The molecular basis of lysine 48 ubiquitin chain synthesis by Ube2K

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1 Supplementary Information The molecular basis of lysine 48 ubiquitin chain synthesis by Adam J. Middleton, Catherine L. Day* Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin 954, New Zealand * catherine.day@otago.ac.nz

2 Supplementary 1 a) : 2 K97R min b) c) A46 H68 A171 A171 F174 M172 I44 L8 T194 M172 E195 UBA e) ~Ub -Ub ~Ub UBA Refractive index (1-5 ) d) R42 E195 R72 UBA I44 L8 V7 L198 E191 L N Elution volume (ml) Molecular mass (kda) F174 H68

3 Supplementary Figure S1. Analysis of the ~Ub conjugate. (a) Multi-turnover time-course assay comparing wild-type and K97R. (b and c) Close up schematic of the UBA:ubiquitin interaction seen in the (b) crystal structure, and (c) ubiquitin complex (PDB ID: 3K9P). Shown in sticks are interacting side chains between and ubiquitin. Oxygen, nitrogen and sulphur atoms are red, blue, and yellow, respectively. (d) Overlay of seven UBA:ubiquitin structures available on the PDB (PDB ID: 1WR1, 2MJ5, 2G3Q, 2MRO, 4UN2, TJY6, 3K9P) against the ubiquitin:uba interface observed in the ~Ub conjugate crystal structure. ~Ub conjugate and UBA:ubiquitin structure (3K9P) are coloured as indicated. (e) MALS analysis of and ~Ub at 3 µm shows that they are monomeric in solution. Calculated molecular mass of (22.4 kda) and ~Ub (31. kda) are shown as dashed lines, while measured mass is represented as green () and red (~Ub) squares.

4 Supplementary S2 a) b) di-ubiquitin / K63 K48 Ub: K11 8L A11 5A L11 1A E12 E12 : L11 1A d) 1N c) 37 E121N ~Ub 25 Ub variant 8L : A11 concentration (ng) 5A 25 L11 2 L11 1A 15 1A 1 E12 5 1N E12.2 Signal (A.U.) x 15 2.

5 Supplementary Figure S2. Mutations targeting α2 of disrupt ubiquitylation. (a) Standard curve of quantified from Coomassie-stained SDS PAGE gels. This standard curve was generated from within the range used in this study (R 2 =.99). (b) variants were assayed in a multi-turnover assay and quantified in triplicate. Vertical bars represent standard deviation. (c) SDS PAGE gels of (top) and charging assays (bottom) of variants. Lane 1 is from Fig 2 (samples on same gel). (d) Single-turnover assay performed with mutant E121N combined with single-lysine containing variants of ubiquitin as Ub A to test chain specificity.

6 Supplementary S3 a) : K97R N132R M172A M172A+ E195R D98S Q126L A128P D98R R74A K97E D127N D124R T88D S85N ~Ub b) : D123R Q126E E121R c) -RNF12 +RNF12 E1 ~Ub Ubn /Ub3 d) : RNF12-Ub E121N D98S D98R D124R Q126L A128P L115A A118L L111A RNF12 N132R K97E S85N R74A D127N T88D M172A K97R M172AE195R RNF12 K / Supplementary Figure S3. Testing catalytic ability of mutants. (a) Charging assay showing that variants analysed for synthesis are properly folded and can interact with the E1 enzyme. (b) Assay of variants that were found to be defective at charging with K ubiquitin. These mutants were not further investigated in this study. (c) Multiturnover ubiquitylation assay (6 min) with wild-type in the presence and absence of RNF12. (d) Charged mutants were spiked with 5 µm RNF12 to check the integrity of their active site by monitoring formation of monoubiquitylated RNF12. Only variants D124R and A118L resulted in no production of ubiquitylated RNF12.

7 Supplementary S4 : T88V T88S Q126L A128P D98S D98R D124R N132R min : K97E S85N R74A E121N A115L A118L L111A min : D127N T88D K97R M172A M172A E195R min Supplementary Figure S4. Time course of initial mutations. Multi-turnover ubiquitylation assay showing time course of formation by and variants. Each assay was performed in parallel with the wild-type at the beginning of the row of gels.

8 Supplementary S5 a) Signal (A.U.) x Incubation time (s) b) Ub ~Ub Ub Supplementary Figure S5. Di kinetics experiments. (a) Wild-type was charged with K and incubated with 1 mm wild-type ubiquitin as Ub A. Samples were removed at the indicated times and were run on SDS-PAGE. Bands corresponding to were quantified and plotted against time. This experiment was performed for all and ubiquitin variant combinations to select a time point within the linear response of formation. For wild-type incubated with wild-type ubiquitin, 4 s was used. (b) Wild-type was charged with K as above and incubated with increasing amounts of wild-type ubiquitin as Ub A. The band was quantified and normalized to ~Ub and time to give apparent k obs (s -1 ).

9 Supplementary S6 a) 75 FITKIWHPNISSVTGAICLDILK...DQWAAAMTLRTVLLSLQALLAAAEPDDPQDAVVAN 132 Ubc1 71 FDTKVYHPNISSVTGAICLDILK...NAWSPVITLKSALISLQALLQSPEPNDPQDAEVAQ 128 Ube2D2 69 FTTRIYHPNIN.SNGSICLDILR...SQWSPALTISKVLLSICSLLCDPNPDDPLVPEIAR 125 Ube2E2 123 FRTRIYHCNIN.SQGVICLDILK...DNWSPALTISKVLLSICSLLTDCNPADPLVGSIAT 179 Ube2R1 77 FLTKMWHPNIY.ETGDVCISILHPPVDDPQSGELPSERWNPTQNVRTILLSVISLLNEPNTFSPANVDASV 146 Ube2G2 73 FTCEMFHPNIY.PDGRVCISILHAPGDDPMGYESSAERWSPVQSVEKILLSVVSMLAEPNDESGANVDASK 142 b) Ube2D2 C92 C85 D127 N79 S85 S86 T88 S8 V86 N81 Supplementary Figure S6. Serine 86 extends the β4- α2 loop. (a) Alignment of and Ubc1 against other E2s. Active site Cys residues are highlighted in yellow, Ub A coordinating Lys/Arg in blue, putative desolvating acidic groups in pink, and critical residues of in green. Identical, highly similar and similar residues are indicated by dark, medium and light grey highlights, respectively. (b) Overlay of and Ube2D2 showing the extension of the β4-α2 loop in due to the presence of Ser86. Side chains of residues of, of Ube2D2 and the active site Cys are shown as sticks. is in blue, and Ube2D2 is in green. Oxygen, nitrogen and sulphur atoms are in red, blue, and yellow, respectively.

10 Supplementary Table S1. Di synthesis kinetics assay (n=3) (Ub A ) Incubation time (s) Mean of apparent K m (µm) (+/- Std. error) Mean of apparent k cat (s -1 ) (+/- Std. error) Wild type Wild type (+/- 196).91 (+/-.17) Wild type E51R (+/- 332).38 (+/-.72) Wild type Y59L >12 N/D a N/D a K97E Wild type 12 >3 b N/D b K97E E51R (+/- 18.9).21 (+/-.15) Q126L Wild type 12 >3 b N/D b Q126L Y59L 6 >3 b N/D b a No significant synthesis activity was observed with 2 mm Ub A b K m was above the measurable range of the experiment, making estimation of apparent K m and k cat impossible