TABLE 1.
Protein mutations and interactions tested
| Mutation in | Mutant tested | Tested for interaction with | Assaya | Figure |
|---|---|---|---|---|
| PIAS1 SP-RING | PIAS1 L337 | UBC9 | BRET (↓) | 1B |
| PIAS1 SIM | PIAS1 SIMmtb | UBC9 | BRET (↓) | 3B, Not shown |
| SUMO1 or SUMO2 | ||||
| PIAS1 serines adjacent to the SIM | PIAS1 3SD | UBC9 | BRET (↑) | 3C and D |
| PIAS1 3SD peptide encompassing the SIMc | SUMO1 SUMO2 UBC9 UBC9 and SUMO1 |
ITC (↑), NMR ITC (↑) NMR NMR |
3F, 4, A–C and F Not shown Not shown 6 |
|
| PIAS1 3SA | UBC9 | BRET (↓) | 3C and D | |
| PIAS1 acidic region recognized by CK2 and adjacent to the SIM | PIAS1 5EA | UBC9 | BRET (↓) | 3D |
| UBC9 loops predicted to interact with PIAS1 SP-RING | UBC9 P69A | PIAS1 | BRET (↓) | 1D |
| UBC9 P105A | PIAS1 | BRET (n.c) | 1D | |
| UBC9 catalytic site | UBC9 C93S or C93A | PIAS1 | BRET( n.c) | 2B |
| UBC9 SUMOylation site | UBC9 K14A | PIAS1 | BRET (n.c) | 2D, Not shown |
| SUMO1 | ||||
| UBC9 backside region forming an interface with β1, 4 and 5 strands of SUMO | UBC9 R13A | PIAS1 | BRET (↓) | 2G, Not shown |
| SUMO1 | ||||
| UBC9 R17A | PIAS1 | BRET (↓) | 2E, Not shown | |
| SUMO1 or SUMO2 | ||||
| UBC9 H20D | PIAS1 | BRET (↓) | 2E, Not shown | |
| SUMO1 or SUMO2 | ||||
| UBC9 F22A | PIAS1 | BRET (↓) | 2F | |
| SUMO surface interacting with UBC9 backside | SUMO1 E67R | UBC9 | BRET (↓) | Not shown |
| SUMO2 D63R | UBC9 | BRET (↓) | Not shown | |
| SUMO1 surface interacting with PIAS SIM sequence | SUMO1mt (Phe-36, Lys-37, Lys-39, Lys-45, Lys-46 mutated to Ala) | PIAS1 | BRET (↓) | Not shown |
a The results relative to wild-type controls in the BRET or ITC signal are indicated in the parentheses: increase (↑), decrease (↓), or no significant change (n.c.).
b SIMmt amino acids 457–464: VEVIDLTI → AEAADATA.
c PIAS1 phosphomimetic peptide amino acids 456–480: KVEVIDLTIDDDDDEEEEEPSAKRT (the SIM is underlined, and the serines mutated to aspartic acid are in bold).