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

Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation1. Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases2, whereas mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B553. Although 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 inhibited4. Inhibition of PP2A:B55 is achieved by the intrinsically disordered proteins ARPP195,6 and FAM122A7. Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the single-particle cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies, both intrinsically disordered proteins bind PP2A:B55, but do so in highly distinct manners, 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 provide a molecular roadmap for the development of therapeutic interventions for PP2A:B55-related diseases.

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 , whereas mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B55 3 .Although 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 .Inhibition of PP2A:B55 is achieved by the intrinsically disordered proteins ARPP19 5,6 and FAM122A 7 .Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown.Here, we report the singleparticle cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A.Consistent with our complementary NMR spectroscopy studies, both intrinsically disordered proteins bind PP2A:B55, but do so in highly distinct manners, 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 provide a molecular roadmap for the development of therapeutic interventions for PP2A:B55-related diseases.
An essential function of the PP2A:B55 holoenzyme is the control of cell cycle progression through mitosis [4][5][6]8,9 . Mittic entry is accomplished via the activation of the CDK1-cyclin B kinase, whose activity enables nuclear envelope breakdown, chromatin condensation and spindle formation 10,11 .Mitotic exit is initiated by cyclin B ubiquitination via the anaphase promoting complex, leading to its degradation 12 , an event accompanied by the dephosphorylation of mitotic substrates by protein phosphatases (PPPs), especially PP2A:B55 13 .Notably, inhibition of PP2A:B55 activity during mitotic onset has been shown to create the necessary dynamic feedback for robust mitotic substrate phosphorylation 14 .The PP2A:B55 holoenzyme also regulates the entry into mitosis at the G2/M checkpoint, as PP2A:B55 inhibition allows normal progression through the checkpoint 8,9 .These essential PP2A:B55-inhibition events are achieved by its interaction with two distinct intrinsically disordered protein (IDP) inhibitors, cAMP regulated phosphoprotein 19 (ARPP19) and family with sequence similarity 122A 6,[15][16][17].The mechanisms by which these inhibitors block PP2A:B55 activity differ, as ARPP19 strictly requires phosphorylation by MASTL kinase to inhibit PP2A:B55 5,6 , whereas FAM122A inhibits PP2A:B55 in a phosphorylation-independent manner 7,8 .The current data suggest that these IDP inhibitors engage PP2A:B55 sequentially during mitotic entry (Fig. 1a). Speciically, PP2A:B55 is initially bound and inhibited by FAM122A.This inhibition results in the full activation of mitotic kinases, including MASTL, which phosphorylates ARPP19 on Ser62 (pS62-ARPP19).Through a currently unknown mechanism, pS62-ARPP19 displaces FAM122A from PP2A:B55.pS62-ARPP19 functions first as an inhibitor of PP2A:B55 but later becomes a substrate 18 .This dephosphorylation reactivates PP2A:B55 and enables progression through mitotic exit. Depite our understanding of the importance of PP2A:B55 in mitosis and knowing the identity of the IDP inhibitors that mediate PP2A:B55 inhibition during mitotic entry, we still lack a detailed understanding of how this inhibition is achieved at a molecular level.

ARPP19 and FAM122A bind PP2A:B55
To determine how ARPP19 and FAM122A bind PP2A:B55, we established a method for producing high yields of active PP2A:B55 from Expi293F cells (Extended Data Fig. 1a-h); using this method, the C-terminal residue of PP2Ac is fully methylated 19,20 (mLeu309).We quantified PP2A:B55 inhibition by ARPP19, thiophosphorylated ARPP19 (full-length (amino acids 1-112) and phosphorylated with ATPγS using MASTL kinase) and FAM122A (N-terminal domain (amino acids 1-124) (FAM122A Nterm )) (Fig. 1b).Whereas PP2A:B55 was only moderately inhibited by ARPP19, it was strongly inhibited by both thiophosphorylated ARPP19 and FAM122A Nterm (Extended Data Table 1 and Extended Data Fig. 2a), with thiophosphorylated ARPP19 inhibiting PP2A:B55 around 250-fold more potently than FAM122A.Fluorescent polarization binding measurements showed that both FAM122A and ARPP19 bind PP2A:B55 tightly, with thiophosphorylation not influencing binding (Extended Data Table 2 and Extended Data Fig. 2b).We then used NMR spectroscopy to identify the residues in ARPP19 and FAM122A that interact with Article (HSQC) spectra of unbound ARPP19 21,22 and FAM122A Nterm confirmed that both are IDPs with multiple regions of amino acids with preferred α-helical propensities (using chemical shift index (CSI) analysis; Extended Data Fig. 3a-f).Overlaying the 2D 1 H, 15 N HSQC spectra of ARPP19 and FAM122A with and without PP2A:B55 identified the residues that bind PP2A:B55 (peaks with reduced intensities are due to either a direct interaction, a dynamic charge-charge interaction or conformational exchange on an intermediate timescale) (Fig. 1c-e).For ARPP19, around 90 N/H N cross-peaks (residues 20-112) showed reduced intensities.Because ARPP19 inhibition of PP2A:B55 strictly requires phosphorylation, we also thiophosphorylated ARPP19 using MASTL kinase.The 2D 1 H, 15 N HSQC spectrum of thiophosphorylated ARPP19 identified two phosphorylated residues, Ser62, the established MASTL phosphorylation substrate, and Ser104, a serine that was previously identified as a protein kinase A (PKA) substrate, and also shows recognition site homology to the MASTL specificity sequence 23 .The NMR data show that MASTL phosphorylation does not alter the preferred ensemble of structures of ARPP19 (Extended Data Fig. 3g-i).Thus, we generated the Ser104 phosphorylation site mutant ARPP19 S104A and repeated the thiophosphorylation step to obtain singly thiophosphorylated ARPP19 (tpS62ARPP19 S104A ; hereafter referred to as tpARPP19).Overlaying the 2D 1 H, 15 N HSQC spectra of tpARPP19 and tpS62tpS104ARPP19 with PP2A:B55 showed that the intensities of the same approximately 90 N/ H N cross-peaks identified with unphosphorylated ARPP19 are reduced with PP2A:B55 (Extended Data Fig. 4a-d).Similar NMR interaction experiments with FAM122A Nterm showed that the intensities of around 85 cross-peaks (residues 30-115) were reduced with PP2A:B55 (Fig. 1e and Extended Data Fig. 4e).On the basis of these data, we created FAM122A ID (amino acids 29-120), which includes all PP2A:B55 interacting residues (Extended Data Figs.3d-f and 4f,g).
ARPP19 and FAM122A inhibit B55-containing PP2A holoenzymes 7,8 .To identify which residues of ARPP19 and FAM122A bind B55, we repeated the NMR experiments using B55 loopless (B55 LL ), a variant that lacks the PP2Aa binding loop (amino acids 126-164 are replaced with NG; Extended Data Fig. 1a) and is thus unable to bind PP2Aa.Overlaying the 2D 1 H, 15 N HSQC spectra of ARPP19 and tpS62tpS104ARPP19 Results representative of n = 3 independent experiments.Unpaired two-tailed t-test with 95% confidence interval was used to compare ARPP19 with tpARPP19 (P < 0.0001) or tpS62tpS104ARPP19 (P < 0.0001).IC 50 values are reported in Extended Data Table 1.c, 2D 1 H, 15 N HSQC spectrum of 15 N-labelled ARPP19 with or without PP2A:B55.d, Plot of peak intensity versus ARPP19 protein sequence for spectra in c; grey shading highlights ARPP19 residues with reduced intensities in the presence of PP2A:B55.Secondary structure elements based on NMR CSI data are indicated.Colour scheme as in c. e, Plot of peak intensity versus FAM122A protein sequence for FAM122A Nterm alone (black) and with PP2A:B55 (green); grey shading highlights FAM122A residues with reduced intensities in the presence of PP2A:B55.Secondary structure elements based on NMR CSI data are indicated.f, 2D 1 H, 15 N HSQC spectrum of 15 N-labelled ARPP19 with (pink) and without (black) B55 LL ; grey shading highlights ARPP19 residues with reduced intensities in the presence of B55 LL .g, Plot of peak intensity versus ARPP19 protein sequence for spectra in f; grey shading highlights ARPP19 residues with reduced intensities in the presence of B55 LL .h, Plot of peak intensity versus FAM122A ID protein sequence for FAM122A ID alone (black) and with B55 LL (blue); grey shading highlights FAM122A ID residues with reduced intensities in the presence of B55 LL .
with and without B55 LL showed that the identity and number of N/H N cross-peaks with reduced intensities are similar, but not identical, to those observed with PP2A:B55 (Fig. 1f,g and Extended Data Fig. 5a,b).Specifically, the peaks corresponding to residues 20-75 and 105-112 show significant reductions in intensities, whereas ARPP19 residues 75-104 show little or no intensity loss with B55 LL .This shows that two distinct ARPP19 domains-20-75 and 105-112-bind B55.An overlay of the 2D 1 H, 15 N HSQC spectrum of FAM122A ID with and without B55 LL (Fig. 1h and Extended Data Fig. 5c) showed that N/H N cross-peaks with reduced intensities correspond to FAM122A residues 73-95, which bind solely to B55.Both ARPP19 and FAM122A bind B55 LL with reduced affinities compared with PP2A:B55 (Extended Data Table 2 and Extended Data Fig. 2b).These B55 interaction regions of more than 20 residues were longer than expected (most PPPs, including PP2A:B56, bind their substrates and regulators using short linear motifs (SLIMs), that are typically 4-8 residues long, and bind their cognate PPP in an extended fashion [24][25][26][27][28] ).This suggests that ARPP19 and FAM122A bind B55 via a different non-SLIM-based mechanism.Consistent with this, our NMR data showed that these IDP inhibitors exhibit helical propensities in solution (Extended Data Fig. 3c,f), suggesting they may bind B55 as helices.

PP2A:B55-inhibitor cryo-EM structures
Following extensive sample optimization, we determined the structures of PP2A:B55-tpARPP19 and PP2A:B55-FAM122A ID using cryo-EM at global resolutions of 2.77 and 2.80 Å, respectively (Fig. 2a,b and Extended Data Figs.6-8).The previously solved PP2A:B55 crystal structure aided the modelling of the PP2Aa, B55 and PP2Ac subunits 29 .Compared with the PP2A:B55 crystal structure, the horseshoe-shaped conformation of PP2Aa contracted upon inhibitor binding (Fig. 2c).In both PP2A:B55-inhibitor maps, we observed continuous sections of density not accounted for by the PP2A:B55 crystal structure.The density common to both maps belongs to the PP2Ac C terminus (Fig. 2d, amino acids 294-309), which was not modelled in the PP2A:B55 crystal structure 29 .The C terminus extends across the PP2Aa central cavity to bind an extended pocket at the B55:PP2Aa interface, positioning mL309 C to bind a hydrophobic pocket in PP2Aa (Extended Data Fig. 9a-d; subscripts denote residues corresponding to the different subunits of the complexes as follows: A, PP2Aa; B, B55; C, PP2Ac; R, ARPP19; F, FAM122A).Overlaying the PP2A:B55-inhibitor complexes with PP2A:B56 (Protein Data Bank (PDB) ID: 2IAE 30 ; superimposed using PP2Ac) showed that the PP2Ac mL309 C residues are more than 36 Å apart (Fig. 2e).The C-terminal interaction buries around 1,900 Å 2 of solvent-accessible surface area, explaining the importance of this post-translational modification for PP2A:B55 complex formation and stability 19 .

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span the front surface of B55 and the PP2Ac active site.Furthermore, in a highly unusual conformation, tpARPP19 loops back on itself to form a stable, overlaid cross at the B55:PP2Ac interface.tpARPP19 binding buries more than 5,200 Å 2 of solvent-accessible surface area.FAM122A also binds exclusively to B55 and PP2Ac and does so, again, using helices (pre-populated in free FAM122A; Extended Data Fig. 3d,f).FAM122A binds a short surface on B55 (the B55 binding platform) and across the PP2Ac active site; the interaction buries 2,700 Å 2 solvent-accessible surface area.Both inhibitors bind the most conserved surface of B55 (Fig. 2f).Despite their common function, these structures show that tpARPP19 and FAM122A bind and inhibit PP2A:B55 using distinct mechanisms.
Because these residues form a helix, amino acids separated in sequence are adjacent in space.F48 R and R52 R form a π-stack and hydrophobic contacts with B55 L4/5 and L5/6 (I284 B /Y337 B /F343 B ), whereas L49 R and L53 R form hydrophobic contacts with L2/3 and L3/4 (Y178 B /L198 B / L225 B /V228 B ). R50 R and R52 R also stabilize L3/4 or L5/6, respectively, via a salt bridge (E223 B /D340 B ) (Fig. 3c).The key interactions between B55 and ARPP19 are mediated by ARPP19 residues F-L-R-X-R-L-X-K.Because these residues in ARPP19 are helical, and not extended, we refer to this sequence as a short helical motif (SHELM).To test whether these ARPP19 residues contribute to B55 binding in cells, we generated 5-Ala and single point variants for the ARPP19 α2-α3 loop and helix α3 and tested their ability to pull down PP2A:B55 from cells.Although B55 binding to the 42 AAAAA 46 variant was unchanged, the 47 AAAAA 51 and 52 AAAAA 56 variants could not pull down B55 (Fig. 3b).Multiple single alanine mutations for ARPP19 residues 47-56 also exhibited reductions in B55 and PP2Ac binding, particularly L53A and R52A (less than 25% compared with wild type), R50A and F48A (around 50% compared with wild type) and L49A and K55A (approximately 75% compared with wild type) (Fig. 3e and Extended Data Fig. 9g).These data are fully consistent with the structure, as mutating residues that interact with B55 (F48, L49, R50, R52, L53 and K55) reduce binding, whereas those that are mostly solvent-accessible (D47, K51, Q54 and G56) do not.
Next, ARPP19 extends towards the B55 L1/2 loop, where it kinks by 180° to bind to PP2Ac (Fig. 3a,f).Here, Y59 R , F60 R and D61 R are splayed apart, with Y59 R binding B55, F60 R binding PP2Ac and D61 R binding both B55 and PP2Ac.These interactions position tpS62 directly above the metal ions in the PP2Ac active site, where it forms bipartite salt bridges with the substrate-coordinating arginine residues, R89 C and R214 C (Fig. 3g).This interaction is further stabilized by D61 R and D64 R , which form salt bridges with R268 C and R89 C , resulting in an extended network of ionic interactions between ARPP19 and PP2Ac that stabilize the tpS62 conformation.ARPP19 helix α4 ( 62 tpSGDYNMAKAKMKNK 75 ) extends from the PP2Ac active site towards the PP2Ac C terminus.In this way, ARPP19 helices α3 and α4 interact with B55 (α3) and PP2Ac (α4) using a helix-turn-helix 'V' conformation.ARPP19 residues 76 QLPTAAPD 83 remain mobile when bound to PP2A:B55.Consistent with the structure, pull-down experiments using YFP-ARPP19 5-Ala variants, in which the kink and helix α4 residues are mutated to alanine ( 57 AAAAA 61 , 62 AAAAA 66 , 67 AAAAA 71 and 72 AAAAA 76 ), weaken B55 binding, albeit not to the same extent as 5-Ala variants of α2 or α3 (Fig. 3b).

ARPP19-mediated inhibition of PP2A:B55
MASTL-phosphorylated ARPP19 is both an inhibitor and substrate of PP2A:B55 [4][5][6]15,18 . To unerstand why MASTL-phosphorylated ARPP19 is only slowly dephosphorylated by PP2A:B55, we overlaid the structures of PP2A:B55-tpARPP19 via the PP2Ac subunit with the PPP subunit of both a PPP product complex (PP1 with a phosphate bound at the active site, PDB ID: 4MOV 26 ) and a PPP pre-dephosphorylation complex (phosphorylated eIF2α trapped by the catalytically deficient PP1 D64A variant, PDB ID: 7NZM 33 ).The thiophosphate and phosphate in the pre-dephosphorylation and product complexes overlap nearly perfectly.By contrast, the thiophosphoryl group of tpARPP19 is approximately 3 Å further away from both metal ions, in a position that is unproductive for dephosphorylation (Fig. 3k).This inhibitory conformation is stabilized by the interactions between the ARPP19 MASTL recognition residues ( 58 KYFDSGDY 65 ) with B55 and PP2Ac and the ARPP19 crossover ( 86 EVTGDHIPTPQDL 98 ), which is secured in place by the interaction of the ARPP19 C terminus at B55 site 3. Consistent with this, previous data showed that mutating either the MASTL recognition residues (K58A, Y59A, F60A, D61A, D64A and Y65A) or deleting the C terminus converts ARPP19 into a substrate 15 (resulting in faster dephosphorylation; Fig. 3k).Similarly, comparing the half-maximal inhibitory concentrations (IC 50 ) of ARPP19 and a C-terminal deletion (ARPP 19-75 ) with and without thiophosphorylation shows that the interaction of the C terminus with B55 is essential for the potent inhibition of PP2A:B55 (Fig. 3l and Extended Data Table 1), as the C-terminal deletion variants either do not inhibit (non-phosphorylated ARPP19 versus ARPP19 19-75 ) or become a more than 50-fold weaker inhibitor (tpARPP19 versus tpARPP19 19-75 ).These overlapped structures also suggest that the mechanism by which ARPP19 becomes a substrate involves a shift of pS62 in the active site to a position in which the metal ion-activated nucleophilic water can mediate dephosphorylation.Our data suggest this is most probably achieved by the release of the ARPP19 C-terminal tail from B55.

Inhibition of PP2A:B55 by FAM122A
The interaction of FAM122A with PP2A:B55 is different to that of ARPP19, with FAM122A residues 81-111 binding PP2A:B55 with two helices (Fig. 4a).Helix α1 (the B55-binding helix) binds B55 and helix α2 (the inhibition helix) binds PP2Ac and blocks the active site.Although FAM122A residues 29-66 were not sufficiently ordered to be modelled, our NMR and binding data suggest that they contribute to binding via a dynamic (fuzzy) charge-charge interaction 24,31,32 (Extended Data Table 2 and Extended Data Fig. 2b).The FAM122A B55-binding helix binds the B55 platform, with its N terminus pointing towards the centre of B55 and its C terminus pointing towards PP2Ac (Fig. 4b,c).Residues L85 F and I88 F are adjacent in space, which enables them to bind the same hydrophobic pockets on B55 used by ARPP19 (L49 R /L53 R ) (Fig. 4d).Residue R84 F forms intramolecular polar and ionic interactions with Q87 F and E91 F that stabilize the helix (Fig. 4e).These interactions also allow R84 F to form a bidentate salt bridge with D197 B. Lys89 F binds the carbonyls of L3/4 residues M222 B , E223 B and L225 B (Extended Data Fig. 9h).Finally, E92 F binds a deep, basic pocket below the B55 platform where it coordinates residues from L1/2, L2/3 and L3/4 (Fig. 4f).The key interactions between B55 and FAM122A are mediated by FAM122A residues R-L-X-X-I-K-X-E-E, four of which (R84 F /K89 F /E91 F /E92 F ) are highly conserved (Extended Data Fig. 1a).Mutating the basic-hydrophobic residue pairs-that is, 84 RLHQIKQEE 92 to 84 AAHQIKQEE 92 ( 84 AA 85 ) and 84 RLHQAAQEE 92 ( 88 AA 89 )-reduced FAM122A binding by 1.6-and 2.0fold, respectively (Fig. 4g and Extended Data Table 2).Pull-down assays using PP2A:B55 lysates incubated with 84 AA 85 and 88 AA 89 FAM122A showed similar reductions in binding compared with the wild-type protein (Fig. 4h).Consistent with their weaker affinities, the IC 50 values of the 84 AA 85 and 88 AA 89 variants increased by 38-and 48-fold, respectively Article (Fig. 4g and Extended Data Table 1).Because the FAM122A E92K mutation was identified in cancer tissues (cBioPortal), we also generated E91K and E92K variants and showed they also bound PP2A:B55 less strongly and were less potent inhibitors of PP2A:B55 (Extended Data Tables 1 and 2 and Extended Data Fig. 2a,b).
Like ARPP19, FAM122A also binds PP2Ac (Fig. 4a,i).FAM122A residues C-terminal to the B55 helix form a sharp turn with helix α2 ( 97 INRET-VHEREVQTAM 111 , the inhibition helix; Extended Data Fig. 9i) binding and blocking the PP2Ac active site.This interaction is stabilized by hydrophobic and electrostatic interactions.L96 F and I97 F bind a hydrophobic pocket, positioning E100 F and E104 F to bind substrate-coordinating residues R268 C /R89 C and R214 C /R89 C , respectively (Fig. 4j).The E104A variant only modestly weakens FAM122A inhibition (twofold; as a control, the inhibitory capacity of E106A, a solvent-accessible residue, was not affected; Extended Data Table 1).This suggests that interactions at the active site are not essential for PP2Ac inhibition, but instead may be due to interactions, such as those of R105 F and V107 F , that stabilize the helix across the active site (Fig. 4k).cBioPortal 34 highlighted that FAM122A R105L, V107G variants are present in different cancers (FAM122A is a tumour suppressor, as patients with cancer who express low levels of FAM122A have significantly worse overall survival than those with high levels of expression 8 ).FAM122A R105L and V107G variants showed 11-and 6-fold less inhibition than the wild-type protein, respectively (Fig. 4l and Extended Data Table 1), demonstrating that the probable mode of action of these cancer variants is due to a weaker inhibition of PP2A:B55, thereby disrupting PP2A:B55 cellular functions.

Substrate recruitment via B55
PP2A:B55 dephosphorylates hundreds of substrates 35,36 , including p107 and p130, whose binding domains share sequence similarity with the B55 helix of FAM122A 37 (Fig. 5a).To test whether their B55 binding sites overlap, we performed an NMR competition assay (Fig. 5b).First, we formed a complex between 15 N-labelled p107 and B55 LL and identified all p107 N/H N cross-peaks that lost intensity due to B55 LL binding.We then added an excess of unlabelled FAM122A Nterm and monitored for p107 displacement from B55.All p107 residues that had reduced N/H N cross-peaks intensities due to B55 binding regained their intensities in the presence of excess FAM122A, showing that FAM122A displaced p107 from B55 (Fig. 5b and Extended Data Fig. 10a-c).These results establish that, in addition to ARPP19 and FAM122A, p107 (and probably other substrates) uses the B55 platform to bind B55, demonstrating that ARPP19 and FAM122A, in addition to inhibiting the active site, also block substrate binding to PP2A:B55 (Fig. 5c).

Simultaneous binding of both inhibitors
ARPP19 and FAM122A share two PP2A:B55 interaction sites: the B55 platform and the PP2Ac active site (Fig. 5d,e).However, their detailed interactions differ.Overlaying both complexes via B55 shows that L53 R and I88 F bind the B55 platform central hydrophobic pocket, whereas L49 R and L85 F bind an adjacent, shallower hydrophobic pocket (Fig. 5f).The number of intervening residues between these two corresponding hydrophobic amino acids are not identical (ARPP19 has four, whereas FAM122A has three) as the orientations of the bound helices differ.Similarly, whereas R52 R forms a π-stacking interaction and salt bridge with D340 B , R84 F binds in a pocket nearly 10 Å away to form a salt bridge with D197 B .These differences are again due to the distinct binding orientations of these helices.The second shared binding site is the PP2Ac active site (Fig. 5g).Although both inhibitors use helices to bind PP2Ac, these helices project in opposite directions, with ARPP19 helix α4 extending towards the PP2Ac C terminus whereas the FAM122A inhibitory helix extends towards the PP2Ac hydrophobic  2).h, Pull-down assay with SHELM variants.expi293F lysates were co-transfected with GFP-B55 and PP2Ac was incubated with purified PP2Aa and FAM122A variants and immunopurified.FAM122A variant binding efficiency was determined by western blot.Quantification based on three independent experiments (mean ± s.d.).Unpaired two-tailed t-test with 95% confidence interval was used to compare wild-type FAM122A with SHELM variants (P < 0.0005).i, FAM122A residues I97-M111 (magenta) bind PP2Ac (cyan).j, Intermolecular interactions of E100 and E104 with PP2Ac (ionic interactions are shown as dashed lines).k, Intermolecular interactions of R105 and V107 with PP2Ac.l, PP2A:B55 inhibition by FAM122A ID variants (mean ± s.d.; n = 3 experimental replicates).Results representative of n = 3 independent experiments.Unpaired two-tailed t-test with 95% confidence interval was used to compare wild-type FAM122A with E104A (P < 0.0001), E106A (P = 0.013), R105L (P < 0.0001) or V107G (P < 0.0001).IC 50 value reported in Extended Data Table 1.
groove.The only area of overlap is at the active site itself, where tpS62 R projects deeply into the active site, whereas E100 F or E104 F bind at the periphery.
The remainder of the interactions are unique, with ARPP19 binding B55 at additional interaction sites via helix α2 and its C terminus.This suggests that ARPP19 and FAM122A, which have similar affinities for PP2A:B55 (Extended Data Table 2) may bind PP2A:B55 simultaneously.To test this, we used NMR and pull-down assays.We first formed the complex between B55 LL and 15 N-labelled FAM122A Nterm.(Fig. 5h) and then added tpARPP19 (Fig. 5i).Despite an excess of around 2.5-fold of tpARPP19, no change in the 2D 1 H, 15 N HSQC spectrum of the FAM122A Nterm was observed, demonstrating that FAM122A Nterm was not displaced by tpARPP19 (Fig. 5i).We also performed a pull-down competition assay by affinity purification of PP2A:B55 (using GFP-B55) in the presence of FAM122A alone or FAM122A with a fivefold excess of tpARPP19 (Extended Data Fig. 10d).Not only were both FAM122A and tpARPP19 pulled down with PP2A:B55, but the amounts of FAM122A pulled down in the absence or presence of tpARPP19 were identical.Finally, we performed the reverse NMR experiment (B55 LL bound to 15 N-labelled ARPP19 and then adding excess unlabelled FAM122A Nterm ), which showed that ARPP19 stays bound to B55, predominantly via helix α2, in the presence of FAM122A (Extended Data Fig. 10e).Together, these experiments show that FAM122A Nterm and tpARPP19 can bind PP2A:B55 simultaneously (Fig. 5j), and thus, that B55 uses its multiple, distinct interaction surfaces to differentially engage B55-specific regulators and/or substrates.

Discussion
The inhibition of PP2A:B55 by two B55-specific inhibitors, FAM122A and ARPP19, is essential for mitotic entry 7,8,38,39 .Our data reveal their unexpected modes of PP2A:B55 binding and inhibition, providing a detailed understanding of their function.These data show that PP2A:B55 binds its regulators in a different manner to other PPPs.PP1 24,27,40 , PP2A:B56 28,41 , calcineurin 25,42 (PP2B-PP3) and PP4 43 recruit their cognate regulators and substrates using PPP-specific SLIMs 44 .By contrast, PP2A:B55 recruits its regulators ARPP19 and FAM122A using α-helices.Different to PPP-SLIM interactions, which are anchored by hydrophobic residues that bind to deep hydrophobic pockets, the B55 platform is comparatively flat with shallow hydrophobic pockets.The lack of pocket depth allows hydrophobic residues to approach and bind via multiple orientations (Fig. 5f), rather than the single orientation observed in SLIM interactions 45 .In addition, the B55 platform is bordered by charged residues, especially acidic residues.Because basic residues (arginine and lysine) are long, when present in B55 binding helices, they form salt bridges using an array of conformations, as necessitated by the helical binding orientation (Figs. 3 and 4).Thus, and in contrast to PP2A:B56 or calcineurin, in which PPP-specific g, Overlay of tpARPP19 (orange) or FAM122A (magenta) at the PP2Ac catalytic pocket.PP2Ac residues from the ARPP19 complex are labelled in cyan and those from the FAM122A complex are labelled in grey.Ionic interactions are shown as dashed lines.Metal ions in PP2Ac are shown as spheres.h, 2D 1 H, 15 N HSQC spectrum of 15 N-labelled FAM122A with and without B55 LL .i, 2D 1 H, 15 N HSQC spectrum of 15 N-labelled FAM122A with and without B55 LL and unlabelled tpARPP19.j, Model of FAM122A and ARPP19 binding B55 LL simultaneously.

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SLIM sequences have been used to identify putative substrates using sequence alone, our data suggests that the analogous strategy is not readily applicable for identifying novel PP2A:B55-specific substrates.For example, the sequences used by ARPP19 and FAM122A to bind the same pockets of the B55 platform are not highly conserved (for ARPP19, F-L-R-X-R-L-X-K; for FAM122A, R-L-X-X-I-K-X-E-E) and although some substrates, such as p107/p130, may share similarities with known binding sequences (that is, FAM122A), our data suggest that different B55 substrates may bind the platform via mechanisms not yet observed.Finally, B55 belongs to the WD40 propeller family, and thus adopts a fold that is an established protein interaction domain that has been demonstrated to bind other proteins using a diverse range of interactions 46 .These observations, coupled with the discovery that ARPP19 also binds B55 using its C terminus, suggests that B55 may recruit a set of substrates via interaction surfaces outside the B55 platform used by ARPP19, FAM122A and p107.
In addition to blocking substrate recruitment, our structures also show that FAM122A and tpARPP19 inhibit PP2A:B55 by blocking the PP2Ac active site (Figs. 3 and 4).This combined mechanism of inhibition (blocking substrate recruitment and inhibiting catalytic site access) is also used by other members of the PPP family, in particular PP1.Like FAM122A, protein phosphatase 1 inhibitor-2 (I-2) is an IDP inhibitor of PP1 47 that both blocks PP1-specific substrate and regulator recruitment (by binding PP1-specific SLIM interaction sites, the SILK and the RVxF binding pockets) and blocks catalytic site access 47,48 (by using a long helix to bind over the PP1 active site in a phosphorylation-independent manner) (Extended Data Fig. 10f-m).This shows that PPP family members PP1 and PP2A:B55 both have endogenous IDP inhibitors that use a common mechanism to potently inhibit their ability to dephosphorylate their cognate substrates, suggesting that this may be a mechanism present throughout the PPP family.
The current literature supports a model in which PP2A:B55 is initially inhibited by FAM122A and later by phosphorylated ARPP19 5,6,8,18,35 (Fig. 1a), with the assumption that inhibitor binding is mutually exclusive.However, our NMR and pull-down data show that FAM122A and ARPP19 can bind PP2A:B55 simultaneously, with FAM122A binding the B55 platform, and ARPP19, leveraging its multiple B55 interaction sites, binding B55 predominantly via helix α2.The ability of two regulators that share a subset of interaction sites to bind simultaneously to their cognate PPP has been observed for other PPPs (that is, the PP1-spinophilin-I-2 complex 49,50 ).In this case, spinophilin binds the PP1 RVxF SLIM interaction pocket, and the I-2 RVxF sequence releases from PP1; it is the extensive interactions of I-2 at the PP1 SILK binding pocket and active site that allows the I-2 RVxF sequence to be dispensable for PP1 binding.Here we show that ARPP19, FAM122A and PP2A:B55 form a similar complex, in which-in the presence of FAM122A-the interactions of ARPP19 at sites 2 and 3 are dispensable for binding.Whether and how these ternary interactions contribute to the regulation of PP2A:B55 activity during mitosis remain to be elucidated.These data also suggest that the stable dissociation of FAM122A from PP2A:B55 is needed for formation of the full inhibitory PP2A:B55-pARPP19 complex.One possibility is that a currently unidentified post-translational modification dissociates FAM122A from PP2A:B55 (such as phosphorylation; phosphorylation of FAM122A S37 has already been shown to quantitatively dissociate FAM122A from PP2A:B55 to activate the G2/M checkpoint 8 ).This would enable the formation of the full PP2A:B55-pARPP19 inhibitory complex and, once formed, serve as a 'timer' to facilitate mitotic exit via the slow transition of pARPP19 from an inhibitor to a substrate.The molecular bases for these events are under active investigation.Together, these studies provide a molecular understanding of regulator and substrate recruitment of the PP2A:B55 holoenzyme.Because of the key regulatory functions of PP2A:B55 in mitosis and DNA damage repair, these data provide a roadmap for characterizing disease-associated mutations and pursuing new avenues to therapeutically target this complex, by individually blocking a subset of regulators that use different B55 interaction sites.

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Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41586-023-06870-3.

Mammalian protein expression
Full-length B55 1-477 was cloned into pcDNA3.4including an N-terminal green fluorescence protein (GFP) followed by a TEV cleavage sequence.Full-length PP2Ac 1-309 was cloned into pcDNA3.4with 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 pcDNA3.4with 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% CO 2 under shaking (125 rpm).On the day of transfection, the cell density was adjusted to 2.8 × 10 6 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.

Phosphorylation of ARPP19
Purified 15 N-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 MgCl 2 ) 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 1 H, 15 N 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 MnCl 2 ), 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 .

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 Article 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.

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 1.2/1.3300 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 4.0 53 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 4.1.14 54on 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 Topaz 55 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 SIDESPLITTER 56 .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 Coot 57 and ISOLDE 58 , and automated global real-space refinement in Phenix 59 .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 maps 60 .
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.

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 (http://www.cara.nmr.ch).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 database 62 .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 structure 63 .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: For each interaction, an excess of unlabelled B55 LL of PP2A:B55 complex (min 25% surplus ratio) was added to the 15 N-labelled FAM122A or ARPP19 construct under investigation and incubated on ice for 10 min before the 2D 1 H, 15 N HSQC spectrum was collected.FAM122A and ARPP19 concentrations ranged from 2-6 µM.NMR data were processed using nmrPipe 64 and the intensity data were analysed in Poky 65 .Each dataset was normalized to its respective most intense peak and the difference between each free 2D 1 H, 15 N HSQC spectrum FAM122A or ARPP19 residue was compared to its respective peak, if present, on the 2D 1 H, 15 N HSQC spectrum of FAM122A or ARPP19 in complex with B55 LL or PP2A:B55.Any overlapping peaks were omitted for this analysis.

Fig. 3 |
HSQC and CSI plots of ARPP19, FAM122A and MASTL-phosphorylated ARPP19.a. Fully annotated 2D [ 1 H,15 N] HSQC spectrum of15 N-labeled ARPP19.b.Chemical Shift Index (CSI) and c.Secondarystructure propensity (SSP) data for ARPP19 plotted vs. residue numbers.(SSP > 0, α helix; SSP < 0, β strand).Cα and Cβ chemical shifts were used to create the CSI and SSP plots (RefDB database62 ).Preferred secondary structure indicated above SSP data.d.Fully annotated 2D [ 1 H,15 N] HSQC spectrum of15 N-labeled FAM122A ID .e. CSI and f.SSP data for FAM122A ID plotted vs. residue numbers; same as c. g.Fully annotated 2D [ 1 H,15 N] HSQC spectrum of15 N-labeled tpS62tpS104ARPP19.h.CSI; same as c. i. CSI comparison between ARPP19 (black) and tpS62tpS104ARPP19 (red).Extended Data Fig.4| NMR data supporting the interaction of phosphorylated ARPP19 and FAM122A with PP2A:B55.a. 2D [ 1 H,15 N] HSQC spectrum of15 N-labeled tpARPP19 alone (black) and in complex with PP2A:B55 (green).pS62 labeled for clarity.b.Peak intensity vs ARPP19 protein sequence plot for tpARPP19 alone (black) and when bound to PP2A:B55 (green).Secondary structure elements based on NMR CSI data are indicated.c. 2D [ 1 H,15 N] HSQC spectrum of15 N-labeled tpS62tpS104ARPP19 alone (black) and in complex with PP2A:B55 (green).pS62 and pS104 labeled for clarity.d.Peak intensity vs ARPP19 protein sequence plot for tpS62tpS104ARPP19 alone (black) and when bound to PP2A:B55 (green).Secondary structure elements based on NMR CSI data are indicated.e. 2D [ 1 H,15 N] HSQC spectrum of15 N-labeled FAM122A Nterm alone (black) and in complex with PP2A:B55 (green).f. 2D [ 1 H,15 N] HSQC spectrum of15 N-labeled FAM122A ID alone (black) and in complex with PP2A:B55 (green).g.Peak intensity vs FAM122A ID protein sequence plot for FAM122A ID alone (black) and when bound to PP2A:B55 (green).Secondary structure elements based on NMR CSI data are indicated.Extended DataFig.9 | Intra-and intermolecular PP2A:B55-inhibitor interactions.a.The methylated C-terminal tail of PP2Ac (cyan) extends to the opposite side of PP2A (grey) where it interacts at the interface between PP2Aa and B55 (lavender).b.Close-up of (a) with the C-terminus shown as sticks and PP2Aa, B55 and the rest of PP2Ac shown as a surface.c.Different view of a,b with the PP2Ac C-terminus shown as sticks and PP2Aa and B55 interacting residues also shown as sticks.Polar/ionic inter-subunit interactions indicated by black dashed lines and the participating residues underlined.d.Detailed interactions between the methylated C-terminal tail of PP2Ac (cyan, highlighted by purple box in (a)) with PP2Aa (grey) and B55 (lavender).e. Alanine scanning mutagenesis of ARPP19 amino acids L32-Q41.The indicated YFP-ARPP19 constructs were transfected into HeLa cells and immunopurified.Binding efficiency of the YFP-ARPP19 derivatives to B55 and PP2Ac was determined by Western blotting.f.Quantification of (e) based on two independent experiments.g.Alanine scanning mutagenesis of ARPP19 amino acids D47-G56.The indicated YFP-ARPP19 constructs were transfected into HeLa cells and immunopurified.Binding efficiency of the YFP-ARPP19 derivatives to B55 and PP2Ac was determined by Western blotting.Quantification of (g) based on two independent experiments shown in Fig. 3e.h.FAM122A K89 makes polar/ionic interactions (dashes) with multiple residues from B55. i. Helical wheel N→C view of the B55 inhibition helix highlighting residues that interact with PP2Ac and those that are solvent exposed.