Detecting small charge movements in biological membrane systems Benoit Roux University of Chicago...

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

€

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.