Protein-protein Interactions June 6, 2017

Why PPI? Protein-protein interactions determine outcome of most cellular processes Proteins which are close homologues often interact in the same way Protein-protein interactions place evolutionary constraints on protein sequence and structural divergence Pre-cursor to networks

PPI classification Strength of interaction Specificity Permanent or transient Specificity Location within polypeptide chain Similarity of partners Homo- or hetero-oligomers Direct (binary) or a complex Confidence score

Determining PPIs Small-scale methods Co-immunoprecipitation Affinity chromatography Pull-down assays In vitro binding assays FRET, Biacore, AFM Structural (co-crystals)

PPIs by high-throughput methods Yeast two hybrid systems Affinity tag purification followed by mass spectrometry Protein microarrays Microarrays/gene co-expression Implied functional PPIs Synthetic lethality Genetic interactions, implied functional PPIs

Yeast two hybrid system Gal4 protein comprises DNA binding and activating domains Binding domain interacts with promoter Activating domain interacts with polymerase Measure reporter enzyme activity (e.g. blue colonies)

Yeast two hybrid system Gal4 protein: two domains do not need to be transcribed in a single protein If they come into close enough proximity to interact, they will activate the RNA polymerase Two other protein domains (A & B) interact Activating domain interacts with polymerase Binding domain interacts with promoter A B Measure reporter enzyme activity (e.g. blue colonies)

Yeast two hybrid system This is achieved using gene fusion Plasmids carrying different constructs can be expressed in yeast Binding domain as a translational fusion with the gene encoding another protein in one plasmid. Activating domain as a translational fusion with the gene encoding a different protein in a second plasmid. A B If the two proteins interact, then GAL4 is expressed and blue colonies form

Yeast two hybrid Advantages Limitations Fairly simple, rapid and inexpensive Requires no protein purification No previous knowledge of proteins needed Scalable to high-throughput Is not limited to yeast proteins Limitations Works best with cytosolic proteins Tendency to produce false positives Necessity for nuclear localization for interactions to occur Can get activation of the reporter gene in the absence of a true PPI

Mass spectrometry Need to purify protein or protein complexes Use a affinity-tag system Need efficient method of recovering fusion protein in low concentration

TAP (tandem affinity purification) PCR product Spacer TEV site Protein A CBP Homologous recombination Chromosome CBP calmodulin binding protein ProtA protein A Both allow efficient recovery of a fusion protein (80 % 50% respectively) from low concentration TEV (tobacco etch virus) protease cleavage site to allow proteolytic release of material under native conditions Fusion of TAP tag to target protein Introduction of target into host cell, maintain expression of fusion protein at close to natural level Fusion protein and associated components are recovered from cell extracts by affinity purification on IgG matrix After washing, TEV protease added to release bound material Eluate incubated with Calmodulin-coated beads in presence of Ca. This is required to remove TEV protease and other minor contaminants Bound material then released with EDTA Fusion protein Protein Spacer TEV site Protein A CBP Calmodulin binding peptide

TAP process "Taptag simple" by Chandres - Own work. Licensed under CC BY-SA 3.0 via Wikimedia Commons

TAP Advantages No prior knowledge of complex composition Two-step purification increases specificity of pull-down Limitations Transient interactions may not survive 2 rounds of washing Tag may prevent interactions Tag may affect expression levels Works less efficiently in mammalian cells TAP tag is 21 kDa; large enough to affect folding, activity or interaction of the fusion protein with other proteins in the cell

Other tags HA, Flag and His Streptavidin binding peptide (SBP) Anti-tag antibodies can interfere with MS analysis Streptavidin binding peptide (SBP) High affinity for streptavidin beads 10-fold increase in efficiency of purification compared to conventional TAP tag Successfully used to identify components of complexes in the Wnt/b-catenin pathway HA, FlAG and HIS tags are smaller than TAP, which may eliminate some issues of interference

Used Dsh-2 and Dsh-3 as bait proteins The KLHL12-Cullin-3 ubiquitin ligase negatively regulates Wnt-b-catenin pathway by targeting Dishevelled for degradation Nature Cell Biology 4:348-357 (2006)

Binding partners of Bruton’s tyrosine kinase Role in lymphocyte development & B-cell maturation Protein Science 20:140-149 (2011)

Databases of protein-protein interactions MINT – Molecular Interaction Database >240,000 interactions with 35,000 proteins Covers multiple species DIP -- Database of Interacting Proteins (UCLA) >79,000 interactions with >27,000 proteins CCSB – Proteomics base interactomes (Harvard) Human, viruses, C. elegans, S. cerevisiae Some unpublished data IntAct – EBI molecular interaction database Curated data from multiple sources

EBI IntAct Submit single or lists of proteins Provides method and reference for interactions List format, can download easily

TLR4 PPI at IntAct Click on the # for interaction to find more information

STRING database Search Tool for the Retrieval of Interacting Genes Integrates information from existing PPI data sources Provides confidence scoring of the interactions Periodically runs interaction prediction algorithms on newly sequenced genomes v.10 covers >2000 organisms http://string-db.org/

Networks in STRING database Starting protein Nice graphical view Interactive, can expand Not so easy to download lists of data

Networks can be expanded 3 indirect interactions

Information about the proteins

Accessing Interaction data From a UniprotKB (reviewed record):

Transferring PPI annotation Most of the high-throughput PPI work is done in model organisms Can you transfer that annotation a homologous gene in a different organism?

Defining homologs Orthologue of a protein is usually defined as the best-matching homolog in another species Candidates with significant BLASTP E-value (<10-20) Having ≥80% of residues in both sequences included in BLASTP alignment Having one candidate as the best-matching homologue of the other candidate in corresponding organism

Interologs If two proteins, A and B, interact in one organism and their orthologs, A’ and B’, interact in another species, then the pair of interactions A—B and A’—B’ are called interologs Align the homologs (A & A’, B & B’) to each other. Determine the percent identity and the E-value of both alignments Then calculate the Joint identity and the Joint Evalue Joint E-value Joint identity

Transfer of annotation Compared interaction datasets between yeast, worm and fly Assessed chance that two proteins interact with each other based on their joint sequence identities Performed similar analysis based on joint E-values All protein pairs with JI ≥ 80% with a known interacting pair will interact with each other More than half of protein pairs with JE  E-70 could be experimentally verified. Yu, H. et. al. (2004) Genome Res. 14: 1107-1118 PMID: 15173116

Examples of Protein-Protein Interologs In C. elegans, mpk-1 was experimentally shown to interact with 26 other proteins (by yeast 2-hybrid) Ste5 is the homolog of Mpk-1 in S. cerevisiae Based on the similarity between the interaction partners of mpk-1 and their closest homologs in S. cerevisiae, the interolog approach predicted 5 of the 6 subunits of the Ste5 complex in S. cerevisiae

This paper has been cited >100 times Why the interest in predicting protein-protein interactions? Determining protein-protein interactions is challenging and the high-throughput (genome-wide) methods are still difficult and expensive to conduct Identifying candidate interaction partners for a targeted pull-down assay is a more viable strategy for most labs

Today in computer lab Explore your gene lists using STRING Finding PPIs in your sublist using IntAct Continue Exercise 5 on protein analyses by exploring possible PPI for your select genes