Supplementary information. Specific recognition of linear polyubiquitin by A20 zinc finger 7 is involved in NF-κB regulation

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1 Supplementary information Specific recognition of linear polyubiquitin by A20 zinc finger is involved in NF-κ regulation Fuminori Tokunaga, Hiroshi Nishimasu, Ryuichiro Ishitani, Eiji Goto, Takuya Noguchi, Kazuhiro Mio, Kiyoko Kamei, Averil Ma, Kazuhiro Iwai and Osamu Nureki This document includes: Supplementary Figures S16

2 A20 CYLD A20 CYLD Cezanne M1 K63 K48 M1 K63 K48 (min) (h) I: I: I: C -NEMO Linear --NEMO (min) A20 CYLD I: Supplementary Figure S1 DU activities of A20, CYLD and Cezanne. (A) DU activities of A20, CYLD and Cezanne for linear polyubiquitin chains. FLAG-tagged A20, CYLD and Cezanne were expressed in HEK293T cells, and the cell lysates were incubated with anti-flag beads to capture the proteins. LUAC-generated linear polyubiquitin chains were reacted with the beads for the indicated times. Linear polyubiquitin degradation was detected by western blotting, using an anti-ubiquitin antibody. () In vitro DU activities of A20 and CYLD for linear, K63- and K48-linked polyubiquitin chains. aculovirally expressed recombinant A20 and CYLD were reacted with linear (M1)-, K48- or K63-linked polyubiquitin, and analyzed as in (A). (C) DU activities of A20 and CYLD for NEMO-conjugated linear polyubiquitin chains. The -tagged C-terminal region of NEMO (residues ) was linearly ubiquitinated by LUAC and purified using glutathione Sepharose beads (left panel). DU activities of A20 and CYLD for NEMO-conjugated linear polyubiquitin chains were examined as in (A) (right panel).

3 HEK293T 0 Relative NF-κ (%) LUAC LUAC CYLD C601A Supplementary Figure S2 Expression of the catalytically inactive CYLD C601A mutant enhances LUAC-mediated NF-κ activation. Luciferase reporter assays were performed as in Figure 1, and the relative luciferase activities are shown as mean ± s.d. (n = 3).

4 E1 ch5c LUAC -ZF4 - E1 ch5c LUAC -ZF4 - NEMO I: I: NEMO Supplementary Figure S3 A20 does not inhibit LUAC E3 activity. (A, ) A20 does not inhibit the LUAC-catalysed generation of either free linear polyubiquitin (A) or NEMO-conjugated linear polyubiquitin (). In vitro ubiquitination experiments were performed using E1 (100 ng), ch5c (200 ng), recombinant LUAC (1 μg) and increasing amounts of proteins (0.3, 1, 3 μg), in the absence (A) and presence () of recombinant FLAG-His6-tagged full-length human NEMO. Polyubiquitin chains were detected by western blotting, using the indicated antibodies.

5 A20 linear di- A20 linear tetra- Proximal Distal 2 2 Form I C Form II D Form I Form II dist dist in parallel filaments dist in parallel filaments Gly6 Met1 prox in antiparallel filaments Gly6 Met1 prox Gly6 Met1 prox Supplementary Figure S4 Two types of crystal packing of A20 in complex with linear ubiquitin chains. (A) In both Forms I and II, A20 (orange) molecules intercalate between each pair of two adjacent ubiquitin molecules (grey) in the same manner, forming a straight, filamentous structure (stereoview). The N-terminal of A20 projects outward from the filamentous structure, suggesting that full-length A20 can bind linear polyubiquitin in a manner similar to that of A20 alone. The schematic drawings show the crystal packing in the diubiquitin and tetraubiquitin complexes. (, C) All of the filaments formed by A20 (orange) and ubiquitin (grey) molecules run parallel in Form I (), whereas one filament is surrounded by six filaments that run antiparallel to it in Form II (C). The filamentous structures run perpendicular to the paper, and the unit cells are indicated by black lines. (D) A20 (orange) interacts with three and four adjacent ubiquitin molecules (grey) in Forms I and II, respectively. Forms I and II commonly contain one A20 linear diubiquitin complex with a virtually identical conformation.

6 Absorbance at 2 nm (mau) 20 WT A64K A66K H69A/F0A N2A NA E81A F85A Y89A F85A/Y89A 10 A Elution volume (ml) Turbo3C protease Turbo3C protease WT A64K A66K H69A/F0A N2A NA E81A F85A Y89A F85A/Y89A WT A64K A66K H69A/F0A N2A NA E81A F85A Y89A F85A/Y89A Turbo3C -A A20 Supplementary Figure S5 Size-exclusion chromatography of WT and mutants of A20. -tagged A20 proteins were expressed in E. coli and purified by glutathione Sepharose chromatography. The purified -tagged A20 proteins (0.1 mg) were treated with Turbo3C protease (0.6 μg) (Nacalai Tesque) overnight at 4ºC to cleave the -tag, and then were analyzed by size-exclusion chromatography on a Superdex5 10/0 column (GE Healthcare), equilibrated with PS buffer supplemented with 1 mm TCEP. The proteins before and after the protease treatment were analyzed by 1020% SDS-PAGE, and stained with Simplylue SafeStain (Invitrogen).

7 Lysate IP: HOIP Myc-HOIP/HOIL-1L-HA/T-SHARPIN FLAG-A20 WT FLAG-A20 OTU FLAG-A20 ZF FLAG-A20 ZF13 FLAG-A20 ZF4 I: Myc, HA, T I: Myc Lysate IP: HOIP Myc-HOIP/HOIL-1L/T-SHARPIN FLAG-A20 WT FLAG-A20 C103A FLAG-A20 ZF4CA FLAG-A20 CA FLAG-A20 I: Myc, HOIL-1L, T I: Myc, HOIL-1L, T A20 HOIP HOIL-1L SHARPIN Supplementary Figure S6 A20 is crucial for association with LUAC. (A) A20 ZF4 participates in the association with LUAC. () Intact A20 is required for the association with LUAC. Cell lysates and immunoprecipitates from HEK293T cells expressing the indicated proteins were detected by western blotting, using the indicated antibodies (A and ).