Extended Data Fig. 10: NMR displacement and pull-down studies show that FAM122A displaces p107 and that FAM122A and tpARPP19 can bind simultaneously, with similarities to PP1.
From: Cryo-EM structures of PP2A:B55–FAM122A and PP2A:B55–ARPP19
a. 2D [1H,15N] HSQC spectrum of 15N-labeled p107 alone (black) and in complex with B55LL (red) shows that specific p107 residues bind B55LL. b. The addition of unlabeled FAM122A replaces 15N-labeled p107 in the identical B55LL binding surface and now starts to appear in the 2D [1H,15N] HSQC spectrum (light blue) in an identical position as in the 2D [1H,15N] HSQC spectrum of 15N-labeled p107 alone (black). c. Further addition of unlabeled FAM122A replaces 15N-labeled p107 in the identical B55LL binding surface and now nearly fully 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). Black boxes highlight inserts shown in Fig. 5b in the manuscript. d. Pulldown assay demonstrating that tpS62-ARPP19 does not displace FAM122A when bound to PP2A:B55. Expi293F lysates co-transfected with GFP-B55 and PP2Ac-strep were incubated with purified PP2Aa and FAM122A alone and with a 5-fold surplus of tpS62-ARPP19, pulled-down using a GFP-TRAP and immunoblotted for the indicated proteins. Results representative of 3 independent experiments. e. 2D [1H,15N] HSQC spectrum of 4.5 μM 15N-labeled ARPP19 alone (black) and in complex with 6.9 μM unlabeled B55LL and 22.5 μM unlabeled FAM122A (red) shows that mostly ARPP19 helix α2 HN/N cross peaks stay bound to B55LL (annotated in orange); compare with Fig. 1f without FAM122A. f. tpARPP19 (orange) bound to PP2Ac (cyan). g. FAM122A (magenta) bound to PP2Ac (light pink). h. I-2 (light green) bound to PP1 (grey; pdbid 2OG8). i. Overlay of PP2Ac and PP1 bound to tpARPP19, FAM122A and I-2, respectively. Colors as in a-c. PP2Ac shown as surface. j. Zoom view of (i) with the residues near the active site shown as sticks. Bound metals are shown. k. PP2Ac:tpARPP19 in same orientation as j. l. PP2Ac:FAM122A in same orientation as j. m. PP1:I-2 in same orientation as j. n. Same as j but without the PPPs.
