Structure- and modeling-based identification of the adenovirus E4orf4 binding site in the protein phosphatase 2A B55α subunit

Abstract

Background: The adenovirus E4orf4 protein must bind protein phosphatase 2A (PP2A) for its functions.

Results: The E4orf4 binding site in PP2A was mapped to the α1,α2 helices of the B55α subunit.

Conclusion: The E4orf4 binding site in PP2A-B55α lies above the substrate binding site and does not overlap it.

Significance: A novel functional significance was assigned to the α1,α2 helices of the PP2A-B55α subunit. The adenovirus E4orf4 protein regulates the progression of viral infection and when expressed outside the context of the virus it induces nonclassical, cancer cell-specific apoptosis. All E4orf4 functions known to date require an interaction between E4orf4 and protein phosphatase 2A (PP2A), which is mediated through PP2A regulatory B subunits. Specifically, an interaction with the B55α subunit is required for induction of cell death by E4orf4. To gain a better insight into the E4orf4-PP2A interaction, mapping of the E4orf4 interaction site in PP2A-B55α has been undertaken. To this end we used a combination of bioinformatics analyses of PP2A-B55α and of E4orf4, which led to the prediction of E4orf4 binding sites on the surface of PP2A-B55α. Mutation analysis, immunoprecipitation, and GST pulldown assays based on the theoretical predictions revealed that the E4orf4 binding site included the α1 and α2 helices described in the B55α structure and involved at least three residues located in these helices facing each other. Loss of E4orf4 binding was accompanied by reduced contribution of the B55α mutants to E4orf4-induced cell death. The identified E4orf4 binding domain lies above the previously described substrate binding site and does not overlap it, although its location could be consistent with direct or indirect effects on substrate binding. This work assigns for the first time a functional significance to the α1,α2 helices of B55α, and we suggest that the binding site defined by these helices could also contribute to interactions between PP2A and some of its cellular regulators.

Keywords: Adenovirus; Bioinformatics; PP2A; Protein-Protein Interactions; Signal Transduction.

FIGURE 1.

Prediction of the B55α protein-protein interaction sites based on structure conservation. A, results of the analysis of the PP2A holoenzyme by the ConSurf program are shown. The scaffolding A and the catalytic C subunits are represented by ribbons colored green and yellow, respectively. B55α is represented by a space-filling model, colored by the following conservation scale: dark purple residues are the most conserved; white residues are the average on the conservation scale; cyan residues are variable. Known protein-protein interaction sites are marked by arrows. B, the surface of B55α was colored according to the ProMate prediction of protein-protein interaction sites as follows; residues with a higher probability for involvement in protein-protein interactions are marked in darker red, and residues with a low probability are in blue. In addition to the correctly predicted interfaces with the scaffolding (green) and catalytic (yellow) subunits, two more sites have been predicted to be involved in protein-protein interactions and are marked by arrows as potential E4orf4 interacting sites.

FIGURE 2.

Modeling the E4orf4 structure. A, the first model of E4orf4 structure proposed by the Quark program is shown. The N terminus and C terminus loops are marked as well as residue 104, which is the last residue retained in the models used for further analysis. B, ProSA analysis of the first E4orf4 model offered by the Quark program is shown, demonstrating the positive energy for the C-terminal loop, marked with an arrow, and a satisfying negative energy for the rest of the protein. The analysis was done using two window sizes of 10 or 40 aa. This analysis has been repeated for all E4orf4 models with similar results. C, superposition of three Quark models of E4orf4: Q1, Q3, Q5. The structural alignment of Q1, Q3, and Q5 presents a high similarity in the α-helical structure yet shows differences in the inner structure, such as inner angels and the position of the N-terminal loop. D, E4orf4 models share the same α-helical structure with highly stabilizing H-bonds. These models (Blue, Q1; red, Q3; green, Q5) are different in the orientation of the helices, as can be seen by the Arg-81 residue, protruding out of the helix at different angels with stabilizing H-bonds in Q1 and Q5 or without them in Q3.

FIGURE 3.

ClusPro predicts that E4orf4 may bind the PP2A B55α subunit at one of two major sites. The B55α surface is represented in the ProMate color-coded prediction of protein-protein interacting sites, as shown in Fig. 1B, whereas the surfaces of the A and C subunits are represented in colored dots: green for A and yellow for C. The E4orf4 docking solutions, shown in various colors for the different solutions, are clustered in two sites, marked as Left and Right sites.

FIGURE 4.

A histogram of B55α residues recurring in the E4orf4 docking predictions. The histogram summarizes the percentage of recurrence of B55α residues in ClusPro docking solutions for E4orf4 binding by PP2A-B55α. The residues shown here received percentage of appearance higher than the set 50% threshold and were divided according to their attributed site: left in black, and right in gray lines.

FIGURE 5.

B55α mutants bind E4orf4 at significantly reduced levels. A, clone 13 cells were transfected with an empty vector (Vec) or with plasmids expressing HA-tagged wild type B55α (WT) or various mutant B55α proteins. Cell lysates were immunoprecipitated with HA-specific antibodies, and immune complexes as well as input lysates were separated by SDS-PAGE and subjected to Western blot analysis with E4orf4- or PP2A-B55-specific antibodies. Input protein levels represent 10% of total protein amounts used for the immunoprecipitation (IP). The asterisk marks the endogenous PP2A-B55 band, which is detected similarly in all lanes, thus serving as a loading control. B, the levels of B55α and E4orf4 proteins in the immune complexes were quantified by densitometry, and E4orf4 binding levels were normalized to B55α expression levels. E4orf4 binding by WT B55α was defined as 1. Mutants L2 and R1, which were expressed at significantly reduced levels, were not subjected to the quantitative analysis. The graph summarizes the results of three independent experiments, and error bars represent S.E. A paired one-tailed t test indicated that E4orf4 binding to R4 and R5 was reduced significantly relative to its binding to WT B55α (*, p = 0.009 for R4; **, p = 0.0005 for R5), whereas there was no statistically significant difference between the binding of the other three well expressed mutants and the WT protein. C, experiments with additional B55α mutants were carried out as in A. Western blots were stained with antibodies to the HA tag, PP2A-C, and E4orf4. Double mutants were R4,6: R4+R6; R4,5: R4+R5. D, GST fused to the WT B55α subunit of PP2A or to B55α mutants as well as GST alone and His-E4orf4 were expressed in bacteria. The GST proteins were purified on glutathione beads and incubated with His-E4orf4. Bound E4orf4 and input GST proteins were detected by a Western blot.

FIGURE 6.

Loss of E4orf4 binding reduces the ability of B55α mutants to enhance E4orf4-induced cell death. IRBα cells were transfected with an empty vector or a vector expressing E4orf4 together with plasmids expressing WT or mutant B55α proteins or the corresponding empty vector (Vec). Cell death was determined 24 h later by measuring the percentage of transfected cells exhibiting nuclear condensation and fragmentation. The average of two independent experiments with two duplicates each is shown. Error bars represent the S.E. *, p < 0.003.

FIGURE 7.

The E4orf4 binding site in PP2A-B55α. A, the location of the B55α residues Phe-280, Tyr-337, and Phe-343 required for E4orf4 binding is shown. The B55α subunit is colored according to the Promate protein-protein interaction prediction as described in Fig. 1B, and the B55α residues Phe-280, Tyr-337, and Phe-343 are colored in yellow. The PP2A A subunit is mesh-surfaced in light blue, and the ribbon representing the C subunit is colored in green. B, the E4orf4 binding site in B55α does not overlap with the Tau binding site. B55α residues Phe-280, Tyr-337, and Phe-343 are colored in yellow, and the Tau protein binding residues Glu-27, Lys-49, and Asp-197 (28) are colored in magenta. The B55α subunit is colored in green. The PP2A A and C subunits are represented as described in A, except that the C subunit is colored red. C, the B55α residues Phe-280, Tyr-337, and Phe-343 are part of the α1 and α2 helices and face each other. The B55α structure including the core β-propeller (ribbons) and the α-helices (cylinders) is shown. Residues Phe-280, Tyr-337, and Phe-343 are colored in green. A close-up of the α1,α2 helices is shown on the right, displaying residues Phe-280, Tyr-337, and Phe-343 as they protrude from the α1 and α2 helices, facing each other.