1 / 41

DNA/Protein structure-function analysis and prediction

Protein-protein Interaction (PPI) and Docking: Protein-protein Interaction Interfaces Solvation Energetics Conformational change Allostery. Examples Arfaptin – Rac Ribosome Docking Search space Docking methods. DNA/Protein structure-function analysis and prediction.

taro
Download Presentation

DNA/Protein structure-function analysis and prediction

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Protein-protein Interaction (PPI) and Docking: Protein-protein Interaction Interfaces Solvation Energetics Conformational change Allostery Examples Arfaptin – Rac Ribosome Docking Search space Docking methods DNA/Protein structure-function analysis and prediction

  2. PPI Characteristics • Universal • Cell functionality based on protein-protein interactions • Cyto-skeleton • Ribosome • RNA polymerase • Numerous • Yeast: • ~6.000 proteins • at least 3 interactions each • ~18.000 interactions • Human: • estimated ~100.000 interactions • Network • simplest: homodimer (two) • common: hetero-oligomer (more) • holistic: protein network (all)

  3. Interface Area • Contact area • usually >1100 Å2 • each partner >550 Å2 • each partner loses ~800 Å2 of solvent accessible surface area • ~20 amino acids lose ~40 Å2 • ~100-200 J per Å2 • Average buried accessible surface area: • 12% for dimers • 17% for trimers • 21% for tetramers • 83-84% of all interfaces are flat • Secondary structure: • 50% a-helix • 20% b-sheet • 20% coil • 10% mixed • Less hydrophobic than core, more hydrophobic than exterior

  4. Complexation Reaction • A + B AB • Ka = [AB]/[A]•[B] association • Kd = [A]•[B]/[AB] dissociation

  5. Experimental Methods • 2D (poly-acrylamide) gel electrophoresis  mass spectrometry • Liquid chromatography • e.g. gel permeation chromatography • Binding study with one immobilized partner • e.g. surface plasmon resonance • In vivo by two-hybrid systems or FRET • Binding constants by ultra-centrifugation, micro-calorimetry or competition • experiments with labelled ligand • e.g. fluorescence, radioactivity • Role of individual amino acids by site directed mutagenesis • Structural studies • e.g. NMR or X-ray

  6. Surface Plasmon Resonance 1 • Evanescent field

  7. Surface Plasmon Resonance 2 • sensitivity limit for layer thickness ~0.2-0.4 nm. http://www.biochem.mpg.de/oesterhelt/xlab/spfs.html#monitoring

  8. plasmon curve Reflexion intensity as function of angle of incidence Q dotted line: after addition of adsorbing molecules. kinetics during adsorption process Reflexion intensity as function of time at fixed angle Q. Surface Plasmon Resonance 3

  9. PPI Network http://www.phy.auckland.ac.nz/staff/prw/biocomplexity/protein_network.htm

  10. Protein-protein interactions • Complexity: • Multibody interaction • Diversity: • Various interaction types • Specificity: • Complementarity in shape and binding properties

  11. Binding vs. Localization strong Non-obligatetriggered transient e.g. GTP•PO4- Non-obligatepermanente.g. antibody-antigen Obligateoligomers Non-obligateco-localised e.g. in membrane Non-obligateweak transient weak co-expressed different places

  12. Some terminology • Transient interactions: • Associate and dissociate in vivo • Weak transient: • dynamic oligomeric equilibrium • Strong transient: • require a molecular trigger to shift the equilibrium • Obligate PPI: • protomers not stable structures on their own • (functionally obligate)

  13. Strong – medium – weak • Nanomolar to sub-nanomolar • Kd < 10-9 • Micromolar to nanomolar • 10-6 > Kd > 10-9 • Micromolar • 10-3 > Kd > 10-6 • A + B AB • Kd = [A]•[B]/[AB] dissociation

  14. Analysis of 122 Homodimers • 70 interfaces single patched • 35 have two patches • 17 have three or more

  15. Patches • Cluster in different domains • (structurally defined units often with specific function) two domains anticodon-binding catalytic

  16. Interfaces • ~30% polar • ~70% non-polar

  17. Interface • Rim is water accessible rim core

  18. Interface composition • Composition of interface essentially the same as core • But % surface area can be quite different!

  19. Propensities • Interface vs. surface propensities • as ln(fint/fsurf)

  20. Conformational Change • Chaperones • extreme conformational changes upon complexation • ligand unfolds within the chaperone GroEL/GroES • Allosteric proteins • conformational change at 'active' site • ligand binds to 'regulating' site • Peptides • often adopt 'bound' conformation • different from the 'free' conformation

  21. Allostery 1 • Regulation by 'remote' modulation of binding affinity (complex strength) www.blc.arizona.edu/courses/181gh/rick/energy/allostery.html

  22. Allostery 2 • Substrate binding is cooperative • Binding of first substrate at first active site • stimulates active shape • promotes binding of second substrate

  23. Allostery 3 • Committed step of metabolic pathway • regulated by an allosteric enzyme • Pathway end product • can regulate the allosteric enzyme for the first committed step • Inhibitor binding favors inactive form

  24. Protein-protein Interaction (PPI) and Docking: Protein-protein Interaction Interfaces Solvation Energetics Conformational change Allostery Examples Arfaptin – Rac Ribosome Docking Search space Docking methods DNA/Protein structure-function analysis and prediction

  25. Small G-proteins cross-talk • Mediated by Arfaptin

  26. Arfaptin and Rac • Micromolar Kd • But specific

  27. Arfaptin Interacting surface

  28. Arfaptin Interacting patches

  29. Arfaptin Exposed surface

  30. Rac Interacting patch

  31. Arfaptin + Rac Exposed surface

  32. 70S structure at 5.5 Å (Noller et al. Science 2001)

  33. 70S structure

  34. 30S-50S interface • Overall buried surface area ~8500 Å2 < 37.5 Å2 37.5 Å2 – 75 Å2 > 75 Å2

  35. Protein-nucleic acid Interactions

  36. Interactions in the Ribosome

  37. Protein-protein Interaction (PPI) and Docking: Protein-protein Interaction Interfaces Solvation Energetics Conformational change Allostery Examples Arfaptin – Rac Ribosome Docking Search space Docking methods DNA/Protein structure-function analysis and prediction

  38. Docking - ZDOCK • Protein-protein docking • 3-dimensional (3D) structure of protein complex • starting from 3D structures of receptor and ligand • Rigid-body docking algorithm (ZDOCK) • pairwise shape complementarity function • all possible binding modes • using Fast Fourier Transform algorithm • Refinement algorithm (RDOCK) • top 2000 predicted structures • three-stage energy minimization • electrostatic and desolvation energies • molecular mechanical software (CHARMM) • statistical energy method (Atomic Contact Energy) • 49 non-redundant unbound test cases: • near-native structure (<2.5Å) for 37% test cases • for 49% within top 4

  39. Protein-protein docking • Finding correct surface match • Systematic search: • 2 times 3D space! • Define functions: • ‘1’ on surface • ‘r’ or ‘d’ inside • ‘0’ outside d r

  40. Protein-protein docking • Correlation function: Ca,b,g = 1/N3SoSpSqexp[2pi(oa + pb + qg)/N] •Co,p,q

  41. Docking Programs • ZDOCK, RDOCK • AutoDock • Bielefeld Protein Docking • DOCK • DOT • FTDock, RPScore and MultiDock • GRAMM • Hex 3.0 • ICM Protein-Protein docking • KORDO • MolFit • MPI Protein Docking • Nussinov-Wolfson Structural Bioinformatics Group • …

More Related