V9: Protein-Protein-Wechselwirkung

V9: Protein-Protein-Wechselwirkung - Consurf - Russell & Aloy Server - Webseite mit Proteindomänen - Beispiel zu Protein-Protein Wechselwirkung Cytoch...
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V9: Protein-Protein-Wechselwirkung - Consurf - Russell & Aloy Server - Webseite mit Proteindomänen - Beispiel zu Protein-Protein Wechselwirkung Cytochrom c: Cytochrom c Oxidase Barnase:Barstar - Klausurvorbereitung

9. Vorlesung WS 2004/05

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Consurf

- 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

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

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

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V9: Protein-Protein-Wechselwirkung

http://www.mshri.on.ca/pawson/research.html 9. Vorlesung WS 2004/05

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Pawson Lab 4

SH2 domains – universally used protein family

http://www.mshri.on.ca/pawson/research.html 9. Vorlesung WS 2004/05

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Pawson Lab 5

Proteins using SH2 domains

http://www.mshri.on.ca/pawson/research.html 9. Vorlesung WS 2004/05

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Pawson Lab 6

Binding properties of SH2 domains

http://www.mshri.on.ca/pawson/research.html 9. Vorlesung WS 2004/05

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Pawson Lab 7

Binding properties of SH2 domains

http://www.mshri.on.ca/pawson/research.html 9. Vorlesung WS 2004/05

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Pawson Lab 8

WW domain

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

Pawson Lab

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14-3-3 domain

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

Pawson Lab

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14-3-3 domain

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

Pawson Lab

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14-3-3 domain

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

Pawson Lab

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Introduction: Photosynthesis

+

light

cytosol

plasma membrane

lumen

3-

H + ADP + Pi cytochrome b6f -complex + H

light reaction harvesting centre + complex 2H QA Ph

2-

ATP

+

4H

QB QB

QH2

2H

+

4H

+

F0 F1 complex

cytochrome c

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Docking strategy No X-ray structure available for complex cyt c552:COX

docking.

Flöck, Helms (2002) Proteins 47, 75 9. Vorlesung WS 2004/05

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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 9. Vorlesung WS 2004/05

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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).

Flöck, Helms (2002) Proteins 47, 75 9. Vorlesung WS 2004/05

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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).

Flöck, Helms (2002) Proteins 47, 75 9. Vorlesung WS 2004/05

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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.

Flöck, Helms (2002) Proteins 47, 75 9. Vorlesung WS 2004/05

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kinetic on-rates of protein-protein complexes from Brownian Dynamics simulations

McCammon group website (UCSD)

9. Vorlesung WS 2004/05

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kinetic on-rates: exp data Mutation

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

Wild type oxidase D135N N160D

3.7 0.3 2.8 P.d. cyt c552 : COX

Ionic strength [mM] 10 35 200 refs:

4.1 1.5 0.1 Drousou, Malatesta, Ludwig, Eur J Biochem (2002) 269, 2980 Maneg, Ludwig, Malatesta, J Biol Chem (2003) 278, 46734

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brownian dynamics details Electrostatics computed with programs UHBD (McCammon group) APBS (Baker, McCammon, Holst) Atomistic brownian dynamics simulations with program SDA (Gabdoulline & Wade, 1997) Simulation parameters: time step translat. diffusion constant rotational diffusion constant Radius b Radius c

2 ps – 10 ps 0.02 Å2 ps-1 4.0 × 10-5 radian2 ps-1 115 Å 540 Å

for each system

4 × 4000 runs Flöck & Helms, Biophys.J. 87, 65 (2004)

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Iteration Algorithm

yi = yi + 0

6N

j =1

D ij0 t + 1 / KT yi

6N

j =1

D ij0 F j0 t +Ri (D 0 , t )

Generalized coordinate vector

y = ( ( r1 )1 ,..., ( rN )3 , (

Generalized force vector

F = (F1 )1 ,..., (FN )3 , (T1 )1 ,..., (TN )3

Diffusion matrices

D

Random displacements

Ri (D 0 , t ) with: < Ri >= 0 < Ri R j >= 2Dij t

(

ij

=

1

(

1

)

1 1

,..., (

N

)3 )

)

)

ij

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

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

exp. rate with solubilized COX

fulfil 1, 2, or 3 contact pairs among D156:K79

D135:K86

A259:K73

D135:K86

S124:K86

Y122:G84 Flöck & Helms, Biophys.J. 87, 65 (2004)

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Simulated mutation effects on on-rates

N160D (2.8) wild-type COX (3.7)

D135N (0.3)

Association of horse heart cyt c and „naked“ COX at 140mM. Data for 3 reaction criteria. Flöck & Helms, Biophys.J. 87, 65 (2004)

Nice agreement with exp. trends! 9. Vorlesung WS 2004/05

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Simulated ionic strength effects on on-rates

10 mM (4.1)

35 mM (1.5)

200 mM (0.1)

Association of P.d. cyt c552 and „naked“ wild-type COX. Data for 3 reaction criteria. Nice agreement with exp. trends!

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

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model of membrane environment

Flöck & Helms, Biophys.J. 87, 65 (2004) 9. Vorlesung WS 2004/05

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Inclusion of Membrane Environment Start diffusion proteins only in spherical cap above COX.

This scheme makes no difference for „naked“ COX: comparison of original and spherical-cap starting positions. Flöck & Helms, Biophys.J. 87, 65 (2004)

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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 horse heart cyt c : COX at 140 mM Large effect! Flöck & Helms, Biophys.J. 87, 65 (2004) 9. Vorlesung WS 2004/05

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Effect of membrane embedding for P.d. cyt c552

with membrane

„naked“ COX

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? Flöck & Helms, Biophys.J. 87, 65 (2004) 9. Vorlesung WS 2004/05

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Computational studies: gain insight by switching isolated interactions off

membrane charges off (cyt c552)

COX charges off (cyt c552)

all charges on membrane charges off (cyt c)

COX charges off (cyt c)

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

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G

Protein – protein association to form complex, interfaces need to be desolvated + sidechains oriented protein B enters into protein A‘s zone of electrostatic attraction directed diffusion free diffusion bound complex

A and B form „encounter complex“ - electrostatically entangled, - no bound complex

idealized reaction coordinate 9. Vorlesung WS 2004/05

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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)

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Aim of this study Aim: clarify nature of encounter complex

Means: statistical analysis of brownian dynamics trajectories

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Coordinate frame

Center of mass coordinates of second protein

Rotational coordinates of second protein

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Different definitions of distance variable

For global view

For local view

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how does the encounter state look like? representative structures of the encounter state ensemble

blue: red: green: purple:

cd1-2 cdmin cdavg cdcenter

black balls: reaction atoms of barstar in crystal structure

barnase

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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 2004/05

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