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V9: Protein-Protein-Wechselwirkung

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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

2 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

3 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.

4 V9: Protein-Protein-Wechselwirkung
Pawson Lab

5 SH2 domains – universally used protein family
Pawson Lab

6 Proteins using SH2 domains
Pawson Lab

7 Binding properties of SH2 domains
Pawson Lab

8 Binding properties of SH2 domains
Pawson Lab

9 WW domain Pawson Lab

10 domain Pawson Lab

11 domain Pawson Lab

12 domain Pawson Lab

13 Introduction: Photosynthesis

14 Docking strategy No X-ray structure available for complex cyt c552:COX  docking. Flöck, Helms (2002) Proteins 47, 75

15 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

16 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

17 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

18 Two favorable docking positions
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

19 kinetic on-rates of protein-protein complexes
from Brownian Dynamics simulations McCammon group website (UCSD)

20 kinetic on-rates: exp data
Mutation kon  106 [M-1s-1] horse heart cyt c : COX Wild type oxidase 3.7 D135N 0.3 N160D 2.8 Ionic strength [mM] P.d. cyt c552 : COX refs: Drousou, Malatesta, Ludwig, Eur J Biochem (2002) 269, 2980 Maneg, Ludwig, Malatesta, J Biol Chem (2003) 278, 46734

21 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 2 ps – 10 ps translat. diffusion constant 0.02 Å2 ps-1 rotational diffusion constant 4.0  10-5 radian2 ps-1 Radius b Å Radius c Å for each system 4  4000 runs Flöck & Helms, Biophys.J. 87, 65 (2004)

22 Iteration Algorithm Generalized coordinate vector
Generalized force vector Diffusion matrices Random displacements Dickinson, E., Allison, S.A. and McCammon, J.A. (1985) J. Chem. Soc. Farad. Trans. 2 81, 591

23 “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)

24 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. Nice agreement with exp. trends! Flöck & Helms, Biophys.J. 87, 65 (2004)

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

26 model of membrane environment
Flöck & Helms, Biophys.J. 87, 65 (2004)

27 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)

28 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)

29 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)

30 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)

31 Protein – protein association
to form complex, interfaces need to be desolvated + sidechains oriented G 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 1

32 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 ~ M binding stabilized by favorable electrostatic binding energy (Wang et al. 2004) association rates extensively studied by Gabdoulline & Wade (1997) Dong et al. (2003) 1

33 Aim of this study Aim: clarify nature of encounter complex
Means: statistical analysis of brownian dynamics trajectories 1

34 Coordinate frame Center of mass coordinates of second protein
Rotational coordinates of second protein 1

35 Different definitions of distance variable
For global view For local view 1

36 how does the encounter state look like?
representative structures of the encounter state ensemble blue: cd1-2 red: cdmin green: cdavg purple: cdcenter black balls: reaction atoms of barstar in crystal structure barnase 1

37 Zusammenfassung 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 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 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. 1


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