.beck_at_uc.edu
Acknowledgments
NSF AFOSR DoD/MURI
University of Pittsburgh
People
John Cuppoletti, Danuta Malinowska Rob Coalson,
Guogang Feng Achi Brandt, Jian Yin, Zhifeng
Kuang, Uma Mahankali, Anping Liu, David
Rogers, Nimal Wijesekera, Lawrence Pratt, Mike
Paulaitis
Army Research Office
2
Mammalian Acid secretory mechanism
Stomach (106 proton gradient)
Pump
K
Cl- (hClC-2)
H
pH sensor
C terminus
Cell
K
H/K-ATPase
K channel
Cl channel
3
Bacterial ClC Structure
1H/2Cl- Antiporter (Miller)
Dutzler, et al. (2002)
4
StClC Dimer and hClC-2 models (front)using
Modeller v6.2
StClC X-ray
pH Sensor loop
(Channel, also ClC-0)
hClC-2 model (model3_07AB_BL020001)
hClC-2 model (model3_07AB_BL020002)
5
Sbs
R147
Cl
One pore bClC
1H
?
Sext
E148(neutral or -)(H path) or Cl ion
Scen
Cl
S107,Y445?
15 Ang
H
E203(neutral or -) Miller, divergent paths
Sint
Cl
2Cl-
6

• Bacterial ClC (Miller) and ClC-4,5 (Jentsch,
  Pusch) are H/Cl- antiporters
• ClC-0,2 (etc) are Cl channels
• What alterations switch from transporter to
  channel?
• Millers proposal of degraded transporter?
  channel.

7
Computational Methods for Ion Channels

• Full MD
• Brownian dynamics (hybrid)
• Electrodiffusion Poisson-Nernst-Planck
  (continuum theory, PMF?) Multigrid solvers
• Multiscale MC simulation and gating?
• Homology models
• Transport pathway search (TransPath)
• The potential distribution theorem for ion PMFs
  and pKas quasi-chemical theory

Length and time scales? ? Multiscale methods for
solving PDEs or simulation of large-amplitude
molecular motions (folding)
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TLB Rev. Mod. Phys. 72, 1041 (2000)
Multiscale Methods Multigrid V-cycle
MG accelerates convergence by decimating error
components with all wavelengths!
2 relaxations per level
Correct, relax
Restrict, relax
Coarsen
FMG
Same principle for polymers ? Hc
9
Molecular Coarsening Example
Modeling of large loop conformational transitions
with Monte Carlo simulations
Coarse point Center of gravity of 4 fine
points, e.g.
Hc on coarse scale ? PMF
Bai and Brandt (2001)
Fine scale corrections?
10
The Potential Distribution Theorem a theory for
m
Ideal
Excess ? r(r)
Uncoupled
Probability of observing interaction energy e,
fully coupled
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The chemical potential is a very (the most?)
important thermodynamic variable for chemical
phenomena

• Chemical equilibrium
• Solute partitioning
• Phase equilibrium
• Free energy of association (drug binding)
• Potential of mean force or free energy profile
  input for dynamical studies
• pKas, acid-base chemistry
• Gradients result in transport, diffusion
• It is a relatively local property (intensive)
• and

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Chemical potential at a point r
Radial distribution function
Potential of mean force (PMF)
Can directly handle multi-ion effects
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• Goal coarse?fine to try to unravel molecular
  driving forces/mechanism in the bacterial ClC
  antiporter
• Path searching and electrostatics
• MD simulations and efficient free energy
  calculations PDT

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Electrostatic Potential Contours of X-ray Dimer
EcClC in water

Using Connolly Surface, grid size
140 (0.5 Angstrom) x 199 x 139
E148 Gate?
Proton path in prokaryotes? 3 acidic residues.
Antiport behavior (Miller). This domain removed
in ClC-0. Recently shown ClC-4, 5 are
antiporters also (Pusch, Jentsch, Nature, 2005).
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New Pathway Search Algorithm TransPath

• Utilize Monte Carlo methods to generate
  transmembrane spanning trajectories
• Incorporate both geometric and electrostatic
  information to bias the random walks
• Once the trajectories are generated, then anneal
  to find the geometric pore center of the
  protopath
• Obtain pore radius and potential profile
• No real Cl ion searches geometry and
  electrostatics
• No prior pore information required
• Searches for transit paths for positive or
  negative ions

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TransPath
OTT mutant (open)
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1OTT Cl Pathway (OPEN)
In
Out
Radius
Potl
S(bs)
R120 Helix D
S(int)
S(ext)
S(cen)
Agrees with proposed MacKinnon pathway
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1OTS closed structure (gating and proton access?)
Green Cl- Purple H Blue H
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pKa shift of Glu148
under different Cl ion binding conditions Chen
and Chen pKa 5.3, i.e. shift of 1
Shifts from roughly 4.3
Sbs
Cl- ion required at Scen for physical pKa of
E148, and possibly at Sbs
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Charge states of E148 affect chloride conductance
Mackinnon (2002, 2003), Glu148 is the fast gate
controlling the chloride conductance. When E148
is protonated, the side chain swings upwards to
open the pore.
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The open state vs. closed state
Closed
Open
E148p
PMF for opening/closing of E148p and E148- ?
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Charge states of Glu148affect chloride
conductance?

• MD Simulation System 100100100 Å3 Cubic,
  Periodic boundary condition

95803 Atoms (206 DMPCs 19275 TIP3s 885 Amino
Acids 24 Na 49 Cl-) Protonation states of
acidic residues were determined by UHBD.
E148neutral at left monomer, charged at right.
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MD Simulation supports Mackinnons observation
E148- (closed)
E148p(opens in simln)
A
B
Sint
Scen
Scen
Sint
Prob. Distr. Of Z locations of Sint and Scen in
monomer A and monomer B.
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Free energy calculation explains the movement of
chloride ions

• Free Energy Calculation (kcal/mol, electrostatic
  part) by MD
• The interaction free energy
• between a single chloride
• ion and the environment
• was calculated when the
• ion has charge of e,
• -0.5e, and 0.0e (see below).

(Single ions?No other Cls)
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Adaptive Biasing Force Simulations of
1OTS(Darve, Pohorille, Chipot, 2001)
Open
Closed

• PMF profile of the dihedral rotation of the
  Glu148 side chain
• The side chain is rotated from the closed
  conformation, 1OTS (dihedral angle CA-CB-CG-CD
  between 70 to 80 degrees) to open conformation,
  1OTU (dihedral angle CA-CB-CG-CD -50 to -60
  degrees)

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Conclusions on pathways and gating

• Glu148 is gate as proposed by MacKinnon, 1OTT and
  1OTU are open relative to wild-type structure
  (recent Chung simulations?)
• Single Cl path (possible alternative on
  intracellular side)
• Multiple proton access paths from external side?
  (NoMiller, proton enters through vestibule)
• External Cl affects gating through binding near
  Arg147
• Cl at bind site shifts Glu148 pKa and alters
  proton path potentials
• Internal Cl resides in selectivity filter,
  required for necessary pKa shift of Glu148
• pH and Cl dependent gating mechanisms ? Chen
• MD simulations, if E148p ? starts to open within
  100 ps

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1H/2Cl- Transporter Interaction Outline

• How do the charge states of Glu148 affect
  chloride conductance (above)?
• How does a proton pass between Glu148 and Glu203?
• How do Cl- and H motions couple to yield
  1H/2Cl- stoichiometry? How does the transporter
  work?

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How does a proton pass from E148 to E203?

• TransPath

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• Two pathways were found

Viewed from dimer interface
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The radius and potential profiles

• Two chloride ions are required for proton
  transport

No Cl ions
P1
Z(Scen) -2.8Ang Z(Sint) -8.4 Ang Z(E203) -9
Ang Z(E148) 1.2 Ang
P2
P1
P2
Add Cls
2Cl
2Cl
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Proton pathway E148?E203?

• Two possible proton paths
• Follow the black dashed line. With the help of
  three water molecules (yellow spheres)
• Hopping along the ring of Tyr445

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• We predict Y445 is involved in proton transport
• Mutate Y445? This has not been done yet

DeCoursey review hydrogen-bonding chains for
proton transport through proteins
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Possible mechanism

• E148 gets protonated due to Cls at Scen and Sbs,
  opens
• Cl at Scen moves up to Sext. Cls at Sext and
  Sbs move out ? 2 Cls exit. Cl fills Scen again,
  Sint occupied
• pKa of E148 shifts downward approx 1 unit due to
  Sext exit, proton released
• Proton attracted towards Scen, interacts with
  Y445, goes directly to E203 (P2) or along water
  chain (P1)
• E203 releases proton, perhaps coupled to nearby
  basic residue
• At some point along this process the E148(-) gate
  residue closes

Net result 2Cl- exit, 1H enters
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Free Energy Perturbation Method by Molecular
Dynamics Simulation

• Free energy methods
• Thermodynamic integration(TI) slow but accurate
• Overlap methods (need overlap of distributions)
• Free energy perturbation(FEP) fast and accurate
  when
• proper states and order are chosen. ( split
  dominant electrostatic part and vdw part)

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Electrostatic part in FEP
Where is a vector with
integer components of is a
convergence parameter is the cubic size
is used for a cubic cell
is the middle point charge and
erfc(x) is the complementary error function.
Can partition mex exactly into vdW and ES parts
(Pratt et al)
(These are 4th ord formulas)
(Hummer et al, us)
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Interaction energy between chloride ion and 256
TIP3 Water, 2ns NAMD
Coupled
Probability

Gaussian model
Energykcal/mol
Lit. -66 to 79 kcal/mol
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Coupled
Uncoupled
P(e) for Na, sampling with full charge and no
charge
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Ion results

Results of ions in SPC water in 4ns molecular
dynamics simulation in (NVT) ensemble. PME and
SHAKE are used in the calculation. Post-analysis
of Ewald electrostatic interaction energy. The
unit is KJ/mol. the number inside brace is the
error estimated by block averages TI results are
from G. Hummer, L. Pratt and A. Garcia, J. Phys.
Chem. 100, 1206 (1996).
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Time effects on Ion simulations
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Molecular results
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Time effects on molecule simulations
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PDTPreliminary results for gA K PMF (ES part)
Condition 1JNO gA dimer in 20DMPC, 2300 TIP3, 19
K-Cl pairs with constraining
mass center and the side-chain of Trp9 in the
rotamer basin at constant pressure
and temperature. 600ps
simulations on each of 3 states. T.W. Allen, O.
S. Andersen and B. Roux, PNAS,104,117-122,2004
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Roux et al 2006
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StClC Dimer and hClC-2 models (front)using
Modeller v6.2
StClC X-ray
pH Sensor loop
hClC-2 model (model3_07AB_BL020001)
hClC-2 model (model3_07AB_BL020002)
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Coarse-graining methods for modeling large
protein loops

• Coarse graining method PMFs
• Simulate on coarse level
• Provide correction back on molecular level
• Interaction between coarse and fine back and
  forth

F
CBMC
PMF
C
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No solvent
Radial distribution function
Potential of mean force (PMF)
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The Distance PMF

• The coarse scale is only physically relevant if
  probability distributions match fine scale.
• Use PMFs
• Sampling distance PMFs is as difficult as system
  rearrangement.
• Sample distance PMFs offline and add as a
  correction to the coarse energy.

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Application to Polypeptides

• T600 K

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Temperature effect on the non-bonded interactions

• for every pair of residues (I,J) at each
  temperature LJ-interaction is derived
• Temperature dependence as important as specifics
  of the residue

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Villin headpiece (1vii.pdb, 36 residues)
Fine scale simulation with PT
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Our results Hansmann
et al
ProteinsStruct,Funct,Gen, 52 436-445(2003)
Total speedup 60 times for 30000 MC
sweeps Speedup with known CG potentials 300
times
J.Chem.Phys. 124,174903 (2006)
Parallel tempering simulations (10 temperatures)
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In progress

• Proton access to the gate and detailed gating
  mechanism of transporter two gates yes E148,
  E203?
• pKas and Cl/H coupling? Quantum/classical
  calculations.
• Experiment/theory test, does mutating Y445
  abolish proton transport?
• What if we took ClC-0 and mutated homologous
  residue to E203? Is it then a transporter?
• Test each step in proposed H pathway/mechanism
  with PDT methods
• Cl ion solvation in water vs. in the channel
  quasi-chemical theory for Cl PMFs and pKas of
  acidic residues. Effects of polarizability?
• PMF studies and CG protein conformational
  changes and folding
• Extracellular loop in ClC-2 pH sensor domain
  conformation charged and neutral in presence of
  channel folding problem

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pH, voltage, Cl dependence of gating?
Chen and Chen, J. Gen. Physiol. 118, 23 (2001)
Extracellular Cl dependence of gating prob (ClC-0)
hi Cl
lo Cl
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pH dependence of gating
5.6
7.1
9.6
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Sbs
P1
P2
P3
E414
pK3.4 epsp20 pK6.2 epsp4
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• Read Pohorille force free energy paper
• Review Hummer 3 pt method
• Review Roux gA PMF