Cryo-EM structures of PP2A:B55-FAM122A and PP2A:B55-ARPP19

Abstract

Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation.¹ Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases,² while mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B55.³ While the role of kinases in mitotic entry is well-established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited.⁴ For PP2A:B55, inhibition is achieved by the two intrinsically disordered proteins (IDPs), ARPP19 (phosphorylation-dependent)^6,7 and FAM122A⁵ (inhibition is phosphorylation-independent). Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies both IDPs bind PP2A:B55, but do so in highly distinct manners, unexpectedly leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provides a molecular roadmap for the development of therapeutic interventions for PP2A:B55 related diseases.

Figure 1.. ARPP19 and FAM122A inhibit PP2A:B55.

a. Current understanding of the roles of ARPP19 and FAM122A in regulating PP2A:B55 activity during the cell cycle. b. IC₅₀ curves for PP2A:B55 inhibition by ARPP19 (with and without phosphorylation), FAM122A_Nterm and FAM122A_ID. IC₅₀ values are reported in Table 1. Data are presented as mean values ± SD, n = 3 experimental replicates. c. 2D [¹H,¹⁵N] HSQC spectrum of ¹⁵N-labeled ARPP19 alone (black) and in complex with PP2A:B55 (green). d. Peak intensity vs ARPP19 protein sequence plot for ARPP19 alone (black) and when bound to PP2A:B55 (green); grey box highlights ARPP19 residues with reduced intensities when bound to PP2A:B55. Secondary structure elements based on NMR CSI data are indicated. e. Peak intensity vs FAM122A protein sequence plot for FAM122A_Nterm alone (black) and when bound to PP2A:B55 (green); grey box highlights FAM122A residues with reduced intensities when bound to PP2A:B55. Secondary structure elements based on NMR CSI data are indicated. f. 2D [¹H,¹⁵N] HSQC spectrum of ¹⁵N-labeled ARPP19 alone (black) and in complex with B55_LL (pink). g. Peak intensity vs ARPP19 protein sequence plot for ARPP19 alone (black) and when bound to B55_LL (pink); grey box highlights ARPP19 residues with reduced intensities when bound to B55_LL. h. Peak intensity vs FAM122A_ID protein sequence plot for FAM122A_ID alone (black) and when bound to B55_LL (blue); grey box highlights FAM122A_ID residues with reduced intensities when bound to B55_LL.

Figure 2.. Structure of the PP2A:B55-tpARPP19 and PP2A:B55-FAM122A complex.

a. Cryo-EM map and model of the PP2A:B55-tpARPP19 complex. Two views of the map (top) are shown next to the corresponding view of the molecular model (bottom). b. Cryo-EM map and model of the PP2A:B55-FAM122A complex. Same views as in a. c. Overlay of PP2Aa from the PP2A:B55 crystal structure (pdbid 3DW8; beige) and the PP2A:B55-inhibitor model (grey), superimposed using heat repeats 1–6. d. Rotated view of the experimental map; PP2Aa is transparent to show the extended PP2Ac C-terminal tail. e. Overlay of the PP2Ac catalytic domains from PP2A:B55-FAM122A (cyan) and PP2A:B56 (brown; pdbid 2IAE), with the C-terminal tails (aa R294-mL309) shown as a surface. The presence of B55 or B56 in the PP2A holoenzyme results in distinct binding sites for the PP2Ac C-terminal tail. f. B55 shown as a surface and colored by sequence conservation using the scale shown; ARPP19 (orange) and FAM122A (grey) bind to the most conserved B55 surface. The location of the B55 platform is also indicated.

Figure 3.. tpARPP19 binding to PP2A:B55.

a. Cartoon describing the binding of tpARPP19 to PP2A:B55; terminology for the multiple binding sites. b. 5 alanine mutational scan through amino acids L32-E86 of ARPP19. The indicated YFP-ARPP19 constructs were transfected into HeLa cells and subsequently immunopurified. Binding efficiency of the YFP-ARPP19 derivatives to B55a was determined by Western blotting. c. Site 1: ARPP19 helix α3 interaction with the B55 binding platform. d. Helical wheel of ARPP19 helix α3. Residues that interact with B55 are colored orange; solvent exposed residues are light orange. Colors of the labels indicate residue within the same helical turn. Primary sequence colored as helix and key interacting residues are underlined. e. Single amino acid change to alanine pull-down analysis of ARPP19 helix α3. f. ARPP19 α3-α4 kink; dramatic splaying of Y59_R, F60_R and D61_R allow for this conformation to occur. g. Phosphorylated S62 binding to the PP2Ac active site. h. ARPP19 folds back on itself and binds B55 at site 3. ARPP19 helices α3/α4 are shown in orange; ARPP19 backwards folding is shown in yellow (all surface), highlighting the crossover above the α3-α4 kink (see e.) and the B55 binding site 3 (over 50 Å distant from helix α2). i. Intramolecular interactions at the ARPP19 crossover. j. ARPP19 binding at B55 binding site 3 k. Overlay of the PPP active site residues of PP2A:B55-tpARPP19 (orange, tpS62 ARPP19, cyan PP2Ac), PP1:phosphate (4MOV; PP1, green) and a pre-dephosphorylation complex (7NZM; tpS51 eIF2α, pink; PP1, light pink) binds 2.9 Å away from the phosphate moieties of the pre- and post-dephosphorylation complexes; below, residues adjacent to S62_R that, when mutated, result in faster dephosphorylation of S62¹⁵. l. IC₅₀ curves for PP2A:B55 inhibition by different ARPP19 (with and without phosphorylation) constructs. IC₅₀ values are reported in Table 1. Data are presented as mean values +/− SD, n = 3 experimental replicates.

Figure 4.. FAM122A binding to PP2A:B55.

a. Cartoon describing the binding of FAM122A to PP2A:B55. b. FAM122A residues R84_F-E92_F form a 3-turn helix when bound to B55. c. Helical wheel of the B55 binding helix. Residues that interact with B55 are colored magenta; solvent exposed residues are light pink; colors of the labels indicate residue within the same helical turn. d. L85_F and I88_F bind into shallow pockets on B55 (electrostatic potential surface). e. Intra- and intermolecular interactions with Arg84_F (polar/ionic interactions shown as dashes). f. E92_F makes polar/ionic interactions (dashes) with multiple residues from B55. g. Mutations in the FAM122A SHelM negatively impact PP2A:B55 binding and inhibition (Table 2). h. Pulldown assay with SHelM variants shown in (e). Expi293F lysates co-transfected with GFP-B55 and PP2Ac-Strep were incubated with purified PP2Aa and FAM122A variants, pulled-down using a GFP-TRAP and immunoblotted for the indicated proteins. i. FAM122A residues Ile97_F-Met111_F form the inhibition helix that binds over the PP2Ac active site. j. Helical wheel N→C view of the B55 inhibition helix highlighting residues that interact with PP2Ac and those that are solvent exposed. k. Orientation of E104_F in the PP2A:B55 active site. l. FAM122A residues R105 and V107. m. IC₅₀ curves for PP2A:B55 inhibition by FAM122A_ID E104A, E106A (blue) and R105L, V107G (orange). R105L and V107G are clinical cancer variants (Table 1). Data are presented as mean values +/− SD, n = 3 experimental replicates.

Figure 5.. PP2A:B55 platform recruitment.

a. PP2A-B55 SHelM from interactors experimentally confirmed to bind the B55 platform. b. FAM122A displaces p107 from the B55 platform. 2D [¹H,¹⁵N] HSQC spectrum of ¹⁵N-labeled p107 alone (black) and in complex with B55_LL (red) shows that specific p107 residues bind B55_LL. The addition of an excess of unlabeled FAM122A displaces ¹⁵N-labeled p107 from B55_LL and now appears in the 2D [¹H,¹⁵N] HSQC spectrum (violet) in an identical position as in the 2D [¹H,¹⁵N] HSQC spectrum of ¹⁵N-labeled p107 alone (black). c. Model how FAM122A displaces substrates that bind the B55 platform in a p107 like manner. d. Overlay of PP2A:B55 bound to tpARPP19 (orange) or FAM122A (magenta). e. Zoom-in of the overlapping regions shown in (d). f. Overlay of tpARPP19 (orange) or FAM122A (magenta) on the B55 platform. g. Overlay of tpARPP19 (orange) or FAM122A (magenta) at the PP2Ac catalytic pocket (cyan, PP2Ac residues from the ARPP19 complex; grey, PP2Ac residues from the FAM122A complex); ionic interactions indicated by dashed lines. PP2Ac metals are shown as spheres. h. 2D [¹H,¹⁵N] HSQC spectrum of ¹⁵N-labeled FAM122A alone (black) and in complex with B55_LL (orange). i. The addition of unlabeled tpARPP19 does not alter the FAM122A:B55_LL complex spectrum (green; identical as orange 2D [¹H,¹⁵N] HSQC spectrum in d), highlighting that tpARPP19 and FAM122A can bind simultaneously. j. Model of FAM122A and ARPP19 binding B55 simultaneously.