Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network

Abstract

In eukaryotic cells the stability and function of many proteins are regulated by the addition of ubiquitin or ubiquitin-like peptides. This process is dependent upon the sequential action of an E1-activating enzyme, an E2-conjugating enzyme, and an E3 ligase. Different combinations of these proteins confer substrate specificity and the form of protein modification. However, combinatorial preferences within ubiquitination networks remain unclear. In this study, yeast two-hybrid (Y2H) screens were combined with true homology modeling methods to generate a high-density map of human E2/E3-RING interactions. These data include 535 experimentally defined novel E2/E3-RING interactions and >1300 E2/E3-RING pairs with more favorable predicted free-energy values than the canonical UBE2L3-CBL complex. The significance of Y2H predictions was assessed by both mutagenesis and functional assays. Significantly, 74/80 (>92%) of Y2H predicted complexes were disrupted by point mutations that inhibit verified E2/E3-RING interactions, and a approximately 93% correlation was observed between Y2H data and the functional activity of E2/E3-RING complexes in vitro. Analysis of the high-density human E2/E3-RING network reveals complex combinatorial interactions and a strong potential for functional redundancy, especially within E2 families that have undergone evolutionary expansion. Finally, a one-step extended human E2/E3-RING network, containing 2644 proteins and 5087 edges, was assembled to provide a resource for future functional investigations.

Figure 1.

Binary human E2/E3-RING protein interaction networks. (A) Previously known interactions derived from the MINT, IntAct, BioGRID, and HPRD databases at the time of this study. (B) Predicted human E2/E3-RING interactions including Interologs (purple edges) or non-Interolog predicted interactions from Hi-map and IntNet databases (orange edges). (C) Increased coverage within the human E2/E3-RING interaction space as a result of this study. Novel interactions are shown as red edges. Bold edges represent interactions confirmed by our data. Blue nodes represent E2 ubiquitin conjugating enzymes, while yellow nodes represent E3-RING proteins. To aid network analysis and node identification, all networks are provided as ready-to-view Cytoscape files (Supplemental File 3).

Figure 2.

Analysis of data from targeted Y2H experiments. (A) The novelty of Y2H data was determined relative to known interactions contained within the MINT, IntAct, BioGRID, and HPRD databases, known Interolog interactions, or non-Interolog predicted interactions (derived from the Hi-map or IntNet databases). (B) Reconfirmation of known or predicted E2/E3-RING interactions is highly dependent upon the source of the predicted data. (C) Strategic mutagenesis studies were performed to establish the proportion of positive Y2H interactions that conform to the known molecular/structural requirements for E2/E3-RING complex formation (see experimental data presented in Supplemental File 5). (D) To assess the efficiency with which Y2H data can predict functionally active E2/E3-RING complexes, 51 different E2/E3-RING combinations were systematically tested for ubiquitination activity in vitro (see experimental data presented in Supplemental File 6). Activity profiles for specific E2/E3-RING complexes were then compared with Y2H data to establish the percentage correlation between the two data sets.

Figure 3.

Comparison of data from targeted Y2H studies and true homology modeling methods. (A) To assess the ability of true homology modeling methods to predict the probability of detecting E2/E3-RING complexes in Y2H assays, the predicted free-energy values for 3150 E2/E3-RING complexes were compared with experimental profiles detected in Y2H studies. A binary score (1/0 = interaction was/was not observed) was assigned for each complex tested in Y2H assays. SigmaPlot 10.0 (Systat) was then used to fit the set of free-energy/binary score data pairs to the logistic model: p(Y2H interaction | free-energy score = x) = a / (1 + exp (−(x + b) / c)), with values for the parameters a, b, and c obtained by nonlinear regression. The resulting regression curve is shown in black. Black vertical lines indicate the distribution of the predicted free-energy values for interacting and noninteracting pairs on the upper and lower horizontal axes, respectively. Predicted free-energy values were ordered and split into bins of 200, and the frequency of Y2H interactions within each bin is shown as a gray horizontal bar (bar width indicates the free-energy values covered by each bin). (B) Distribution of free-energy values for all predicted human E2/E3-RING complexes (open bars); “strong” Y2H interactions selected on −Ade and −His + 2.5 mM 3AT plates (black bars); “weaker” Y2H interactions selected on −His + 2.5 mM 3AT plates (dark gray bars); interactions tested but not detected in Y2H studies (light gray bars). Dashed line indicates the approximate predicted free-energy values for the structurally defined UBE2L3–CBL complex (−7.87 ΔG int kcal/mol).