9. vorlesung ws 2004/05softwarewerkzeuge1 v9: protein-protein-wechselwirkung - consurf - russell...

9. Vorlesung WS 2004/05

Softwarewerkzeuge 1

V9: Protein-Protein-Wechselwirkung

- Consurf

- Russell & Aloy Server

- Webseite mit Proteindomänen

- Beispiel zu Protein-Protein Wechselwirkung

Cytochrom c: Cytochrom c Oxidase

Barnase:Barstar

- Klausurvorbereitung


Softwarewerkzeuge 2

Consurf

http://consurf.tau.ac.il/

- degree of conservation at each amino acid site

is inversely related to its rate of evolution.-aim: identify functional regions on protein surface

by mapping degree of sequence conservation

within protein family-Use phylogenetic trees instead of MSA: by a

weighting scheme reduce influence of redundant

sequences


Softwarewerkzeuge 3

InterPreTS

http://www.russell.embl.de/interprets/

Protein-protein interactions are structurally

conserved for > 30% sequence identity.

Predict complexes of A:B based on

sequence homology to A‘ and B‘ when

structure of A‘:B‘ complex is available.


Softwarewerkzeuge 4

V9: Protein-Protein-Wechselwirkung

http://www.mshri.on.ca/pawson/research.html Pawson Lab


Softwarewerkzeuge 5

SH2 domains – universally used protein family



Softwarewerkzeuge 6

Proteins using SH2 domains



Softwarewerkzeuge 7

Binding properties of SH2 domains



Softwarewerkzeuge 8

Binding properties of SH2 domains



Softwarewerkzeuge 9

WW domain

http://www.mshri.on.ca/pawson/research.html

Pawson Lab


Softwarewerkzeuge 10

14-3-3 domain


Pawson Lab



14-3-3 domain


Pawson Lab



Ph

QQ

A

B

QHQ

B

2

ATPH + ADP + P+ 3- 2-

i

plasmamembrane

cytosol

lumen

lightharvestingcomplex

reactioncentre

cytochromeb f -complex

F F complex0 1

6H

+

light

cytochrome c

+2H

+2H 4H

4H+

+

Introduction: Photosynthesis



Docking strategy

No X-ray structure available for complex cyt c552:COX docking.

Flöck, Helms (2002)Proteins 47, 75



Protein-protein docking of cyt c552 and COX

Exercise on model system:

Complex of

yeast Cytochrome c Peroxidase

with iso-1-Cytochrome c

X-ray structure (Kraut et al. 1992)

Heme positions of crystal complex

and 19 best docked and

energy-minimized complexes.

Crystal complex has lowest energy.

Docked complex with second-best

energy has RMSD of only 2.0 Å.Flöck, Helms (2002)Proteins 47, 75



Protein-protein docking of cyt c552 and COX

Superposition of complexes

of Cyt c / Cyt c552 with

COX (bovine)

COX (P.d.)

Additonal loop of bovine COX

collides with c552 (grey).




Protein-protein docking of horse heart cyt c and COX

Best predicted complex of

horse heart cytochrome c with

cytochrome c oxidase from

Paracoccus denitrificans.

Almost identical with best

structure of Roberts et al. for

complex of horse heart

cytochrome c with bovine

cytochrome c oxidase. Docking

with DOT-program (1999).




Two favorable docking positions

Two favorable docking

positions for cyt c552

with COX.

The conformation of

the flexible linker and

of the N-terminal

helical anchor are

fictitous.




kinetic on-rates of protein-protein complexesfrom Brownian Dynamics simulations

McCammon group website (UCSD)



kinetic on-rates: exp data

Mutation kon 106 [M-1s-1] horse heart cyt c : COX

Wild type oxidase 3.7D135N 0.3N160D 2.8

Ionic strength [mM] P.d. cyt c552 : COX

10 4.135 1.5200 0.1

refs: Drousou, Malatesta, Ludwig, Eur J Biochem (2002) 269, 2980Maneg, Ludwig, Malatesta, J Biol Chem (2003) 278, 46734



brownian dynamics details

Electrostatics computed with programsUHBD (McCammon group)APBS (Baker, McCammon, Holst)

Atomistic brownian dynamics simulations with programSDA (Gabdoulline & Wade, 1997)

Simulation parameters:time step 2 ps – 10 pstranslat. diffusion constant 0.02 Å2 ps-1 rotational diffusion constant 4.0 10-5 radian2 ps-1 Radius b 115 ÅRadius c 540 Å

for each system 4 4000 runs

Flöck & Helms, Biophys.J.87, 65 (2004)



Iteration Algorithm

06 6

0 0 0 0

1 1

1/ ,N N

iji i ij i

j ji

Dy y t KT D t R D t

y

jF

1 11 3 1 3,..., , ,...,N NF F T TF

1 1ij ij

D

0

2i

i j ij

R

RR D t

Generalized force vector

Diffusion matrices

Random displacements 0, with:iR D t

Generalized coordinate vector

Dickinson, E., Allison, S.A. and McCammon, J.A. (1985) J. Chem. Soc. Farad. Trans. 2 81, 591

1 11 3 1 3,..., , ,...,N Ny r r



fulfil 1, 2, or 3 contact pairs among

D156:K79 D135:K86

A259:K73 D135:K86

S124:K86 Y122:G84

“naked” wild-type COX : horse heart cyt c 140mM

exp. rate with solubilizedCOX




Association of horse heart cyt c and „naked“ COX at 140mM.

Data for 3 reaction criteria.

Nice agreement with exp. trends!

Simulated mutation effects on on-rates

wild-type COX (3.7)

N160D (2.8)

D135N (0.3)




Simulated ionic strength effects on on-rates

Association of P.d. cyt c552 and „naked“ wild-type COX.

Data for 3 reaction criteria.

Nice agreement with exp. trends!

10 mM (4.1)

35 mM (1.5)

200 mM (0.1)




model of membrane environment




Start diffusion proteins only in

spherical cap above COX.

Inclusion of Membrane Environment

This scheme makes no difference for „naked“

COX:

comparison of original and spherical-cap

starting positions.




association rates with and without membrane environment for

horse heart cyt c : COX at 140 mM

Large effect!

Effect of membrane embedding for horse heart cyt c

„naked“ COX

with membrane potentials from APBS,

UHBD




association rates with and without membrane environment for

P.d. cyt c552 : COX at 140 mM

Association to „naked“ COX slower than for horse heart cyt c.

Small effect of membrane! Physiological relevance?

Effect of membrane embedding for P.d. cyt c552

„naked“ COX

with membrane




Computational studies: gain insight by switching isolated interactions off

all charges on

membrane charges off (cyt c552)

COX charges off (cyt c552)

membrane charges

off (cyt c)

COX charges off

(cyt c)




Protein – protein association

idealized reaction coordinate

G

free diffusion

protein B enters intoprotein A‘s zone of

electrostatic attraction directed diffusion

A and B form„encounter complex“

- electrostatically entangled,- no bound complex

to form complex,interfaces need tobe desolvated +

sidechains oriented

bound complex



Barnase:Barstar complex

extensively studied by Fersht group

barnase is an extracellular ribonuclease,

barstar its intracellular inhibitor

fast binding kon ~ 108 M-1s-1

high affinity kD ~ 10-14 M

binding stabilized by favorable electrostatic

binding energy (Wang et al. 2004)

association rates extensively studied by

Gabdoulline & Wade (1997)

Dong et al. (2003)



Aim of this study

Aim: clarify nature of encounter complex

Means: statistical analysis of brownian dynamics trajectories



Coordinate frame

Center of mass coordinates

of second protein

Rotational coordinates of

second protein



Different definitions of distance variable

For global view

For local view



how does the encounter state look like?

representative structures of the

encounter state ensemble

black balls: reaction atoms of

barstar in crystal structure

barnase

blue: cd1-2

red: cdmin

green: cdavg

purple: cdcenter



Zusammenfassung(1) Charakterisierung von Protein-Protein-Wechselwirkung heutzutage am

besten möglich per wissensbasierten Ansätzen (Sequenzhomologie).

(2) Energetische Charakterisierung noch schwierig

- Problem bei Protein-Protein Docking die beste Lösung zu finden

(3) Kinetik kann mittels Brownscher Dynamik charakterisiert werden.

Versuche, die 6-dimensionale G-Oberfläche durch Mapping der

Trajektorien zu erhalten.

So kann man den Encounter-Zustand als Minimum der freien Enthalpie

entlang einer geeigneten Reaktionskoordinate beschreiben

(4) Evolutionäre Relevanz: können verschiedene Teile der Proteinoberfläche für

verschiedene Phasen der Proteinassoziation verantwortlich sein?- Geladene Patches: langreichweitige Attraktion- Hydrophobe Patches: bilden Bindungsinterphase

Dann ergibt sich ein „evolutionärer Druck“ auf die ganze Oberfläche, nicht nur

auf das Bindungsinterface.

9. vorlesung ws 2004/05softwarewerkzeuge1 v9: protein-protein-wechselwirkung - consurf - russell...

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