Disease mutations and phosphorylation alter the allosteric pathways involved in autoinhibition of protein phosphatase 2A

Abstract

Mutations in protein phosphatase 2A (PP2A) are connected to intellectual disability and cancer. It has been hypothesized that these mutations might disrupt the autoinhibition and phosphorylation-induced activation of PP2A. Since they are located far from both the active and substrate binding sites, it is unclear how they exert their effect. We performed allosteric pathway analysis based on molecular dynamics simulations and combined it with biochemical experiments to investigate the autoinhibition of PP2A. In the wild type (WT), the C-arm of the regulatory subunit B56δ obstructs the active and substrate binding sites exerting a dual autoinhibition effect. We find that the disease mutant, E198K, severely weakens the allosteric pathways that stabilize the C-arm in the WT. Instead, the strongest allosteric pathways in E198K take a different route that promotes exposure of the substrate binding site. To facilitate the allosteric pathway analysis, we introduce a path clustering algorithm for lumping pathways into channels. We reveal remarkable similarities between the allosteric channels of E198K and those in phosphorylation-activated WT, suggesting that the autoinhibition can be alleviated through a conserved mechanism. In contrast, we find that another disease mutant, E200K, which is in spatial proximity of E198, does not repartition the allosteric pathways leading to the substrate binding site; however, it may still induce exposure of the active site. This finding agrees with our biochemical data, allowing us to predict the activity of PP2A with the phosphorylated B56δ and provide insight into how disease mutations in spatial proximity alter the enzymatic activity in surprisingly different mechanisms.

FIG. 1.

C-terminal SLiM loosening in MD simulations. (a) Overview of the PP2A holoenzyme structure with the B56δ subunit (white). The catalytic subunit is colored green, and the scaffold subunit is colored blue. The N-arm and C-arms of the B56δ are colored yellow and magenta, respectively. The serine residues phosphorylated in WT4P are labeled. The top inlay shows that a bound substrate (from PDBID: 2NPP) is incompatible with the closed C-arm due to steric clashes. The magnitude of the C-arm SLiM fluctuation is depicted in the bottom inlay. (b) Root mean square fluctuation of the terminal 20 C-arm residues. The SLiM residues are underlined. The error bars correspond to the standard deviation of the mean calculated by bootstrapping the trajectories 30 times.

FIG. 2.

The E198K mutation and phosphorylation of B56δ loosen the allosteric connection to SLiM. (a) Allosteric pathways from mutation to the SLiM in the WT, E198K, E200K, and WT4P. The C-arm is colored magenta, and the SLiM residues are labeled in WT. The percentage labels reflect the number of paths in channel 1 or channel 2 (labeled blue and orange, respectively). (b) The mean weight of allosteric channels beginning at the mutation sites and ending on the SLiM normalized by the WT. The confidence interval corresponds to the standard deviation of the mean based on networks derived from ten samples of bootstrapped trajectories. (c) The number of contacts between the C-arm SLiM residues and the core of B56δ. (d) Effects of mutations on substrate-SLiM binding assessed by the pulldown of WT and mutant holoenzymes by GST-tagged B56 SLiM in the substrate. Each data point is normalized by the mean value for WT. See Sec. II for details of the GST-mediated biophysical pulldown assay. The P values were obtained with Welch’s t-test.

FIG. 3.

Optimal pathways starting from the mutation sites and ending on the C-arm SLiM residues. The source residues (E198 and E200) are colored yellow, and sink residues (C-arm SLiM residues, L595, S598, and E600) are colored red. Residues belonging exclusively to channel 1 are colored blue. Residues belonging exclusively to channel 2 are colored orange. Residues shared among channels are green. The thickness of the arrows corresponds to the number of pathways connecting two residues.

FIG. 4.

Modifications of B56δ loosen the allosteric pathway to the active site. (a) Allosteric pathways from mutation to the active site residues in the WT, E198K, E200K, and WT4P. The paths belonging to each variant are colored blue, orange, green, and red, respectively. (b) Mean and standard deviation of path weights from the mutation sites to the active site residues D57 and D58 obtained from networks of ten bootstrapped trajectories normalized by the WT. (c) Enzyme kinetics of the WT and mutant PP2A-B56δ holoenzyme activity toward a SLiM-independent substrate, KRpTIRR.

FIG. 5.

Exposition of S573 to the solvent and its phosphorylation rates. (a) SASA of S573 in variants of PP2A. (b) Effects of B56δ disease mutations on S573 phosphorylation of the holoenzyme by PKA in vitro. The data are normalized to the maximum phosphorylation of WT in a 40-min time course. The number of repeats, their scatter plots, averages, and SEM are shown. The P values for the comparison of WT and disease variants, E198K and E200K, are 0.002 and 0.3, respectively (calculated using two-sided Wilcoxon rank sum test).