CG990 – Methods in proteomics Protein-protein interaction analysis Assoc. Prof. Jan Paleček Laboratory of Structural proteins of eukaryotic chromosoms, NCBR jpalecek@sci.muni.cz Information sources Golemis a Adams: Protein-protein interactions … … the newest methods from current literature … Databases of protein-protein interactions: http://string-db.org/newstring_cgi ... http://www.ebi.ac.uk/intact/?conversationContext=1 Protein-protein interaction analysis - Matrix/beads-based: - pull-down assay - co-purification – gel filtration - co-immunoprecipitation - Analysis of protein domains - Analysis of interaction surfaces - Peptide libraries - Hybrid-based: Y2H (yeast 2-hybrid), BiFC … - Proximity-based: PLA, BioID … - MS-based: crosslink, D/H-exchange … - Quantitative methods: SPR, ITC … - Structural methods: co-crystalization, NMR … - Genetic methods: synthetic lethality … - Bioinformatics methods: databases, docking … Y Protein A-antibody Co-immunoprecipitation beads/matrix/particles are used to precipitate bait protein with its bound partners Common tags for coimmunoprecipitation assay: GFP, FLAG, myc, HA, T7, PK These tags are recognized by specific antibodies (commercially available) TAP-tag (can be used as well): „Tandem-affinity purification“ = immunoglobulin tag + calmodulin tag (usually used to purify complexes) Risk of indirect interactions (in same organism) Protein Tag Vassylyeava et al, PNAS, 2017 TAG co-purification Strong interactions (protein complexes) can be recognized during the purification of the proteins (similar approach to pulldown assay) – proteins can be co-purified through different tags and using gel filtration Totalextract FT Solublefraction 25 35 40 55 70 1. Input 15 10 25 35 40 55 70 15 10 2. 3. 4. 5. 6. 7. 8. 9. 10. FT 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Elution fractions – 10ml Elution fractions – 1,5ml Nse3 His- Nse1 Strep- Nse4 1. His-tag purification 2. Strep-tag purification Strep Strep 6xHis Nse4Nse3Nse1 Zabrady et al, NAR, 2016 Nse1-Nse3-Nse4 co-purify (interact strongly) Common tags: GST (glutathione) Streptactin (biotin- streptavidin) MBP (maltose) S-tag (protein S) … Pull-down 1. tagged (e.g. GST) protein (bait) is bound to (glutathione) beads/particles (GP) 2. Partner protein (prey) is added - if the bait and prey interact then prey will be pulled down (together with bait protein) on the beads strong interaction – both proteins can be at equal concentrations (expressed/purified from bacteria or expressed/labelled in TnT in vitro expression system) weak interaction – bait overexpressed vs prey from TnT strong Nse4-SMC5 interaction GST strong = nM-pM range weak = mM-µM range Palecek et al, JBC, 2006 Nse4 protein well expressed in TNT (insoluble in cells) Pull-down Strong interaction – both proteins v TNT (nM-pM) PAGE Similar to co-purification/precipitation in vitro TNT (Transcription and Translation) system methionin S35 advantages: - No need to purify proteins - No need for anntibodies - No toxicity in cell - Higher solubility … Palecek et al, JBC, 2006 INPUT UNBOUND BOUND Pull-down Common tags for pull-down assay: GST (glutathione) Streptactin (biotin- streptavidin) MBP (maltose) S-tag (protein S) … + tags recognized by antibodies (see co-immunoprecipitation) Weak Nse3-SMC6 interaction - control nonspecific binding of prey (Smc6 does not bind to GST-bound beads) - GST-Nse3 expressed in bacteria – (pre)purified on glutathione particles – detected with anti-GST - Smc6 expressed in TnT (radiolabeled) – radiogram detection (neither tag nor antibody needed) Palecek et al, JBC, 2006 Protein-protein interaction analysis - Matrix/beads-based: - pull-down assay - co-purification – gel filtration - co-immunoprecipitation - Analysis of protein domains - Analysis of interaction surfaces - Peptide libraries - Hybrid-based: Y2H (yeast 2-hybrid), BiFC … - Proximity-based: FRET, PLA … - MS-based: crosslink, D/H-exchange … - Quantitative methods: SPR, ITC … - Structural methods: co-crystalization, NMR … - Genetic methods: synthetic lethality … - Bioinformatics methods: databases, docking … Characterization of binding domainDoyleetal,MolCell,2010 Proteins interact via their domains (motifs) – analyze domain composition of your protein – prepare fragments of your protein defined by domain boundaries – test them in pull-down, co- … Truncated versions of TRIM28 were used to determine MAGEC2binding domain (only fragments A, C, D overlapping in coiled-coil domain do interact) Characterization of binding regions Proteins interact via their domains (motifs) – (sometimes) only fragments of the domain can interact (can be precipitated) A. Peptide coverage of the protein B. Peptide enrichment after co- immunoprecipitation with the bait protein (red arrow points to enriched/bound peptide in MS spectra) A. B. Reichman, Diploma thesis, 2009 Peptide libraries – region definition Proteins interact via their domains (motifs) – (sometimes) only fragments of the domain can interact (can be precipitated) – peptide library can be synthetized (with conjugated biotin tag) and used in pull-down or ELISA assays (similar to antigen-epitope mapping) wells are coated with streptavidin which anchors biotinylated peptides – binding partner interacts with peptide – antibody against the partner with conjugated enzyme (or 2nd antibody-enzyme) is applied - luminescence or colour detection binding partner ELISA Proteins interact via their domains (motifs) – (sometimes) only fragments of the domain can interact (can be precipitated) - peptide library can be synthetized (with conjugated biotin tag) and used in pull-down or ELISA assays (similar to antigen-epitope mapping) ELISA Guerineau, PhD thesis, 2013 Peptide libraries – region definition wells are coated with streptavidin which anchors biotinylated peptides – binding partner interacts with peptide – antibody against the partner with conjugated enzyme (or 2nd antibody-enzyme) is applied - luminescence or colour detection Peptide libraries 25 amino acids long (18) peptides library with 4 amino acids overlap (covering 90 amino acids region of Nse4 protein) – peptides #6-8 bind with highest affinity, suggesting the core of the binding region Guerineau, PhD thesis, 2013 ELISA Proteins interact via their domains (motifs) – amino acids essential for the interaction can be identified (via mutational analysis – e.g. alanine substitutions = “alanine scan”) - peptide library or yeast two-hybrid system (see below) can be used Guerineau, PLoS One, 2012 Peptide libraries – surface mapping 21 amino acids long (20) peptides library with single amino acid alanine substitution (covering every non-Ala amino acid) Guerineau, PLoS One, 2012 Peptide libraries – alanine scan mappingELISA 197 217 Guerineau, PLoS One, 2012 Peptide libraries – surface mapping ELISA Helical peptide is sitting in the pocket of the partner protein – most peptide residues are in contact (red labeled) with the pocket (so, their mutations reduced the mutant peptide affinity), while the D204 (black labeled) residue is exposed to solvent Guerineau, PLoS One, 2012 - maleimid reacts with sulph-hydryl group of Cys (covalent bond) - directed crosslink (low abundant Cys) - detection on SDS-PAGE Mapování interakcí - crosslinking protein 1 crosslink protein 1 with protein 2 intensity corelates With distance - matrix/beads-based: pull-down (in vitro), coIP … - hybrid-based: - classical systems- domain - transcription 2-hybrid systems - reverse systems - multi-hybrid systems - alternative (membrane) systems - pathway - complementation systems – fold - BiFC, DHFR - proximity-based: FRET, PLA, BioID … - MS-based: crosslink, D/H-exchange … - Quantitative methods: SPR, ITC … - Structural methods: co-crystalization, NMR … - Genetic methods: synthetic lethality …) - Bioinformatics methods: databases, docking … Protein-protein interaction analysis Principal differences in hybrid systems Stynen et al, MMBR, 2012 A. In classical systems, PPI reconnects two separated domains (normally present in one protein) back to one tight complex B. In complementation systems, PPI reconnects fragments of one domain and reconstitutes its fold complete domain hybrid protein hybrid protein hybrid protein hybrid protein UetzandFinley,FEBSlett.,2005 Classical (first) yeast two-hybrid system is based on transcription factor Gal4 function – Gal4 binds promotor regions (sequences) of GAL genes and activates their transcription Classical yeast two-hybrid system Gal4-based two-hybrid system Gal4 transcription factor binds specific DNA sequence through its DNA-binding domain (DBD) - Gal4 transcription activation domain (AD) binds to general TFII factors/RNA polymerase II and activates transcription machinery TFII A. Gal4 (DBD-AD) protein activates reporter gene (lacZ) B. When DNA-binding domain (DBD) and activation domain (AD) are separated, they are not able to activate transcription machinery C. When DBD and AD are fused in frame to interacting proteins (X and Y), then PPI reconnects DBDAD and enables transcription TFII Gal4-based 2-hybrid system Other transcription factors have been employed in two-hybrid variants: Stynen et al, MMBR, 2012 To detect/score transcription activation (i.e. “see” interaction of partner proteins), different reporter genes are used BD-Snf1/- BD-Snf1/AD-Snf4 -/AD-Snf4 Only yeast cells expressing binding partners will turn blue (as the lacZ reporter will be transcribed/expressed and will convert transparent X-gal substrate to blue product) – lacZ enzymatic activity can be measured (thus, the strength of the PPI can be quantified) quantitative auxotrophy (selective) FACSorting antibiotic resistance Reporter genes Stynen et al, Microbiol Mol Biol Rev, 2012 His3 enzyme activity can be titrated by its 3- aminotriazol inhibitor Trp1 and Leu2 genes must be mutated to enable (auxotrophy) selection of plasmids (bearing hybrid genes) - many yeast strains exist; systems adopted to bacterial and mammalian cells exist as well Yeast 2-hybrid strain example AH109 (and other strains) contains His3 and lacZ reporter genes (integrated in LYS2 and URA3 genes, respectively) under different Gal4-binding promotors (GAL1 and MEL1, respectively) genotype Yeast 2-hybrid plasmid example myc Tag HA Tag pGBKT7 and pGADT7 plasmids contain Gal4 BD and AD elements (to make hybrid proteins) as well as selective markers (Trp1 and Leu2 for yeast selection) T7 promoters in front of myc and HA tag, respectively, are suitable for additional pull-down experiments (see previous slides) Uetz et al, Nature, 2000 Y X DBD AD a cellsα cells High-throughput screens can be done as – 1. simple study: one bait is screened against AD-library (e.g. of all human hybrid proteins) - or – 2. interactom study: collection of all BD-proteins is screened against ADlibrary (e.g. 6000x6000 yeast proteins = yeast interactom) Yeast 2-hybrid screens x Reverse systems For detail PPI analysis (e.g. binding surface mapping), mutation (drug) will disturb interaction - it (loss of interaction) is detected by the loss of growth of the yeast cells on selective plate (or inability to turn on the blue colour) – reverse systems were developed to “visualize” loss of interaction … Vidal & Endoh, T in Biotech, 1999 … in reverse systems, PPI results in lethal phenotype – yeast cells will not grow until PPI is disturbed (by mutation or drug) – for example, cells expressing URA3 reporter gene will grow on plates without uracil, but these cells will be killed by 5-flouro-orothic acid (Ura3 enzyme converts FOA to toxic compound); in contrast, when PPI is disturbed, yeast cells will not express URA3 reporter gene (will not grow on plates without uracil), but these cells will not convert 5-flouro-orothic acid and therefore they will be able to grow on plates with FOA Reverse systems Stynen et al, MMBR, 2012 … new reverse system (also called split system) is based on two transcription regulation steps: PPI activates transcription of repressor which blocks transcription of reporter gene (only when PPI is disturbed, the His3 reporter gene is transcribed) Reverse systems Stynen et al, MMBR, 2012 First three-hybrid system was developed to study RNAbinding proteins – DBD-hybrid protein (1) binds one RNA motif (MS2) within the RNA-hybrid molecule (2), while the other part of the RNA-hybrid molecule (X) is recognized by AD-hybrid protein (3) – this RNA-protein complex will switch on lacZ reporter gene transcription – in this way, you can screen ADhybrid library for RNA-X binding proteins (multi) three-hybrid systems Stynen et al, MMBR, 2012 3. hybrid protein 1.hybridprotein 2. hybrid RNA molecule SenGupta et al, PNAS, 1996 DBD-hybrid protein binds one part of bridging protein, while the other part of the bridging (non-hybrid) protein is bound by AD-hybrid protein (several bridging proteins can be used) Three-component 2-hybrid system Stynen et al, MMBR, 2012 2. hybrid protein 1.hybrid protein bridging protein Bednarova, Diploma thesis, 2009 Nse1-Nse3-Nse4 complex DBD AD - matrix/beads-based: pull-down (in vitro), coIP … - hybrid-based: - classical systems- domain - transcription 2-hybrid systems - reverse systems - multi-hybrid systems - alternative (membrane) systems - pathway - complementation systems – fold - BiFC, DHFR - proximity-based: FRET, PLA, BioID … - MS-based: crosslink, D/H-exchange … - Quantitative methods: SPR, ITC … - Structural methods: co-crystalization, NMR … - Genetic methods: synthetic lethality …) - Bioinformatics methods: databases, docking … Protein-protein interaction analysis Stynen et al, Microbiol Mol Biol Rev, 2012 Number of proteins can’t be used in transcription-based hybrid systems (e.g protein can’t be localized to the yeast cell nucleus) – CytoTrap (Ras recruitment) system is based on membrane-anchored Ras pathway reactivation – A. RAS protein is activated only when human hSOS-hybrid, ortholog of yeast cdc25 (guanine exchange factor; cdc25-2 mutant cells are used), is anchored at the cytoplasmic membrane via interaction of myristylated hybrid-protein partner – B. RAS-hybrid protein works when it binds to myristylated hybrid-protein partner Alternative membrane systems - Ras 2. hybrid protein 2. hybrid protein 1.hybridprotein 1.hybridprotein myristyl anchor myristyl anchor Aga2-hybrid protein is localized at the yeast surface – taggedpartner interaction anchors it at the yeast surface – anti-tag antibody recognizes the tagged protein – fluorescence of the antibody (primary or secondary antibody) is detected and can be used for yeast strain selection (by FACS) Yeast surface display system Stynen et al, MMBR, 2012 - matrix/beads-based: pull-down (in vitro), coIP … - hybrid-based: - classical systems- domain - transcription 2-hybrid systems - reverse systems - multi-hybrid systems - alternative (membrane) systems - pathway - complementation systems – fold - BiFC, DHFR - proximity-based: FRET, PLA, BioID … - MS-based: crosslink, D/H-exchange … - Quantitative methods: SPR, ITC … - Structural methods: co-crystalization, NMR … - Genetic methods: synthetic lethality …) - Bioinformatics methods: databases, docking … Protein-protein interaction analysis Johnsson et al, PNAS, 1994 Stynen et al, MMBR, 2012 Complementation systems PPI reconnects fragments of one domain and reconstitutes its fold – original (A) assay based on reconstitution of ubiquitin (western blot analysis of protein degradation) – new alternative versions use different detection approaches – for example (B), in transcription-based approach, reporter gene is transcribed only when LexA-VP16 transcription factor is released from membrane localization Complementation systems Several systems based on complementation of different protein folds have been developed Shekhat & Ghosh, CO in ChB, 2011 Bimolecular fluorescence complementation (BiFC) – PPI reconnects GFP Kodama & Hu, Biotechniques, 2012 Bimolecular fluorescence complementation (BiFC) Kodama & Hu, Biotechniques, 2012 Pekarova et al, Plant J., 2011 Bimolecular fluorescence complementation (BiFC) – PPI reconnects GFP and its fluorescence is detected binding non- binding Bruckner et al, IJMS, 2009 Overview of yeast 2-hybrid systems Protein-protein interaction analysis - matrix/beads-based: pull-down (in vitro), coIP … - Hybrid-based: Y2H (yeast 2-hybrid), BiFC … - Proximity-based: - FRET - PLA - MS-based: crosslink, D/H-exchange … - Quantitative methods: SPR, ITC … - Structural methods: co-crystalization, NMR … - Genetic methods: synthetic lethality … - Bioinformatics methods: databases, docking … FRET (Forster/fluorescence resonance energy transfer) - CFP-hybrid protein emits 480-525nm light when excited (by 458nm light) – when CFP-hybrid protein binds partner YFPhybrid protein, the 480-525nm emitted light excites YFP which then emits 525-575nm light (detected in the fluorescence microscope) Proximity based Proximity ligation assay - PLAWeibrechtetal,ERProt,2010 - Specific antibodies conjugated with oligonucleotides, which are complementary to circular DNA – if the antibodies come close (<16nm) via PPI of they target proteins then – DNA is ligated and polymerase synthesis reaction can run