Figure 2. TA2B and TID form an anti-parallel β-sheet with a polar and a hydrophobic face.

(A) Domain organization of TAp63α and secondary structure elements of TAD and TID. (B) Proposed interaction of TA2 and TID through β-sheet formation. This interaction is thought to be stabilized by hydrophobic amino acids clustered on one face of the β-sheet (bottom) and electrostatic interactions between charged amino acids on the other face (top). Extensive charge swap experiments (see Figure 2—figure supplement 1) revealed interactions between TA2B and TID. Interactions are depicted in green. (C, D) Introduction of negative charges in the TID and charge swaps between TID and TA2B show interaction via β-sheet formation. (C) SEC profiles of TAp63α R604E R608E (orange) and the charge swap mutant TAp63α E51R D55R R604E R608E (blue). (D) SEC profiles of TAp63α R595E R598E (orange) and the charge swap mutant TAp63α D61R D63R R595E R598E (blue).

DOI: http://dx.doi.org/10.7554/eLife.13909.014

Figure 2—figure supplement 1. TA2B and TID form an anti-parallel β-sheet.

(A) Secondary structure prediction of TAD and TID. TA2B and TID are predicted to form β-strands. (B) On validation, charges were swapped between presumably distant amino acids. SEC profiles of TAp63α E51R D55R R595D R598D (orange) and TAp63α D61R D63R R604D R608E (blue). (C) Charge swaps are used to reveal interactions between charged amino acids across the presumed β-sheet formed by TA2B and TID. A destabilizing mutation (arginine to aspartate or glutamate) is introduced into TAp63α. Solely mutation R604D/E leads to the formation of tetramers. Any other arginine in TA2B or TID mutated to aspartate or glutamate does not change the oligomeric state. In order to break the interaction between TA2B and TID, a second destabilizing mutation is introduced that disrupts the dimeric state and leads to the formation of tetramers. Additional compensating mutations in the double charge swap recover the dimeric state. To prove a single interaction between two differently charged amino acids, they are swapped in presence of a destabilizing mutation (R608E) resulting in a triple mutant. Similar SEC profiles of the destabilizing and the triple mutation prove the interaction between the swapped amino acids. (D) Extensive charge swap experiments (shown in E-I) revealed interactions (shown in green) between TA2B and TID. (E) SEC profiles of a single charge swap between R604 and D55 (blue) and the destabilizing mutation R604D show a direct interaction between D55 and R604. (F,G,H,I,J) SEC profiles of double arginine mutants (top, orange) and double charge swaps (top, blue). SEC profiles of triple mutants (bottom, blue) and the R608E mutant (bottom, orange) should be identical to verify the interaction shown in bold (top). For comparison identical western blots / SEC profiles of TAp63α R608E are shown on bottom. (F) SEC profiles of TAp63α R58D R608E (top, orange), charge swap TAp63α E51R R58D D601R R608E (top, blue), triple mutant TAp63α R58D D601R R608E (bottom, blue) and mutant TAp63α R608E (bottom, orange). (G) SEC profiles of TAp63α R598D R608E (top, orange) and charge swap TAp63α E51R D61R R598D R608E (top, blue), triple mutant TAp63α D61R R598D R608E (bottom, blue) and mutant TAp63α R608E (bottom, orange). (H) SEC profiles of TAp63α R595D R608E (top, orange) and charge swap TAp63α E51R D61R R595D R608E (top, blue), triple mutant TAp63α D61R R595D R608E (bottom, blue) and mutant TAp63α R608E (bottom, orange). (I) SEC profiles of TAp63α R58E R608E (top, orange) and charge swap TAp63α E51R R58E E597R R608E (top, blue), triple mutant TAp63α R58E E597R R608E (bottom, blue) and mutant TAp63α R608E (bottom, orange). (J) SEC profiles of TAp63α R598D R608E (left, orange, identical blot/profile as shown in G) and charge swap TAp63α E51R D63R R598D R608E (left, blue), triple mutant TAp63α D63R R598D R608E (right, blue) and mutant TAp63α R608E (right, orange).

DOI: http://dx.doi.org/10.7554/eLife.13909.015