Protein-protein interactions Ia. A combined algorithm for genome-wide prediction of protein function. Edward M. Marcotte, Matteo Pellegrini, Michael J. Thompson, Todd O. Yeates, David Eisenberg(1999) Nature 402, Protein function in the post-genomic era. David Eisenberg, Edward M. Marcotte, Ioannis Xenarios & Todd O. Yeates(2000) Nature 405,

FUNCTIONAL RELATIONSHIPS AMONG PROTEINS: GENOME-WIDE PREDICTION (FUNCTIONAL GENOMICS) Does not rely on DIRECT SEQUENCE HOMOLOGY 3 independent predictions methods & available experimental data.

STRATEGIES USED TO “FUNCTIONALLY LINK” PROTEINS: 6217 yeast proteins Correlated Evolution: Related Phylogenetic Profiles (pattern of presence or absence of a particular protein across a set of organisms whose genomes have been sequenced): proteins, which operate together in a common pathway or complex, are inherited together. Correlated mRNA Expression Patterns: Correlated mRNA Expression Patterns under different growth conditions Correlated Patterns Of Domain Fusion: Link 2 proteins whose homologs are fused into a single gene (Rosetta stone sequences) in another organism.

STRATEGIES USED TO “FUNCTIONALLY LINK” PROTEINS:(continued) Gene Neighbour Method: if in several genomes, the genes that encode 2 proteins are neighbors on the chromosome, the proteins tend to be functionally linked Experimental Evidence: Mass spectrometry, Coimmunoprecipitaion, Yeast 2-hybrid data (DIP, MIPS yeast genome db) Metabolic pathway neighbours: Proteins, which participate in same metabolic pathway, common structural complex or biological process or closely related physiological function: BLAST homology searches and pairwise links were defined between yeast proteins whose E.Coli homologs catalyse sequential reactions in a metabolic pathway (EcoCyc db)

RESULTS: Phylogenetic profiles: 20,749 links mRNA expression patterns: 26,013 links Domain fusion method: 45,502 links 93,750 pairwise functional links among 76% (4,701) of yeast proteins 4130: “HIGHEST CONFIDENCE” links (experimental proof, valid by 2 of 3 prediction methods) 19,251: “HIGH CONFIDENCE”links: (predicted by phylogenetic profiles) Remainder predicted by domain fusion or correlated mRNA expression patterns

VALIDATION : Excellent reliability if 2 or more prediction methods agreed on a link. These methods link many proteins that are already known to function together on the basis of experiments. (Ribosomal proteins, proteins from flagellar motor apparatus and metabolic pathways) “Keyword recovery”: Prediction could be compared to the actual annotation: compare keyword annotation on SwissPDB, for both members of each pair of proteins, linked by one of the methods-possible when the members have known function. “Keyword recovery”: if keywords match. Average signal to noise ratio for “Keyword recovery”: Phylogenetic profiles: 5 mRNA expression patterns: 2 When 2 prediction methods gave same linkage: 8 Direct experimental data: 8

OUTCOME: Functional links between proteins of unknown function: General function assigned to more than half of 2557 previously uncharacterized yeast proteins: 15% from high and highest confidence links, 62% using all links. Functional Links Between Non-Homologous Proteins: beyond traditional “sequence matching”: Sup35, MSH6 Discovery of potential interactions within and across cellular processes and compartments. Connections represent a “gold mine” for experimentally testing specific hypotheses about gene function. Viewing protein-protein interactions globally as a network and not as binary data sets, increases the confidence levels for individual interactions: inspection of interaction web at different steps identifies “unexpected” links between previously unconnected cellular processes.

Ib. A network of protein-protein interactions in yeast. Schwikowski B, Uetz P, Fields S. (2000). Nat Biotechnol. 18,

DATA SOURCE: MIPS site YPD DIPS Yeast-2-hybrid studies Biochemical experimental data

Prediction of function: Annotated functions of all neighbors of P are ordered in a list, from the most frequent to the least frequent. Functions that occur the same number of times are ordered arbitrarily. Everything after the third entry in the list is discarded, and the remaining three or fewer functions are declared as predictions for the function of P. Evaluation of the quality of the links: For unknown protein, test predicted function

RESULTS: Analyzed 2,709 published interactions involving 2,039 yeast proteins Single large network containing 2,358 links among 1,548 individual proteins.Other networks had few proteins. 65% of the interactions in the complete set of networks occur among proteins with at least one common functional assignment. 78% of the 1,432 interactions between proteins of known localization, the proteins share one or more compartments. Correctly predicted a functional category for 72% of 1393 characterised proteins, with at least one partner of known function. Cross-talk between and within functional groups/subcellular compartments. Local function vs Contextual/cellular function (extended web of interacting molecules) Predicted functions of 364 uncharacterised proteins.

Reliability of the generated networks: 1,393 of the 2,039 proteins were annotated with some function and had at least one neighbor annotated with a function. In 1,005 of these 1,393 cases (72.1%), at least one annotated function was predicted correctly by the above method. Performed the same prediction algorithm 100 times on the basis of randomly generated interactions. Only 12.2% of the predictions yielded a prediction that agreed with the known annotation.

PROBLEMS… Interactions of membrane proteins underrepresented: Y2H data Y2H data: lots of false positives. Only 15% agreement between this interaction data and Marcotte’s “high quality” prediction data. Uncertainities remain that WILL require additional experimentation.

CHALLENGES: Protein complexes are not static: change with metabolic state of cell, external stimuli etc. Protein chip technology: used to study transient interactions: amenable to variety of assays like nucleotide-binding, enzymatic activity etc.

II. Mapping protein family interactions: intramolecular and intermolecular protein family interaction repertoires in the PDB and yeast. Park J, Lappe M, Teichmann SA. (2001). J Mol Biol. 307,

Protein DOMAIN interactions: interactions between whole structural families of evolutionarily related domains as opposed to interactions between individual proteins. Types of domain interactions: 1) Domain-domain(intra-chain) interactions in multi-domain polypeptide chains 2) Inter-chain protein interactions in multi- subunit protein complexes. 3) In transient complexes between proteins, which can also exist independently

METHODS: Protein superfamilies from SCOP db Interactions between families in the PDB: (domains of known 3D structure) coordinates of each domain were parsed to check whether there are 5 or more contacts with 5A  to another domain Interactions between families in the yeast genome: by homology: -Protein structures assigned to the yeast proteins using the domains from SCOP as queries in PSI-BLAST. -Yeast sequences also compared to the PDB-ISL with FASTA Assumption: Within polypeptide chains, structural domains interact if there are less than 30 amino acids separating them. If one family F has 2 domains, a and b, and each of these interacts with a domain from a different family, then the number of interaction families for F will be 2.

RESULTS: 1 st attempt at classifying interactions between all the known structural protein domains according to their families. Could classify 8151 interactions between individual domains in the PDB and the yeast in terms of 664 types of interactions between pairs of protein families. Scale free network: Most protein families only interact with 1 or 2 other families. A few families are extremely versatile in their interactions and are connected to many families (Hubs in the graph)-functional reasons. Eg: -Immunoglobulins, P-loop nucleotide triphosphate hydrolases In 45% of all families in the PDB, domains interact with other domains from the same family: internal duplication and domain oligomerisation is favourable. Pairs of families that interact both within and between polypeptide chains belong mostly to 2 types of domains: enzyme domains and domains from the same family.

PROBLEMS: Multi-domain proteins: cannot resolve exactly which domains are interacting: not used Members of 2 families can sometimes interact in different ways, using different types of interface (different modes of oligomerisation of nucleoside diphosphate kinases) Does not take account of symmetric homooligomers, of which only one monomer is in the PDB entry and hence the number of homomultimeric family interactions may be underestimated.

FUTURE: 51 new interactions between superfamilies: potential targets for structure elucidation and experimental investigation of these interacting polypeptides that do not have analogs in the PDB. For interactions in which one partner does not have a structural assignment, possible structures can be picked up from the set of known family interactions Database of domain-domain interfaces