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

Bacterial protein expression

PP2Aa_9–589, FAM122A_1–124 (FAM122A_Nterm), FAM122A_29–120 (FAM122A_ID), FAM122A_67–120, ARPP19, ARPP19_S104A, ARPP19_19–75 and p107_612–687 were subcloned into pTHMT containing an N-terminal His₆-tag followed by maltose binding protein (MBP) and a tobacco etch virus (TEV) protease cleavage site. For expression, plasmid DNAs were transformed into Escherichia coli BL21 (DE3) RIL or BL21 (DE3) cells (Agilent). Freshly transformed cells were grown at 37 °C in LB broth containing kanamycin antibiotics (50 µg ml⁻¹) until they reached an optical density (OD₆₀₀) of ~0.8. Protein expression was induced by addition of 1 mM β-d-thiogalactopyranoside (IPTG) to the culture medium, and cultures were allowed to grow overnight (18–20 h, 250 rpm shaking) at 18 °C. Cells were collected by centrifugation (8,000g, 15 min, 4 °C) and stored at −80 °C until purification. Expression of uniformly ¹³C- and/or ¹⁵N-labelled protein was carried out by growing freshly transformed cells in M9 minimal medium containing 4 g l⁻¹ [¹³C]-d-glucose and/or 1 g l^{−1 15}NH₄Cl (Cambridge Isotopes Laboratories) as the sole carbon and nitrogen sources, respectively. FAM122A_ID variants E92K, R105L, V107G, S120C, E104A/S120C, E106A/S120C, R84A/L85A/S120C, I88A/K89A/S120C, E91K/S120C, E92K/S120C, FAM_67-120S120C and ARPP19/S10C were generated by site-directed mutagenesis, sequence verified and expressed as described above.

Cell culture

Expi293F cells were obtained from ThermoFisher (A14527) and grown in HEK293 Cell Complete Medium (SMM293-TII, Sino Biological M293TII). For transient overexpression of B55 and PP2Ac constructs, cells were transfected using polyethyleneimine (PEI) transfection reagent. For western blot and immunoprecipitation studies, whole-cell extracts were prepared by lysing cells in ice-cold lysis buffer (20 mM Tris pH 8.0, 500 mM NaCl, 0.5 mM TCEP, 1 mM MnCl₂, 0.1% Triton X-100, Phosphatase inhibitor cocktail (ThermoFisher)), sonicating and clearing the lysate by centrifuging at 15,000g for 20 min at 4 °C. Total protein concentrations were measured using the Pierce 660 Protein Assay Reagent (ThermoFisher).

Mammalian protein expression

Full-length B55_1–477 was cloned into pcDNA3.4 including an N-terminal green fluorescence protein (GFP) followed by a TEV cleavage sequence. Full-length PP2Ac_1–309 was cloned into pcDNA3.4 with an N-terminal Strep tag followed by a TEV cleavage sequence. B55 loopless (B55_LL), in which B55 residues 126–164 that interact directly with PP2Aa were removed and replaced with a single NG linker (Fig. 1b), was cloned into pcDNA3.4 with an N-terminal GFP followed by a TEV cleavage sequence. All plasmids were amplified and purified using the NucleoBond Xtra Maxi Plus EF (Macherey-Nagel). B55_WT and B55_LL were individually expressed in Expi293F cells (ThermoFisher). B55_1–477 and PP2Ac_1–309 were co-expressed in Expi293F cells at a 1:2 DNA ratio.

Transfections were performed in 500 ml medium (SMM293-TII, Sino Biological) in 2 l flasks using polyethylenimine (Polysciences) reagent according to the manufacturer’s protocol in an incubator at 37 °C and 8% CO₂ under shaking (125 rpm). On the day of transfection, the cell density was adjusted to 2.8 × 10⁶ cells per ml using fresh SMM293-TII expression medium. DNA of PP2Ac and B55 (2:1 ratio) were diluted in Opti-MEM Reduced Serum Medium (ThermoFisher). Similarly, in a separate tube, PEI (3× the amount of DNA) was diluted in the same volume of Opti-MEM Reduced Serum Medium (ThermoFisher). The DNA and PEI mixtures were combined and incubated for 10 min at room temperature, before being added to the cell culture. Valproic acid (2.2 mM final concentration, Sigma) was added to the cells 4 h after transfection and 24 h after transfection sterile-filtered glucose (4.5 ml per 500 ml cell culture, 45%, glucose stock) was added to the cell culture flasks to boost protein production. Cells were collected 48 h after transfection by centrifugation (2,000g for 20 min, 4 °C) and stored at −80 °C.

FAM122A purification

Cell pellets expressing FAM122A_Nterm, FAM122A_ID and FAM_67–120 and variants were resuspended in ice-cold lysis buffer (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole, 0.1% Triton X-100, EDTA-free protease inhibitor tablet (ThermoFisher)), lysed by high-pressure cell homogenization (Avestin Emulsiflex C3). Cell debris was pelleted by centrifugation (42,000g, 45 min, 4 °C), and the supernatant was filtered with 0.22-µm syringe filters (Millipore). The proteins were loaded onto a HisTrap HP column (Cytiva) pre-equilibrated with buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole) and eluted using a linear gradient (0–60%) with buffer B (50 mM Tris pH 8.0, 500 mM NaCl and 500 mM imidazole). Fractions containing the protein were pooled and dialysed overnight at 4 °C with TEV protease (in house; His₆ tagged) to cleave the His₆–MBP tag. Following cleavage, the sample was either (1) loaded under gravity onto Ni²⁺-NTA beads (Prometheus) pre-equilibrated with buffer A, the flow through and wash A fractions were collected, and then twice heat purified (80 °C, 10 min) or (2) twice heat purified (80 °C, 10 min). Samples were centrifuged at 15,000g for 10 min to remove precipitated protein. Supernatant was concentrated and purified using size-exclusion chromatography (SEC; Superdex 75 26/60 (Cytiva)) in either NMR buffer (20 mM Na₂HPO₄/NaH₂PO₄ pH 6.3, 150 mM NaCl, 0.5 mM TCEP), IC₅₀ assay buffer (20 mM Tris pH 8.0, 150 mM NaCl, 0.5 mM TCEP) or fluorescence polarization assay buffer (20 mM Tris pH 7.0, 150 mM NaCl, 0.5 mM TCEP). Samples were either directly used for NMR data collection or flash frozen and stored at −80 °C.

ARPP19 purification

The protocol is identical for all ARPP19 constructs. Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 5 mM imidazole, 0.1% Triton X-100, EDTA-free protease inhibitor (ThermoFisher)), lysed by high-pressure cell homogenization (Avestin C3-Emulsiflex), cell debris pelleted by centrifugation (42,000g, 45 min), and the supernatant was filtered with 0.22-µm syringe filters (Millipore). The proteins were loaded onto a HisTrap HP column (Cytiva) pre-equilibrated with buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole), and eluted using a linear gradient (0–60%) of buffer B (50 mM Tris pH 8.0, 500 mM NaCl, 500 mM imidazole). Fractions containing the protein were pooled and dialysed overnight at 4 °C with TEV protease to cleave the MBP and His₆ tags. The cleaved protein was incubated with Ni²⁺-NTA resin (Cytiva) and washed with buffer A. The flow through and wash A fractions were collected, and heat purified by incubating the samples at 80 °C for 20 min. The samples were centrifuged at 15,000g for 10 min to remove precipitated protein, concentrated and purified using SEC (Superdex 75 26/60 (Cytiva)) in NMR buffer (20 mM Na₂HPO₄/NaH₂PO₄ pH 6.3, 150 mM NaCl, 0.5 mM TCEP), IC₅₀ assay buffer (20 mM Tris pH 8.0, 150 mM NaCl, 0.5 mM TCEP) or fluorescence polarization assay buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 0.25 mM TCEP). Purified samples were again heat purified (80 °C for 5 min), centrifuged at 15,000g for 10 min to remove any precipitated protein, and were either directly used for NMR data collection or flash frozen and stored at −80 °C.

PP2Aa purification

Cell pellets expressing PP2Aa_9-589 were resuspended in ice-cold lysis buffer (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole, 0.1% Triton X-100, EDTA-free protease inhibitor tablet (ThermoFisher)), lysed by high-pressure cell homogenization (Avestin Emulsiflex C3). Cell debris was pelleted by centrifugation (42,000g, 45 min, 4 °C), and the supernatant was filtered with 0.22-µm syringe filters. The proteins were loaded onto a HisTrap HP column (Cytiva) pre-equilibrated with buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole) and eluted using a linear gradient (0 to 40%) with buffer B (50 mM Tris pH 8.0, 500 mM NaCl and 500 mM imidazole). Fractions containing the protein were pooled and dialysed overnight at 4 °C with TEV protease (in house; His₆-tagged) to cleave the His₆–MBP tag and loaded under gravity onto Ni²⁺-NTA beads (Prometheus) pre-equilibrated with buffer A. Flow through and wash A fractions were collected, concentrated and loaded onto QTrap HP column (Cytiva) for further purification. The proteins were eluted with a 100 mM–1 M salt gradient (buffer A: 20 mM Tris pH 8.0, 100 mM NaCl, 0.5 mM TCEP; buffer B: 20 mM Tris pH 8.0, 1 M NaCl, 0.5 mM TCEP). PP2Aa fractions were concentrated and further purified using SEC (Superdex 200 26/60 (Cytiva)) in assay buffer (20 mM Tris pH 8.0, 150 mM NaCl, 0.5 mM TCEP). Samples were either directly used or flash frozen and stored at −80 °C.

MASTL expression and purification

Expi293F cells were transfected with pcDNA5_FRT_TO_3xFLAG_MASTL as described above. A cell pellet expressing MASTL was resuspended in ice-cold lysis buffer (20 mM Tris pH 8.0, 500 mM NaCl, 0.5 mM TCEP, 0.1% Triton X-100, EDTA-free protease inhibitor tablet (ThermoFisher)), lysed by high-pressure cell homogenization (Avestin Emulsiflex C3). Cell debris was pelleted by centrifugation (42,000g, 45 min, 4 °C), and the supernatant was filtered with 0.22-µm syringe filters (Millipore). Lysates were incubated with Anti-Flag M2 beads (Sigma), pre-equilibrated with wash buffer 1 (20 mM Tris pH 8.0, 500 mM NaCl and 0.5 mM TCEP) and slowly rocked at 4 °C for 2 h. Following, beads were washed 3 times with wash buffer (20 mM Tris pH 8.0, 500 mM NaCl, 0.5 mM TCEP, 1 mM MnCl₂) and bound MASTL protein was eluted by incubating with 150 ng µl⁻¹ 3× Flag peptide (Biosynthesis) for 10 min. Purified, active MASTL was mixed with 10% glycerol and stored at −80 °C.

PKA expression and purification

For expression, PKA (human Cα1 in pet15b) was transformed into E. coli BL21 (DE3) RIL cells (Agilent). Freshly transformed cells were grown at 37 °C in LB broth until they reached an optical density (OD₆₀₀) of ~0.8. Protein expression was induced by addition of 1 mM β-d-thiogalactopyranoside (IPTG) to the culture medium, and cultures were allowed to grow overnight (18–20 h, 250 rpm shaking) at 18 °C. Cells were collected by centrifugation (8,000g, 15 min, 4 °C) and stored at −80 °C until purification. For purification, cell pellets were resuspended in ice-cold lysis buffer (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole, 0.1% Triton X-100, EDTA-free protease inhibitor (ThermoFisher)) and lysed by high-pressure cell homogenization (Avestin Emulsiflex C3). Cell debris was pelleted by centrifugation (42,000g, 45 min, 4 °C), and the supernatant was filtered with 0.22-µm syringe filters (Millipore). The proteins were loaded onto a HisTrap HP column (Cytiva) pre-equilibrated with buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole) and eluted using a linear gradient (0–80%) with buffer B (50 mM Tris pH 8.0, 500 mM NaCl, 500 mM imidazole). Fractions containing the protein were pooled and dialysed overnight in the buffer (20 mM Tris pH 8, 50 mM NaCl, 1 mM EDTA, 2 mM DTT) at 4 °C. Purified sample was centrifuged at 15,000g for 10 min to remove precipitated protein. Supernatant protein sample was mixed with 50% glycerol and stored at −80 °C.

Phosphorylation of ARPP19

Purified ¹⁵N-labelled-ARPP19 (25 μM) was incubated with either PKA or MASTL kinase (10:1 ratio) in phosphorylation buffer (100 mM Tris pH 7.5, 2 mM DTT, 10 mM MgCl₂) with 500 µM of ATP-γ-S or ATP (Sigma) for thiophosphorylation and phosphorylation. The kinase reaction was left at 37 °C for 72−90 h. Phosphorylated ARPP19 was heat purified by incubating the samples at 80 °C for 10 min. The samples were centrifuged at 15,000g for 10 min to remove precipitated kinase and either immediately used for experiments or flash frozen and stored at −80 °C. Complete phosphorylation was confirmed by chemical shift changes of the phosphorylated serine residue(s) using 2D ¹H,¹⁵N HSQC spectra.

Immunoprecipitation and western blot for B55 versus B55_LL interaction with PP2Aa

GFP-tagged B55 or B55_LL and associated endogenous proteins were captured by incubating equal amounts of total protein (~500 µg) for each condition with GFP-Trap nanobody agarose beads (prepared using AminoLink Plus Immobilization Kit; ThermoFisher) at 4 °C for 16 h. Following 3 washes with wash buffer (20 mM Tris pH 8.0, 500 mM NaCl, 0.5 mM TCEP, 1 mM MnCl₂), bound proteins were eluted with 2% SDS sample buffer (90 °C, 10 min), resolved by SDS–PAGE (Bio-Rad) and transferred to PVDF membrane for western blot analysis using indicated antibodies (see Reporting summary). Purified PP2A:B55 complex was used as a positive control. Antibody fluorescence signals were captured using a ChemiDoc MP Imaging System (Image Lab Touch Software 2.4; Bio-Rad) and band intensities quantified using ImageJ 1.53t^51,52.

FAM122A interaction with PP2A:B55 complex

Purified FAM122A and variants (~25 µg, see preparation in ‘FAM122A purification’ in Methods) were mixed with Expi293F whole-cell extracts expressing B55, PP2Ac constructs and purified PP2Aa. Input samples were collected prior to incubation with agarose beads. GFP-tagged B55 and associated proteins were captured by incubating equal amounts of total protein (~500 µg) for each condition with GFP-Trap nanobody agarose beads (prepared as described in ‘eGFP–nanobody protein expression, purification, and immobilization onto agarose beads’ in Methods) at 4 °C for 16 h. Following 3 washes with wash buffer (20 mM Tris pH 8.0, 500 mM NaCl, 0.5 mM TCEP, 1 mM MnCl₂), bound proteins were eluted with 2% SDS sample buffer (90 °C, 10 min), resolved by SDS–PAGE (Bio-Rad) and transferred to PVDF membrane for western blot analysis using indicated antibodies (see Reporting summary) anti-B55 (2290 S, 1:1,000), anti-PP2Ac (MABE1783, 1:1,000), goat anti-rabbi IgG, (12005869, 1:3,000) and goat anti-mouse IgG (12004158, 1:3,000). Antibody fluorescence signals were captured using a ChemiDoc MP Imaging System (Image Lab Touch Software 2.4; Bio-Rad) and band intensities were quantified using ImageJ 1.53t. Uncropped blots are shown in Supplementary Fig. 2.

FAM122A and ARPP19 competition assay

Purified FAM122A_Nterm (~25 µg) and S62 tpARPP19_S104A (~25 µg or 125 µg, see preparation in ‘Phosphorylation of ARPP19’ in Methods) alone or in combination were mixed with Expi293F whole-cell extracts expressing B55, PP2Ac constructs and purified PP2Aa. Input samples were collected prior to incubation with agarose beads. GFP-tagged B55 and associated proteins were captured by incubating equal amounts of total protein (500 µg) for each condition with GFP-Trap nanobody agarose beads (prepared using AminoLink Plus Immobilization Kit; ThermoFisher) at 4 °C for 16 h. Following 3 washes with wash buffer (20 mM Tris pH 8.0, 500 mM NaCl, 0.5 mM TCEP, 1 mM MnCl₂), bound proteins were eluted with 2% SDS sample buffer (90 °C, 10 min), resolved by SDS–PAGE (Bio-Rad) and transferred to PVDF membrane for western blot analysis using indicated antibodies (see Reporting summary) anti-FAM122A (MA5-24510, 1:1,000), anti-ARPP19 (Proteintech, 11678-1-AP, 1:1,000). Antibody fluorescence signals were captured using a ChemiDoc MP Imaging System (Image Lab Touch Software 2.4; Bio-Rad) and band intensities quantified using ImageJ 1.53t. Uncropped blots shown in Supplementary Fig. 2.

Alkaline treatment for PP2Ac methylation

For alkaline treatment, 100 μl PP2A:B55 triple complex fraction from anion exchange was mixed with NaOH to a final concentration of 0.2 M and incubated for 10 min at room temperature. The reaction was neutralized by adding HCl to a final concentration of 0.2 M and diluted to 200 μl with lysis buffer. The control reaction was treated with pre-neutralization solution (0.2 M NaOH and 0.2 M HCl) and diluted to 200 μl with lysis buffer. The samples were boiled with 2% SDS sample buffer (90 °C, 10 min), resolved by SDS–PAGE (Bio-Rad) and transferred to PVDF membrane for western blot analysis using indicated antibodies (see Reporting summary) anti-PP2Ac (MABE1783, 1:1,000), anti-PP2Ac Methyl (Leu309) (828801, 1:1,000). Antibody fluorescence signals were captured using a ChemiDoc MP Imaging System (Image Lab Touch Software 2.4; Bio-Rad) and band intensities quantified using ImageJ 1.53t. Uncropped blots shown in Supplementary Fig. 1.

eGFP–nanobody protein expression, purification, and immobilization onto agarose beads

For expression, pOPIN-eGFP-nanobody plasmid DNA (a gift from M. Bollen) was transformed into E. coli BL21 (DE3) cells (Agilent). Freshly transformed cells were grown at 37 °C in LB broth containing ampicillin antibiotics (50 µg ml⁻¹) until they reached an optical density (OD₆₀₀) of ~0.8. Protein expression was induced by addition of 0.5 mM β-d-thiogalactopyranoside (IPTG) to the culture medium, and cultures were allowed to grow overnight (18–20 h, 250 rpm shaking) at 18 °C. Cells were collected by centrifugation (8,000g, 15 min, 4 °C) and stored at −80 °C until purification. Cell pellets expressing eGFP–nanobody were resuspended in ice-cold lysis buffer (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole, 0.1% Triton X-100, EDTA-free protease inhibitor tablet (ThermoFisher)), lysed by high-pressure cell homogenization (Avestin Emulsiflex C3). Cell debris was pelleted by centrifugation (42,000g, 45 min, 4 °C), and the supernatant was filtered with 0.22-µm syringe filters. The proteins were loaded onto a HisTrap HP column (Cytiva) pre-equilibrated with buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole) and eluted using a linear gradient (0–60% B) with buffer B (50 mM Tris pH 8.0, 500 mM NaCl and 500 mM imidazole). Fractions containing the protein were pooled, concentrated, and further purified at room temperature using SEC (Superdex 75 26/60 (Cytiva)) in PBS pH 7.5 buffer. Purified and concentrated eGFP–nanobody protein was immobilized onto agarose beads (20 mg protein per column) using AminoLink Plus Immobilization Kit (ThermoFisher), following manufacturer’s instructions in PBS pH 7.5 coupling buffer.

B55 and B55_LL purification

Pellets of Expi293F cells expressing eGFP–B55 or eGFP–B55_LL were resuspended in ice-cold lysis buffer (20 mM Tris pH 8.0, 500 mM NaCl, 0.5 mM TCEP, 0.1% Triton X-100, EDTA-free protease inhibitor tablet (ThermoFisher)), lysed by high-pressure cell homogenization (Avestin Emulsiflex C3). Cell debris was pelleted by centrifugation (42,000g, 45 min, 4 °C), and the supernatant was filtered with 0.22-µm syringe filters. Lysates were mixed with GFP–nanobody-coupled agarose beads (see preparation in ‘eGFP–nanobody protein expression, purification, and immobilization onto agarose beads’ in Methods), pre-equilibrated with wash buffer 1 (20 mM Tris pH 8.0, 500 mM NaCl and 0.5 mM TCEP) and slowly rocked at 4 °C for 2 h. After 2 h, lysate–bead mixture was loaded onto gravity columns, the flow through (FT1) was collected and the column was washed 3 times with 25 ml of wash buffer (washes 1–3). The GFP–B55 resin was resuspended in 20 mM Tris pH 8.0, 250 mM NaCl and 0.5 mM TCEP, and TEV was added for on-column cleavage with rocking overnight at 4 °C. The flow through was again collected (FT2) and the resin was washed with 20 ml of wash buffer 2 (20 mM Tris pH 8.0, 250 mM NaCl and 0.5 mM TCEP; wash 4) and 2× 20 ml with the wash buffer 1 (washes 5 and 6). The flow through 2 (FT2) and washes 4–6 were collected, diluted to ~100 mM salt concentration (with 0 mM NaCl wash buffer), and loaded onto QTrap HP column (Cytiva) for further purification. The proteins were eluted with a 100 mM–1 M salt gradient (buffer A: 20 mM Tris pH 8.0, 100 mM NaCl, 0.5 mM TCEP; buffer B: 20 mM Tris pH 8.0, 1 M NaCl, 0.5 mM TCEP). B55 or B55_LL were concentrated and further purified using SEC (Superdex 200 26/60 (Cytiva)) in NMR buffer (20 mM Na₂HPO₄/NaH₂PO₄ pH 6.3, 150 mM NaCl, 0.5 mM TCEP) or assay buffer (20 mM Tris pH 8.0, 150 mM NaCl, 0.5 mM TCEP).

PP2A:B55 complex purification

Expi293F cell pellets expressing StrepII–PP2Ac and eGFP–B55 constructs were resuspended in ice-cold lysis buffer (20 mM Tris pH 8.0, 500 mM NaCl, 0.5 mM TCEP, 1 mM MnCl₂, 0.1% Triton X-100, EDTA-free protease inhibitor tablet (ThermoFisher)), lysed by high-pressure cell homogenization (Avestin Emulsiflex C3). Purified PP2Aa was added to the cell lysate. Cell debris was pelleted by centrifugation (42,000g, 45 min, 4 °C), and the supernatant was filtered with 0.22-µm syringe filters. Lysates were loaded onto a GFP–nanobody-coupled agarose bead (see preparation in ‘eGFP–nanobody protein expression, purification, and immobilization onto agarose beads’ in Methods) column, pre-equilibrated with wash buffer 1 (20 mM Tris pH 8.0, 500 mM NaCl, 1 mM MnCl₂ and 0.5 mM TCEP) and slowly rocked at 4 °C for 2 h. After 2 h, the flow through (FT1) was collected and the column was washed 3 times with 25 ml of wash buffer (washes 1–3). The GFP–B55 resin was resuspended in 20 mM Tris pH 8.0, 250 mM NaCl, 1 mM MnCl₂ and 0.5 mM TCEP, and TEV was added for on-column cleavage rocking overnight at 4 °C. The flow through was again collected (FT2) and the resin was washed with 20 ml of wash buffer 2 (20 mM Tris pH 8.0, 250 mM NaCl, 1 mM MnCl₂ and 0.5 mM TCEP) (wash 4) and 2× 20 ml with the wash buffer 1 (washes 5 and 6). The flow through 2 (FT2) and washes 4–6 were collected, diluted to ~100 mM salt concentration (with 0 mM NaCl Wash buffer), and loaded onto Mono Q column (Cytiva) for further purification. The proteins were eluted with a 100 mM–1 M salt gradient (buffer A: 20 mM Tris pH 8.0, 100 mM NaCl, 1 mM MnCl₂ and 0.5 mM TCEP; buffer B: 20 mM Tris pH 8.0, 1 M NaCl, 1 mM MnCl₂ and 0.5 mM TCEP). PP2A:B55 complex and B55 fractions were pooled, concentrated and further purified using SEC (Superdex 200 26/60 (Cytiva)) in NMR buffer (20 mM Na₂HPO₄/NaH₂PO₄ pH 6.3, 150 mM NaCl and 0.5 mM TCEP) or assay buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM MnCl₂ and 0.5 mM TCEP).

Cryo-EM data acquisition and processing

The PP2A:B55–FAM122A complex was prepared by purifying PP2A:B55 and incubating it with a 1.5 molar ratio of PP2A:B55 to FAM122A_ID at a total concentration of 1.2 mg ml⁻¹. The PP2A:B55–tpARPP19 complex was prepared by purifying PP2A:B55 and incubating it with a 1.5 molar ratio of PP2A:B55 to tpARPP19 at a total concentration of 2.4 mg ml⁻¹. Immediately prior to blotting and vitrification (Vitrobot MK IV, 18 °C, 100% relative humidity, blot time 5 s), CHAPSO (3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate) was added to a final concentration of 0.075% (w/v) for PP2A:B55-FAM122A and 0.125% (w/v) for PP2A:B55–tpARPP19. 3.5 μl of the sample was applied to a freshly glow discharged UltAuFoil 1.2/1.3 300 mesh grid, blotted for 5 s and plunged into liquid ethane. Imaging was performed using a Titan Krios G3i equipped with a Gatan BioQuantum K3 energy filter and camera operating in CDS mode. Acquisition and imaging parameters are given in Supplementary Table 1. All data processing steps were performed using Relion 4.0⁵³ and are summarized in Extended Data Figs. 6–8. For both datasets, micrograph movies were summed and dose-weighted; contrast transfer function (CTF) parameters were estimated using CTFFind 4.1.14⁵⁴ on movie frame-averaged power spectra (~4 e Å⁻² dose). Micrographs were filtered to remove outliers in motion correction and/or CTF estimation results and screened manually to remove micrographs with significant non-vitreous ice contamination. Potential particle locations on the full micrograph set were selected using Topaz⁵⁵ using a model trained on a random subset of the micrographs. Particles on the training subset were selected by a Topaz model trained on previous screening data. Subset picks were subjected to 2D classification, ab initio 3D initial model generation, and 3D classification, and surviving particles used to train an improved Topaz model used to pick the full micrograph set. From these picks, 2D classification and 3D classification (with full angular and translational searches) were used to select particles in classes showing clear secondary structure and representing the full complex. Resolution in both datasets was then further improved by cycles of CTF parameter refinement, particle polishing, and fixed-pose 3D classification, alongside the following elaborations: For PP2A:B55–tpARPP19, particles with well-resolved ARPP19 density were selected by isolating ARPP19 via signal subtraction of the vast majority of the holoenzyme, followed by fixed-pose 3D classification; this process was performed twice in the course of the processing workflow. The final map was refined from 52,934 particles to a resolution of 2.77 Å. For PP2A:B55–FAM122A, multi-body refinement of the B55 and PP2Ac segments of the complex was needed to resolve details of both segments. Within each resulting body alignment, signal subtraction and fixed-pose 3D classification of FAM122A and its surrounding binding groove was used to select for particles for which multi-body refinement was successful and FAM122A was present and well-resolved. This yielded 103,522 particles for which this was simultaneously true in both bodies. Using these particles, a second multi-body refinement was used to generate maps for model building within each body, with final resolutions of 2.55 Å for the B55 body and 2.69 Å for the PP2Ac body. To generate a consensus map, a refinement was run using only the top 25,000 particles with the smallest sum of squared eigenvalues from the multi-body refinement (as reported by relion_flex_analyse). All 3D auto-refinements for both datasets utilized a soft solvent mask and SIDESPLITTER⁵⁶. All global map resolutions reported in this work were calculated by the gold-standard half-maps Fourier shell correlation (FSC) = 0.143 metric. Further validation information is given in Extended Data Figs. 6–8 and Supplementary Table 1.

Cryo-EM model building

All models were built and refined by iterating between manual rebuilding and refinement in Coot⁵⁷ and ISOLDE⁵⁸, and automated global real-space refinement in Phenix⁵⁹. For PP2A:B55–FAM122A, the relevant segments of the model were built into the B55 and PP2Ac body maps, using the previously determined crystal PP2A:B55 holoenzyme crystal structure (PDB ID 3DW8) and the available FAM122A AlphaFold model (UniProt Q96E09) as a starting point. The two body models were then joined, and the regions near the joints further rebuilt, and the entire complex refined against the 25,000-particle consensus subset map. For PP2A:B55–tpARPP19, the holoenzyme portion of the PP2A:B55-FAM122A model and the available ARPP19 AlphaFold model (UniProt P56211) were used as starting points. Model geometry and map–model validation metrics are given in Supplementary Table 1. Maps in Fig. 2 are LAFTER filtered and sharpened maps⁶⁰.

PP2A:B55 activity assay

Phosphatase activity assays were conducted in 96 well plates (Corning). PP2A:B55 holoenzyme was diluted to desired concentration range (0 to 20 nM) in Enzyme buffer (30 mM HEPES pH 7.0, 150 mM NaCl, 1 mM MnCl₂, 1 mM DTT, 0.01% Triton X-100, 0.1 mg ml⁻¹ BSA) and incubated at 30 °C. The reaction was started by the addition of 6,8-difluoro 4-methylumbelliferyl phosphate (DiFMUP) to a final concentration of 50 μM. Assays were read every 15 s for ~50 min on a CLARIOstarPlus (BMG LABTECH) plate reader (using reader control software v. 5.7 R2) and the data was evaluated using GraphPad Prism 9.5.

DiFMUP fluorescence intensity assay for PP2A:B55 IC₅₀ measurements

DiFMUP based IC₅₀ assays were conducted in 384-well plates (Corning, 4411). For ARPP19 and FAM122A IC₅₀ assays, PP2A:B55 holoenzyme in Enzyme buffer (30 mM HEPES pH 7.0, 150 mM NaCl, 1 mM MnCl₂, 1 mM DTT, 0.01% triton X-100, 0.1 mg ml⁻¹ BSA) was pre-incubated with various concentrations of ARPP19 and FAM122A variants for 30 min at room temperature (Extended Data Fig. 2). The reaction was started by adding DiFMUP (final concentration 50 μM) into the PP2A:B55-FAM122A enzymatic reaction (final concentration of PP2A:B55 holoenzyme at 1 nM) and then incubated at 30 °C for 30 min. End-point reads (excitation 360 nm, emission 450 nm) were taken on a CLARIOstarPlus (BMG LABTECH) plate reader (using reader control software version 5.7 R2) after the reaction was stopped by the addition of 300 mM potassium phosphate (pH 10). The experiments were independently repeated ≥ 3 times (each reaction was made in n = 3 to 6) and the averaged IC₅₀ and s.d. values were reported. The data was evaluated using GraphPad Prism 9.5.

Fluorescence polarization PP2A binding assays

Following the instructions of the manufacturer, 100 µM of FAM122A_ID(S120C) (or variants) or ARPP19(S10C) was labelled with Alexa Fluor 488 C5 Maleimide (ThermoFisher) using 1:10 protein to fluorophore ratio. The mixture was incubated for 2 h in the dark at room temperature at pH 7.0 and excess β-mercaptoethanol (1.2× the concentration of the fluorophore) was added to inactivate any unreacted Alexa Fluor 488. Labelled FAM122A_ID(S120C) (or variants) or ARPP19(S10C) was recovered by analytical SEC (Superdex 75 Increase 10/300 (Cytiva)) and used for the fluorescence polarization assays. The labelled FAM122A_ID(S120C) (or variants) or ARPP19(S10C) are hereafter referred to as FAM122A_ID-tracer, or ARPP19-tracer.

The fluorescence polarization assays were standardized using black 384-well low volume round bottom microplates (Corning, 4411) with 15 µl solution per well. The measurements were performed using a CLARIOstarPlus (BMG LABTECH Inc) microplate reader (using reader control software version 5.7 R2) set up to 482 ± 16 nm excitation, 530 ± 40 nm emission, and dichroic long pass filter 504 nm with reflection ranging between 380–497 nm and transmission ranging between 508–850 nm. For the dissociation constant (K_d) binding measurements, all dilutions were made into fluorescence polarization buffer (10 mM HEPES pH 7.0, 150 mM NaCl, 0.5 mM TCEP, 0.01% Triton X-100, 0.1 mg ml⁻¹ BSA). A predilution of FAM122A_ID-tracer/ARPP19-tracer was prepared for 0.3 nM and a serial dilution of PP2A:B55 was made at 3 times the final concentration. Five microlitres of FAM122A_ID-tracer/ARPP19-tracer, 5 µl of serially diluted PP2A:B55 complex and 5 µl of fluorescence polarization buffer were distributed into the 384-well microplate, resulting in a 0.1 nM final concentration of FAM122A_ID-tracer or ARPP19-tracer. All assay experiments were repeated in triplicate and incubated for 30 min in the dark and sealed at room temperature before reading. The experiments were independently repeated ≥3 times and the averaged K_d and s.d. values were reported. The data was evaluated using GraphPad Prism 9.5.

ARPP19 immunoprecipitation

Synthetic DNA encoding the various ARPP19 sequences was purchased from GeneArt, Life Technologies and cloned into the pcDNA5/FRT/TO (Invitrogen) expression vector containing YFP resulting in YFP–ARPP19 fusion proteins. These constructs were transiently transfected into HeLa cells 24 h prior to collecting cells. Cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT and 0.1% NP40). Complexes were immunoprecipitated at 4 °C in lysis buffer with GFP-Trap (ChromoTek) beads as described by the manufacturer. Precipitated protein complexes were washed 3 times in lysis buffer, eluted in 2× SDS sample buffer and subjected to western blotting using the following antibodies: YFP (1:5,000; generated in house), B55α (1:2,000; 5689S, Cell Signaling Technology), PP2Ac (1:2,000; 05-421, Millipore). Uncropped blots are shown in Supplementary Fig. 1.

NMR data collection

All NMR data were collected on either a Bruker Avance Neo 600 MHz or 800 MHz NMR spectrometer equipped with TCI HCN z-gradient cryoprobe at 283 K. (¹⁵N,¹³C)-labelled FAM122A_Nterm (150 µM), (¹⁵N,¹³C)-labelled FAM122A_ID (400 µM), (¹⁵N,¹³C)-labelled ARPP19 (400 µM) and (¹⁵N,¹³C)-labelled pS62pS104ARPP19 (200 µM) were prepared in either FAM122A or ARPP19 NMR buffer with 5-10% (v/v) D₂O added immediately prior to data acquisition. The sequence-specific backbone assignments both proteins were determined by recording a suite of heteronuclear NMR spectra: 2D ¹H,¹⁵N HSQC, 3D HNCA, 3D HN(CO)CA, 3D HNCACB, 3D CBCA(CO)NH, 3D HNCO, and 3D HN(CA)CO, with an additional spectrum, 3D (H)CC(CO)NH, collected for FAM122A_ID (t_m = 12 ms)⁶¹. Spectra were processed in Topspin (Bruker Topspin 4.1.3) and referenced to internal DSS.

Sequence-specific backbone assignment, chemical shift index and chemical shift perturbation

Peak picking and sequence-specific backbone assignment were performed using CARA 1.9.1 ( CSI calculations of FAM122A_Nterm, FAM122A_ID, ARPP19 and pS62pS104ARPP19 were performed using both Cα and Cβ chemical shifts for each assigned amino acid, omitting glycine, against the RefDB database⁶². Secondary structure propensity (SSP) scores were calculated using a weighted average of seven residues to minimize contributions from chemical shifts of residues that are poor measures of secondary structure⁶³. The changes in peak position between different FAM122A or ARPP19 constructs or variants were traced according to nearest neighbour analysis. Chemical shift differences (∆δ) were calculated using the following equation:

NMR interaction studies of FAM122A and ARPP19 with PP2A:B55 and B55_LL

All NMR interaction data of FAM122A_Nterm/ID, ARPP19 or pS62pS104ARPP19 with either PP2A:B55 or B55_LL were recorded using a Bruker Neo 600 MHz NMR spectrometer equipped with a HCN TCI active z-gradient cryoprobe at 283 K. All NMR measurements of FAM122A_Nterm or FAM122A_ID or ARPP19 and pS62pS104ARPP19 were recorded using ¹⁵N-labelled protein in NMR buffer and 90% H₂O/10% D₂O. For each interaction, an excess of unlabelled B55_LL of PP2A:B55 complex (min 25% surplus ratio) was added to the ¹⁵N-labelled FAM122A or ARPP19 construct under investigation and incubated on ice for 10 min before the 2D ¹H,¹⁵N HSQC spectrum was collected. FAM122A and ARPP19 concentrations ranged from 2–6 μM. NMR data were processed using nmrPipe⁶⁴ and the intensity data were analysed in Poky⁶⁵. Each dataset was normalized to its respective most intense peak and the difference between each free 2D ¹H,¹⁵N HSQC spectrum FAM122A or ARPP19 residue was compared to its respective peak, if present, on the 2D ¹H,¹⁵N HSQC spectrum of FAM122A or ARPP19 in complex with B55_LL or PP2A:B55. Any overlapping peaks were omitted for this analysis.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.