Simulating Membrane Channels - University of Illinois at · PDF file ·...

Simulating Membrane Channels -Transport in Aquaporins

12/9/2004

Emad Tajkhorshid 1

Simulating Membrane Channels

Part II. Structure-FunctionRelationship and Transport in

Aquaporins

Theoretical and Computational BiophysicsDec 2004, Boston, MA

http://www.ks.uiuc.edu/Training/

Analysis of Molecular DynamicsSimulations of Biomolecules

• A very complicated arrangement of hundreds of groupsinteracting with each other

• Where to start to look at?

• What to analyze?

• How much can we learn from simulations?

It is very important to getacquainted with your system


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AquaporinsMembrane water channels

Lipid Bilayer Permeability

Water is an exception:•Small size•Lack of charge•Its high concentration


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Water Transport Across CellMembrane

• Diffusion through lipid bilayers

slower, but enough for many purposes

• Channel-mediated

Large volumes of water needed to be transported

(kidneys).

Fast adjustment of water concentration is necessary

(RBC, brain, lung).

Always passive; bidirectional; osmosis-driven

(Animal)

(Animal)

(Bacterial)

(Bacterial)

(Bacterial)

(Bacterial)

(Bacterial)

(Plant)

(Yeast)

(Yeast)

(Yeast)

(Plant)

(Plant)

(Animal)

The Aquaporin Superfamily

Watertransport

Heymann and Engel News Physiol. Sci. 14, 187 (1999)

Glyceroltransport

Discoveredin 1992


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Aquaporins inHuman Body

lenstears

brain

salivary glands

lung

kidney

red blood cells

Aquaporin-10

Fluid balance of thelens

Eye: lens fiber cellsAquaporin-o

LeukocytesAquaporin-9

Testis, pancreas, liverAquaporin-8

Testis and spermAquaporin-7

Very low waterpermeability!

KidneyAquaporin-6

Production of salivaProduction of tears

Salivary glandsLacrimal glands

Aquaporin-5

Reabsorption of waterCSF fluid balanceO smosensingfunction?Bronchial fluidsecretion

Kidney: collecting ductsBrain: ependymal cellsBrain: hypothalamusLung: bronchialepithelium

Aquaporin-4

Reabsorption of waterSecretion of water

Kidney: collecting ductsTrachea: epithelial cells

Aquaporin-3

ADH hormone activityKidney: collecting ductsAquaporin-2

O smotic protectionConcentration of urineAqueous humorProduction of CSFAlveolar hydration

Red blood cellsKidney: proximaltubulesEye: ciliary epitheliumBrain: choriod plexusLung: alveolarepithelial cells

Aquaporin-1

Additional members are suspected to exist.

Kidney

Nephron

Cortex

Collecting ductProximal tubules

Collecting ductlumen

Extracellularfluid

ADH

AQP3

AQP2

Proximal tubulelumen

Extracellularfluid

AQP0AQP0

urineconcentration

•reabsorptionof water intoblood

•ADH activity

Aquaporins inthe Kidney


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Proximal tubulelumen

Extracellularfluid

AQP0AQP0

Collecting ductlumen

Extracellularfluid

ADH

AQP3

AQP2

>200 LitersWater

Everyday!

High Permeation to Water

Nephrogenic diabetes insipidus

apicalBasolateral apicalBasolateral

Monomeric poresWater, glycerol, …

Tetrameric porePerhaps ions???

Aquaporins Aquaporins of known structure:of known structure:GlpF – E. coli glycerol channel (aquaglycerolporin)

AQP1 – Mammalian aquaporin-1 (pure water channel)AqpZ and AQP0 (2004)


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Fu, et al, Science 290, 481 (2000)

N C

Architecture of the Channel

Channel Fold

NPAN- -CNPARE E

RMSD 1.3 Å

Internal gene duplication

Fu, et al, Science 290, 481 (2000)


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• Tetrameric architecture• Amphipatic channel interior• Water and glycerol transport• Protons, and other ions are

excluded• Conserved asparagine-proline-

alanine residues; NPA motif• Characteristic half-membrane

spanning structure

~100% conserved -NPA- signature sequenceNPA NPARN CE E

Functionally ImportantFeatures

A Semi-hydrophobic channel

Fu, et al, Science 290, 481 (2000)


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Complementarityglycerol molecule channel

hydrophobicacceptor

donor

A Semi-hydrophobic channel


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Molecular Dynamics SimulationsProtein: ~ 15,000 atomsLipids (POPE): ~ 40,000 atomsWater: ~ 51,000 atomsTotal: ~ 106,000 atoms

NAMD, CHARMM27, PME

NpT ensemble at 310 K

1ns equilibration, 4ns production

10 days /ns – 32-proc Linux cluster

3.5 days/ns - 128 O2000 CPUs0.35 days/ns – 512 LeMieux CPUs

Protein Embedding in Membrane

Ring ofTyr and Trp

Hydrophobicsurface of the

protein


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Embedding GlpF in Membrane

77 A

122 A

112 A


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A Recipe for Membrane Protein Simulations• Insert your protein into a hydrated lipid bilayer.

• Fix the protein; minimize the rest and run a short“constant-pressure” MD to bring lipids closer to theprotein and fill the gap between the protein and lipids.

• Watch water molecules; if necessary apply constraintsto prevent them from penetrating into the open gapsbetween lipids and the protein.

• Monitor the volume of your simulation box until it isalmost constant. Do not run the system for too longduring this phase.

• Now release the protein, minimize the whole system,and start an NpT simulation of the whole system.

• If desired, you may switch to an NVT simulation, whenthe system reaches a stable volume.

Lipid-Protein Packing During theInitial NpT Simulation


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Adjustment of Membrane Thickness tothe Protein Hydrophobic Surface

Stability of NPA – NPA Interaction

An extremely stable protein


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Glycerol-Saturated GlpF


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Description of full conduction pathway

Complete description of the conduction pathway

Selectivityfilter

Cons

triction

reg

ion


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Details of Protein-SubstrateInteraction Are Always Important

• Identify those groups of the protein that aredirectly involved in the main function of the protein.

• Look at the interaction of these primary residueswith other groups in the protein.

• Look at buried charged residues inside the protein;they must have an important role.

• Backbone hydrogen bonds are mainly responsible forstabilization of secondary structure elements in theprotein; side chain hydrogen bonds could befunctionally important.

Channel Hydrogen Bonding Sites

…

{set frame 0}{frame < 100}{incr frame}{

animate goto $frameset donor [atomselect top“name O N and within 2 of(resname GCL and name HO)”]lappend [$donor get index] list1set acceptor [atomselect top“resname GCL and name O andwithin 2 of (protein and name HN HO)”]lappend [$acceptor get index] list2

}

…


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GLN 41 OE1 NE2TRP 48 O NE1GLY 64 OALA 65 OHIS 66 O ND1LEU 67 OASN 68 ND2ASP 130 OD1GLY 133 OSER 136 OTYR 138 OPRO 139 O NASN 140 OD1 ND2HIS 142 ND1THR 167 OG1GLY 195 OPRO 196 O

LEU 197 OTHR 198 OGLY 199 OPHE 200 OALA 201 OASN 203 ND2

LYS 33 HZ1 HZ3GLN 41 HE21TRP 48 HE1HIS 66 HD1ASN 68 HD22TYR 138 HNASN 140 HN HD21 HD22HIS 142 HD1GLY 199 HNASN 203 HN HD21HD22ARG 206 HE HH21HH22


GLN 41 OE1 NE2TRP 48 O NE1GLY 64 OALA 65 OHIS 66 O ND1LEU 67 OASN 68 ND2ASP 130 OD1GLY 133 OSER 136 OTYR 138 OPRO 139 O NASN 140 OD1 ND2HIS 142 ND1THR 167 OG1GLY 195 OPRO 196 O

LEU 197 OTHR 198 OGLY 199 OPHE 200 OALA 201 OASN 203 ND2

LYS 33 HZ1 HZ3GLN 41 HE21TRP 48 HE1HIS 66 HD1ASN 68 HD22TYR 138 HNASN 140 HN HD21 HD22HIS 142 HD1GLY 199 HNASN 203 HN HD21HD22ARG 206 HE HH21HH22



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The Substrate Pathwayis formed by C=O groups

Non-helical motifsare stabilized bytwo glutamateresidues.

NPA NPARN CE E

The Substrate Pathwayis formed by C=O groups


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Conservation of Glutamate Residue inHuman Aquaporins

Glycerol – water competition forhydrogen bonding sites


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Revealing the Functional Roleof Reentrant Loops

Potassium channel

Single Glycerol per channel


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Note that glycerols moved, but notas extensively as earlier!

We need toenforce an entireconduction event.

initial state

final (1ns) state

constant velocity (30 Å/ns)

constantforce

(250 pN)

Steered Molecular Dynamics


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Trajectory of glycerol pulled by constant force

SMD Simulation of Glycerol Passage

Evidence for Stereoselectivity of Glycerol

Cannot be verified by experimental measurements

H

HH

H

HOH

OHOHHO

HOHO

H

HH

HH


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Free Energy Calculation in SMD

Free energy

displacement

applied force

SMD simulationa non-equilibrium process

One needs to discountirreversible work

TkWTkG BB ee// !"!

=

Jarzynski, PRL 1997Hummer, PNAS, JCP 2001Liphardt, et al., Science 2002

WG !"

4 trajectoriesv = 0.03, 0.015 Å/psk = 150 pN/Å

Constructing the Potential of Mean Force

])([)( 0 vtztzktf !!!=

! ""=t

0)()( tvftdtW


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• Captures major features of the channel• The largest barrier ≈ 7.3 kcal/mol; exp.: 9.6±1.5 kcal/mol

Features of the Potential of Mean Force

phosphorylation

Asymmetry of thePotential of Mean Force

Asymmetric Profile in the Vestibules

Periplas

m

Cyto

plas

m


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MaltoporinOmpF GlpF AQP1

Assymetric structure; biological implication?

Asymmetric structure of maltoporin


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Glycerol-Free GlpF

Water permeation

7-8 watermolecules ineach channel

5 nanosecondSimulation

18 water conducted In 4 monomers in 4 ns

1.125 water/monomer/nsExp. = ~ 1-2 /ns


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Correlated Motion of Water in the ChannelWater pair correlation

The single file ofwater molecules ismaintained.


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The single file ofwater moleculesis maintained.

Correlated Motion of Water inthe Channel

Experimental value for AQP1: 0.4-0.8 e-5

One dimensional diffusion: 2

02 ( )t

Dt z z= !

Diffusion of Water in the channel


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Diffusion of Water in the channel2

02 ( )t

Dt z z= !

0 1 2 3 4Time (ns)

Improvement of statistics

Density of O and H atoms along the GlpF channel


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Water Distribution in Aquaporins

Water Bipolar Configuration in Aquaporins


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Water Bipolar Configuration in Aquaporins


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One of the most useful advantages ofsimulations over experiments is thatyou can modify the system as youwish: You can do modifications thatare not even possible at all in reality!

This is a powerful technique to testhypotheses developed during yoursimulations. Use it!

R E M E M B E R:

Electrostatic Stabilization ofWater Bipolar Arrangement


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H+H

H

O H

H

O H

H

O

H

H

O H

H

O H

H

OH+H+

H+

H+H+

H+

H+

H+H+H+

H+H

H

O H

H

O H

H

OH+

H+H+

Proton transfer through water

K+ channel

Cl- channel

Aquaporins


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Proton Blocking by a GlobalOrientation Mechanism


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Klaus Schulten

Emad Tajkhorshid

Morten Jensen

Sanghyun Park

Fangqiang Zhu

Paul Grayson

Simulating Membrane Channels - University of Illinois at · PDF file ·...

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