Fig. 8.
Long-distance allosteric effects in ANP32B induced by cytochrome c. (A) Ribbon representation of ANP32B complexed with PP2A after 10-ns MD. ANP32B is colored in blue, PP2A catalytic subunit (PP2A-C) is represented in orange and PP2A scaffold and regulatory subunits—PP2A-A and PP2A-B respectively—are colored in grey. Inset — Detailed view of ANP32B Asp31 side chain—labelled in blue—in close contact with the Mn atoms from PP2A catalytic center. PP2A-C residues in close contact with ANP32B Asp31 residue are labelled in black. (B) Upper panel — Analysis of the atomic fluctuations (RMSF) of ANP32B LRR domain—comprising residues from 1 to 161—within the context of ANP32B1-251 either free (black line), in the presence of 2 molecules of Cc bound to the LCAR domain (red line) or in complex with PP2A (blue line). Lower panel — Subtraction of the RMSF values of ANP32B LRR domain in ANP32B1-251 to ANP32B1-251:2Cc (red line) or ANP32B1-251:PP2A (blue line) MD data. The dynamic behavior of ANP32B Asp31 residue is marked with an asterisk. (C) Representation of the LCAR concerted motions with the LRR domain in ANP32B structure either free (left panel) or in the presence of 2 molecules of Cc placed at LCAR (right panel). Red spheroids represent the 3D RMSF values of the oxygen atoms of Glu or Asp sidechains, whereas blue ellipsoids represent 3D RMSF values of nitrogen atoms of Lys and Arg sidechains. A detailed-rotated view of ANP32B Asp31 residue is shown in each panel. Black arrows on left panel point out to those residues exhibiting lower atomic fluctuations than in free ANP32B1-251 due to Cc binding. (D) Relative PP2A enzymatic activity in HEK293T cell extracts transfected with either empty pcDNA 3.1 (colored in green; E.V. stands for Empty Vector), pcDNA 3.1-ANP32B1-251 WT-c-myc (colored in blue) or pcDNA 3.1-ANP32B1-251 D31A-c-myc (colored in orange) upon addition of recombinant Cc at increasing amounts. As a control, additional WT ANP32B1-251 assays were performed in parallel to those for the ANP32B1-251 D31A mutant. Each data is the average value ± S.D. of three independent experiments. Detection by Western-Blot of transfected ANP32B species in cell extracts by using anti-c-myc antibody are included. (E) Upper panel — Analysis of the RMSF of the ANP32B LRR domain within the context of free ANP32B1-251 WT (black line) and D31A mutant (green line), and in the presence of two molecules of Cc bound to the LCAR (red line). Lower panel — Subtraction of the RMSF values of WT ANP32B LRR domain in ANP32B1-251 to ANP32B1-251:2Cc (red line) or ANP32B1-251 D31A mutant (green line) MD data. ANP32B Asp31 is marked with an asterisk. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)