detecting small charge movements in biological membrane systems benoit roux university of chicago...
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Detecting small charge movements in biological
membrane systems
Benoit RouxUniversity of Chicago
March 2015
Sliding helix
Paddle
Transporter-like
Large movement
Small movement
Long, Campbell & Mackinnon. Crystal structure of a mammalian voltage-dependent Shaker family K+ channel.Science. 309(5736):897-903, 2005.
?
Voltage Gating 101…
OC
side II
side I
Vmp
++
+ ---
side II
side I
Vmp
++
+
---
F. Khalili, V. Jogini, V. Yarov, E. Tajkhorshid, B. Roux, K. Schulten
MD of Full-length Kv1.2 in bilayer in open and closed state
Open Closed
V=0
V
+
Define the excess free energy (or PMF) for the system at X arising from the applied membrane voltage V and averaged over all solvent degrees of freedom Y
The constant field in PBC
Displacement charge
There are 3 routes that can be exploited:
• “W” PMF with & without voltage
• “Qd” Average displacement charge (with or without voltage)
• “G” Free energy of charging with & without voltage
Application of the Q-route to the VSD of Kv1.2
Correlation time is about 10 ns
RMS fluctuations are related to capacitance
Application of the Q-route to the VSD of Kv1.2
F. Khalili, V. Jogini, E. Tajkhorshid, K. Schulten, B. Roux
VSD
Application of the Q-route to the Kv1.2 channel
Khalili-Araghi et al. Calculation of the gating charge for the Kv1.2 voltage-activated potassium channel. Biophys J. 98(10):2189-98, 2010.
Application of the G-route to the Kv1.2 channel
Khalili-Araghi et al. Calculation of the gating charge for the Kv1.2 voltage-activated potassium channel. Biophys J. 98(10):2189-98, 2010.
Na+/K+-pump overview
K+
K+
Na+
Na+
ADP + Pi
ATP
Extracellularmatrix
Cytoplasm
• Membrane transporter
• P-type ATPase
• Actively export 3Na+ and import 2K+ per pump cycle.
• Maintain healthy ion concentration gradients across cell membrane.
• Indispensible for excitable cells such as neurons.
Skou, J. C., Biochim. Biophys. Acta. (1957) 23:394
ForwardPump cycle
E1
E2
Na+/K+-pump overview
Extracellularmatrix
Cytoplasm
• “Alternating-access” pump cycle
Post, R. L. et al, J. Gen. Physiol. (1969) 54:306Gadsby, D. C. et al, Nat. Commun. (2012) 3:669
ForwardPump cycle
Na+/K+-pump overview• Crystal structures available
PDBID: 2ZXE2.4 Å
PDBID: 3WGV2.8 Å
Shinoda, T. et al, Nature (2009) 459:446Kanai, R. et al, Nature (2013) 502:201
Na+/K+-pump overview
Extracellular matrix
Cytoplasm
β
α
β
α
A P
N
A P
N
PDBID: 3WGVNa3
.E1.(ADP.Pi)
PDBID: 2ZXEE2(K2)
• Crystal structures available
ForwardPump cycle
??
Extracellular ion binding• Limited structural information on the P-E2 state
(Ca2) E1~P:ADP
Ca2 E2P:ATP
Ca2 E1:ATP
E2P
Hn E2P:ATP
(Hn) E2 ~P:ATP
Hn E2:ATP
• Modeling the P-E2 state based on Ca2+ SERCA pump
PDBID1VFP
PDBID1WPG
PDBID3B9B
Ca2+ SERCAPump cycle
Extracellular ion binding
Ca2 E1:ATP
E2P
(Hn) E2 ~P:ATP
• Modeling the P-E2 state based on Ca2+ SERCA pump
PDBID1VFP
PDBID1WPG
PDBID3B9B
Ca2+ SERCAPump cycle
Extracellular ion binding
• Models for outward facing, ion loaded Na+/K+ pump
P-E2.Na3 P-E2.K2
Extracellularmatrix
Cytoplasm
Extracellular ion binding
Water filled pathway leading to the binding site
• Simultaneous rebinding of ions from the extracellular side
Extracellular ion binding
Na+ rebinding happens at 30-ns time mark in a 120-ns MD simulation.
Gating charge upon ion binding
Instantaneous displacement charge:
Average displacement charge of a trajectory:
Gating charge:
• Estimating gating charge (ΔQD) from MD trajectories
A 82 Å
B 107 Å
C 155 Å
nAtom ~150K
nPOPC 213
nWater 32K
System information
Gating charge upon ion binding
Triple occupancy Double occupancy Single occupancy Empty sites
• MD systems involved in Na+ release
1/3
1/3
1/3
1/3
1/3
1/3
1 1
Gating charge upon ion binding
Empty sites Single occupancy Double occupancy
• MD systems involved in K+ binding
1/2
1/2
11
Na+ release from P-E2.Na3 K+ binding in P-E2.K2
Calc. 0.56 ± 0.10 0.39 ± 0.07 0.01 ± 0.1 0.49 ± 0.12 0.37 ± 0.20
Exp. 0.61-0.71* ~0.3* 0.46** 0.27**
• Models for outward facing, ion loaded Na+/K+ pumpModelP-E2.Na3
ModelP-E2.K2
Table. Gating charge of ion binding/release from P-E2.
Gating charge upon ion binding
* Holmgren, M. et al, Nature (2000) 403:898**Castillo, J. P. et al, Nat. Commun. (under review)
• Titratable residues at crystal structure ion binding sites
αM4αM5
αM8αM6
αM9
αM4αM5
αM8αM6
αM9
IIIIII
III
PDBID: 3WGVNa3
.E1.(ADP.Pi)
PDBID: 2ZXEE2(K2)
RMSDsiteHA = 3.0 Å
Ion binding site protonation state
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exp −ΔGab kT[ ] = dX exp[−Ub /kT]∫dX exp[−Ua /kT]∫
= dX exp[− Ub −Ua( ) /kT] exp[−Ua /kT]∫
dX exp[−Ua /kT]∫
= exp[− Ub −Ua( ) /kT](a)
Gb - Ga = − kT ln exp[− Ub −Ua( ) /kT](a)
⎛ ⎝ ⎜
⎞ ⎠ ⎟
Free Energy Perturbation
Difference in Hydration Free Energy
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GNa −GK = - kT ln exp[−UNa −UK( ) / kT ](K)
⎛ ⎝ ⎜
⎞ ⎠ ⎟
-17.2 kcal/mol
K+ Na+
FEP/MD simulations
Selectivity of the Na+/K+ Pump in state E2P
System Setup Site I Site II
2ZXE: 334,786,811,815 -2.5 -2.7
3B8E: 327,779,804,808 -1.7 -4.5
The sites are selective for Na+ over K+ ?????!!!!!!!
Yu H, Ratheal IM, Artigas P, Roux B. Protonation of key acidic residues is critical for the K⁺-selectivity of the Na/K pump. Nat Struct Mol Biol. 18(10):1159-63, 2011.
Selectivity of the Na+/K+ Pump in the state E2P
System Setup Site I Site II
2ZXE: 334+,786+,811,815+ +4.0 +4.6
3B8E: 327+,779+,804,808+ +3.0 +1.7
2ZXE: 334+,786+,811,815 -0.4 +3.5
2ZXE: 334,786+,811,815+ -0.8 -7.7
2ZXE: 334,786,811,815 -2.5 -2.7
3B8E: 327,779,804,808 -1.7 -4.5
Na+/K+ pump can modulate the local electrostatic environmentof the binding sites to shift the pKa values of the residuesso that it can achieve K+ selectivity at the E2P state.