The Interaction Mechanism of Intrinsically Disordered PP2A Inhibitor Proteins ARPP-16 and ARPP-19 With PP2A

Figures

• 

  FIGURE 1. The structure of PP2A. The 3D structure of PP2A. The scaffolding A-subunit (PR65α) is shown with magenta, regulatory B56γ subunit shown with green and the catalytic Cα-subunit with orange. The coordinates are taken from 2npp.pdb.

• 

  FIGURE 2. ARPP-19 and ARPP-16 are intrinsically disordered proteins having propensity to form three transient α-helices. (A) IUPRED2A analysis and secondary structure propensity (SSP) calculation of ARPPs. IUPred2 and Anchor2 score are represented with black and red curves, respectively. The disordered prediction score in black highlights disordered nature of ARPPs and disordered binding region prediction score in red suggests presence of multiple disordered binding regions which are shown as shaded region. SSP score for ARPPs calculated using 1Hα, 13Cα, and 13Cβ chemical shift shows that both ARPPs have propensity to form three α-helices. The positions of α-helices are highlighted with orange color. (B) The cross peaks in the 2D 15N-HSQC spectra of uniformly 15N labeled ARPPs are highly overlapped because of the degeneracy of the proton spectra dispersion indicating that the ARPPs lacks well-defined 3D structure. (C) The Kratky plot calculated from SAXS data exhibits rising curve with increasing angle, which corresponds to the scattering pattern of IDPs.

• 

  FIGURE 3. ARPPs have flexible N- and C-terminus whereas the core regions have restricted backbone motions. Plots for the spectral density function values at J(0), J(ωN), and J(0.87ωH) frequencies versus the residues of (A) ARPP-16 and (B) ARPP-19. Regions with restricted backbone motion are shaded.

• 

  FIGURE 4. ARPPs obtain both compact and extended conformations. (A,B) The representation of different conformers of ARPP-16 and ARPP-19 calculated by ENSEMBLE program using chemical shifts (Cα, Cβ, and H), R2 relaxation rates from NMR spectroscopy and the SAXS data. The structure of different conformers obtained from ENSEMBLE calculation shows the conformational freedom of ARPPs. The Dmax of each representative structure is shown. (C) Maximum distance (Dmax) distributions of ARPPs obtained from EOM analyses of the SAXS data plotted as function of frequency (a.u.) reveals that with both ARPPs, the compact conformations are more favored than extended ones.

• 

  FIGURE 5. The phosphorylation does not have significant effect on the structural properties of ARPPs. (A,B) The overlay 2D 15N-HSQC spectra of uniformly 15N labeled ARPP WT and phosphomutants reveals that the phosphomimicking mutations have no significant effect on ARPPs’ structural properties. The cross peaks in the 15N-HSQC spectra of both ARPP-16 and ARPP-19 phosphomutants are highly similar to WT spectra, showing low dispersion of amide proton chemical shifts. This indicates that the ARPPs’ phosphomutants do not induce formation of well-defined 3D structure. (A) The spectrum of WT ARPP-16 is shown in magenta, ARPP-16 S46E and ARPP-16 S88E in green and red, respectively. (B) The spectrum of WT ARPP-19 is shown in orange, ARPP-19 S62E and ARPP-19 in gray and maroon, respectively. (C,D) Dmax distributions of ARPPs’ WT and phosphomutants ARPPs obtained from EOM analyses of the SAXS data plotted as function of frequency (a.u.) reveals that with phosphomutants, the extended conformation is less favored than with WT.

• 

  FIGURE 6. Chemical shift perturbation of phosphomimetic mutants of ARPP-16 and ARPP19 (A) and (B) Chemical shift perturbation due to S46E and S88E mutations, respectively. (C,D) Chemical shift perturbation due to S62E and S104E mutations, respectively. Δδ [ppm] refers to the combined HN and N chemical shift changes, according to the equation: Δδ(HN, N) = sqrt[(ΔδHN)2 + 0.2(ΔδN)2]. Red arrow indicates the phosphomimetic mutation site. Positions of α-helices and the A-subunit (PR65) binding site are shown.

• 

  FIGURE 7. The A-subunit provides a scaffold for ARPPs in PP2A whereas B56 subunits regulate the specificity. The binding curves obtained from MST experiments show that both ARPPs interact strongly with the scaffolding A-subunit. The interaction between WT and phosphomimicking mutants with the A-subunit is similar. The ARPPs interact in different way with various B56 isoforms, and the strength of the interactions is dependent on the phosphorylation state and site. MST measurements were performed using fluorescent labeled A- or B56 subunit as a target and unlabeled ARPP as a ligand.

• 

  FIGURE 8. The PP2A A-subunit binds to the 2nd transient α-helices. HSQC spectra of 15N-labeled ARPPs collected before and after the addition of the PP2A A-subunit reveals the A-subunit interacts with the region that based on SSP calculations form a transient α-helices. (A) The overlaid of 15N-HSQC of 15N-labeled free ARPP-16 (magenta), 1:1 (green), 1:2 (red) ARPP-16: PP2A A-subunit. The NH cross peaks that broadened with 1:2 PP2A A-subunit-ARPP-16 are labeled red, whereas the cross peaks that shifted the most and exhibit significant line broadening are labeled with magenta and the NH correlations with small CSPs are labeled blue. Those NH cross peaks whose intensity and position remained intact are labeled with black color. (B) ARPP-16 sequence showing the epitope that interacts with the A-subunit of PP2A as well as the positions of transient α-helices obtained from SSP calculations are shown. (C) The overlaid of 15N-HSQC of 15N-labelled free ARPP-19 (orange), 1:1 (gray) and 1:2 (maroon) ratio with PP2A A-subunit. The effect to the addition of 1:2 ratio of A-subunit into ARPP-19 are labeled similarly as in panel (A). (D) ARPP-16 sequence showing the region that interacts with the A-subunit…