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Protein-Protein Interactions

Protein-Protein Interactions. Genome. Transcriptome. Proteome. Interactome. Atomic NMR spectroscopy X-ray crystallography: co-crystallisation Electron microscopy Molecular modelling: docking  Protein Data Bank. Experimental methods to identify PPI’s. Biochemical/biophysical binary

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Protein-Protein Interactions

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  1. Protein-Protein Interactions

  2. Genome Transcriptome Proteome Interactome

  3. Atomic NMR spectroscopy X-ray crystallography: co-crystallisation Electron microscopy Molecular modelling: docking  Protein Data Bank Experimental methods to identify PPI’s

  4. Biochemical/biophysical binary yeast two-hybrid (interactions are identified by the transcription of reporter gene) surface plasmon resonance (SPR) f.e. BIAcore etc. multiprotein complexes co-immunoprecipitation tagged genes, affinity chromatography …. followed by mass spectroscopy  Various databases Experimental methods to identify PPI’s

  5. correlated mRNA expression (microarray): mostly permanent complexes homologues known to operate sequentially in metabolic pathways, conserved operons (prokaryotes) related phylogenetic profiles / correlated evolution gene fusion: homologues that are fused into a single gene in different organism (Rosetta Stone method)  Some databases Computational methods (Genomics)

  6. homologues of proteins known to interact based on experimental data (e.g. 3D structure) subcellular localisation Medline abstract search if biomolecules are cited in the same abstract, they are likely to be functionally related or to physically interact Computational methods

  7.  Proteomics  Functional genomics

  8. Localisation: co-expression, subcellular, compartmentalisation Affinity and concentration Binary or multimeric (cooperativity) PPI paradigms

  9. PDBStructural characterisation of different PPI’s

  10. Types of PP-interactions lifetime of assembly examples folding/structure Arc repressor homodimer flavoprotein heterodimer permanent obligate 2-state intracellular signalling enzyme-inhibitor antibody-antigen transient/ permanent non-obligate 3-state

  11. Rho-GAP (non-obligate) Arc repressor (obligate) Thrombin-inhibitor (non-obligate)

  12. Crystal contacts of monomers (non-biological) Homo/heterodimers Non-obligate/obligate Transient/permanent Datasets from PDB

  13. Wright (1990) J. Mol. Biol.215, 635. [PDB-code: 9WGA] Wheat Germ Agglutinin

  14. Wright (1990) J. Mol. Biol.215, 635. [PDB-code: 9WGA] Wheat Germ Agglutinin

  15. Protein Data Bank (PDB): PDB file

  16. Crystal symmetry

  17. Monomer, dimer, tetramer …?

  18. Number of chains in Asymmetric Unit (ASU) for data set of homo-multimers Crystal properties Number of chains in ASU multimer type

  19. macromolecular assemblies from PDB sparse PDB annotation of multimers Protein Quaternary Structure (PQS) server http://pqs.ebi.ac.uk (Kim Henrick) crystal contacts  interfaces score interfaces: DASA, solvation free energy of folding, number of salt bridges and di-sulphide bonds add atom-pair frequencies, statistical pair potential Inferring Quaternary Structure

  20. cut-off at -70 cut-off at 856 Å2 Ponstingl et al. (2000) Proteins 41, 47-57

  21. <Counts for homo-multimers> / <counts for hetero-multimers> Performance Contingency table for oligomer classification classified as reference

  22. contact scores - not binding energies long-range interactions (electrostatics), … ligand binding in protein-protein interface different physiological/crystallisation conditions protein concentration, pH, … Oligomeric equilibrium Improve confidence by including sequence conservation Problems & improvements

  23. Crystal contacts of monomers (non-biological Homo/heterodimers Non-obligate/obligate Transient/permanent Datasets from PDB

  24. Accessible Surface Area (ASA) Planarity Protrusion Hydrophobicity Percentage polarity Interface properties

  25. Dataset I: ‘weak’ transient homodimers Both oligomeric forms populated at physiological conditions; dynamic equilibrium Dataset II: functionally validated transient heterodimers Intracellular signalling complexes Transient interactions

  26. ‘weak’ transient interactions have small and flat interfaces: DASA < 1000 Å2, planarity < 3.5Å

  27. transient homodimer miscellaneous homodimer structurally obligate homodimer 60 50 y t i r a l o p 40 e g a t n e 30 c r e p 20 10 0 1000 2000 3000 4000 5000 6000 contact area (A) 2

  28. obligate heterodimer co-expressed co-localised “ intracellular signalling other heterodimer } compartimentalised antibody-antigen enzyme-inhibitor extracellular receptor-ligand 60 50 y t i r a l 40 o p e g a t n 30 e c r e p 20 structural rearrangements likely to occur upon complexation 10 0 1000 2000 3000 4000 5000 6000 2 contact area (A)

  29. Continuum between a monomer and stable homodimer: weak, transient homodimers can be distinguished from more permanent or obligate homodimers: DASA < 1000 Å2, planarity < 3.5Å, polarity > 25% The above correlation is less distinct for non-obligate, non-colocalised heterodimers Co-expressed/co-localised, obligate complexes are generally more hydrophobic than non-obligate complexes (co-translation and co-folding) PP complexes with interfaces larger than about 1000 Å2 are likely to undergo structural rearrangements upon association (e.g. co-folding homodimers, ‘strong’ transient complex) Interface properties diverse PP-interactions

  30. effector X further oligomerisation/ cooperativity competition effector X X = D protein concentration, i.e. level of gene expression/secretion, degradation, temporary storage, diffusion (viscocity, steric environment) X = D pH, D temperature, D ionic strength X = molecular (cooperative/allosteric) binding i.e. D concentration of metabolite, protein or ions ( e.g. ATP, Ca2+) or covalent modification through enzymatic activity ( e.g. PO4-) strong transient complexes weak transient complexes large interface small interfaces CONTINUUM large D conformation no/minor D conformation

  31. Homology of PPI’s

  32. Structural data PPI ? Homologous sequences (paralogues and orthologues) residue level PPI ? PPI ? PPI Experimental data PPI ? Homologous sequences (paralogues and orthologues) protein level PPI ? PPI ?

  33. Defining the PPI interface Outer Zone Exposed accessible in monomer and does not lose asa on complexation asa in dimer < asa in monomer Central Zone accessible in monomer and inaccessible in dimer Core inaccessible in monomer Central Zone Outer Zone Total interface “Accessible” = relative accessible surface area of > 5% Total Interface Exposed Surface

  34. Alignments and conservation Use PSI-Blast alignment PSI-Blast NR database Inclusion E value = 10E-40 Max iterations = 20 1 = highly conserved Assess significance of contact conservation Score conservation for each position in the alignment 0 = unconserved

  35. Residue conservation, consider: physical and chemical properties of amino acids frequency distribution of amino acids observed -> use PAM matrix similarity score normalise against redundancy in the alignment

  36. Interface conservation: Central zone Significantly conserved Not significantly conserved

  37. Interface conservation: Total interface Significantly conserved Not significantly conserved

  38. Strong tendency for biological contacts to be more conserved Contact conservation Conserved Variable

  39. Tertiary structure – function Quaternary structure – function OR happenstance?

  40. sequence conservation of ‘weak’ transient interactions in homologous family protein no of mean conservation mean conservation ratio mean conservation sequences score surface score interface interface / surface cro repressor 4 0.78 0.88 1.20 dimerization and SH3-like 14 0.40 0.50 1.32 domain of tox repressor 51 0.60 0.77 1.47 dimerization domain RHR NFkB IL-8 54 0.61 0.55 1.08 MCP-1 56 0.62 0.62 1.19 a 10 0.99 1.00 1.01 SDF1 SCF 29 0.84 0.92 1.19 CNTF 10 0.76 0.78 0.98 insulin 28 0.81 0.89 1.20 cytochrome c’, type I 1 - - - cytochrome c’, type II 1 - - - b 44 0.57 0.52 0.97 -lactoglobulin Ecad12 243 0.47 0.44 1.30 LC8 24 0.73 0.85 1.28 lysin 25 0.71 0.81 1.23 galectin I 60 0.45 0.18 0.39 average 0.68 0.71 1.11 other obligate homodimers (Valdar et al.) 0.63 0.79 1.30

  41. human MCP-1 dimer human interleukin-8 dimer different interfaces

  42. Lost rest of slides data management: databases: total 50-60, specific to species, biological process or experimental methods BIND, DIP, MINT, etc. data management: data integration: figures from Bork review IntAct database (http//:www.ebi.ac.uk/intact) OEPS ...

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