Uncovering Quantitative Protein Interaction Networks for Mouse PDZ Domains Using Protein Microarrays

Abstract

One of the principal challenges in systems biology is to uncover the networks of protein-protein interactions that underlie most biological processes. To date, experimental efforts directed at this problem have largely produced only qualitative networks that are replete with false positives and false negatives. Here, we describe a domain-centered approach--compatible with genome-wide investigations--that enables us to measure the equilibrium dissociation constant (K(D)) of recombinant PDZ domains for fluorescently labeled peptides that represent physiologically relevant binding partners. Using a pilot set of 22 PDZ domains, 4 PDZ domain clusters, and 20 peptides, we define a gold standard dataset by determining the K(D) for all 520 PDZ-peptide combinations using fluorescence polarization. We then show that microarrays of PDZ domains identify interactions of moderate to high affinity (K(D) < or = 10 microM) in a high-throughput format with a false positive rate of 14% and a false negative rate of 14%. By combining the throughput of protein microarrays with the fidelity of fluorescence polarization, our domain/peptide-based strategy yields a quantitative network that faithfully recapitulates 85% of previously reported interactions and uncovers new biophysical interactions, many of which occur between proteins that are co-expressed. From a broader perspective, the selectivity data produced by this effort reveal a strong concordance between protein sequence and protein function, supporting a model in which interaction networks evolve through small steps that do not involve dramatic rewiring of the network.

A Network Analysis of Changes in Molecular Interactions in Cellular Signaling

“…Other recent examples for microarray-based approaches in the highly parallel analysis of molecular interactions have inferred possible signal transduction networks from interactions obtained with recombinant proteins (24,25). In contrast, the interaction network derived by our approach provides functional information on activation-dependent changes in molecular interactions.…”

“…In contrast to networks inferred from a systematic mapping of molecular interactions using e.g. yeast two-hybrid screens (4) or microarrays with immobilized recombinant protein domains (24,25), this network carries information on changes in the pattern of molecular interactions in the signaling-active cells themselves, expressing endogenous proteins only. PAK, p21-activated kinase, serine/threonine-protein kinase; WIP, WASP-interacting protein; FYB, FYN-binding protein; pY, phosphotyrosine.…”

Microarray-based enzyme profiling: Recent advances and applications (Review)

“…20,37,38 These experiments are maturing to a point that they are not just being carried out with purified targets, but even with enzymes within whole proteomes or cellular lysates, [39][40][41] in order to interrogate protein-protein interactions and analyze the pathways involved in regulating protein function. [42][43][44][45] Enzymes are an integral part of every cellular process and metabolic exchange. They are proteins that not only sustain life but are also implicit to its regulation and evolution.…”

Large-Scale Protein-Protein Interaction Detection Approaches: Past, Present and Future

“…In addition, expressing domains rather than entire proteins increases the chances of producing and isolating soluble proteins in suffi cient quantities in bacteria (92). Up to date, a number of seminal PPI studies took that route (42,63,91,92).…”