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Deciphering the substrate specificity of ubiquitin conjugating enzymes

Deciphering the substrate specificity of ubiquitin conjugating enzymes. Fábio M. Marques Madeira Supervisor: Professor Ronald T. Hay. 24 th July 2013. Protein ubiquitylation. Hochstrasser , M. (2009) Nature 458 , 422–9. 1. The ubiquitin modification cascade. RNF4.

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Deciphering the substrate specificity of ubiquitin conjugating enzymes

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  1. Deciphering the substrate specificity of ubiquitin conjugating enzymes Fábio M. Marques Madeira Supervisor: Professor Ronald T. Hay 24th July 2013

  2. Protein ubiquitylation Hochstrasser, M. (2009) Nature458, 422–9 1

  3. The ubiquitin modification cascade RNF4 STUbL having a key role in DNA damage response Woelk, et al. (2007) Cell Division2:11 2

  4. RNF4 RING bond to ubiquitin-loaded UbcH5a Plechanovová, et al. (2012) Nature489, 115–20 3

  5. RNF4 RING bond to ubiquitin-loaded UbcH5a pKa 10.5 ± 1.1 ε-amino group of lysine Tetrahedral transition state intermediary Plechanovová, et al. (2012) Nature489, 115–20 4

  6. Ube2W conjugates ubiquitin to α-amino groups of protein N-termini pKa 7.7 ± 0.5 α-amino group of the substrate N-terminus Tatham, et al. (2013) The Biochemical Journal453, 137–45 5

  7. Aims Investigate what are the features of the active site of UbcH5a and Ube2W that enable them do discriminate between N-terminal α-amino groups and Lys ε-amino groups • Sequence and structure-informed mutational analysis of key residues • Protein expression and purification of the mutant proteins • Biochemical characterization of the proteins and in vitro ubiquitin conjugation assays 6

  8. Structural analysis of UbcH5a and Ube2W Helix 1 D117 * N77 Helix 2 Ubiquitin (4AP4:F) Ube2W model (I-TASSER) UbcH5a (4AP4:E) Ube2W model (Phyre2) Ube2W (2A7L:A) N 7

  9. Multiple alignment analysis of UbcH5a and Ube2W Helix 2 Helix 1 UbcH5a~Ubiquitin (4AP4:E~F) Model of Ube2W~Ubiquitin (4AP4:F) 8

  10. Multiple alignment analysis of UbcH5a and Ube2W Helix 2 Helix 1 Ube2W M1 – H94N M2 – K133P/R134D/R135D M3 – M1/M2 M4 – S129D/delC130/K131P/E132N/K133P/R134D/R135D M5 – M1/M4 UbcH5a M1 – N77H M2 – P115K/D116R/D117R M3 – M1/M2 M4 – D112S/insC/P113K/N114E/P115K/D116R/D117R M5 – M1/M4 8

  11. Protein expression and purification E. coli BL21 (DE3) - + M2 - + M2 - + wt M1 - + - + M3 M3 - + M2 - + M4 - + - + M4 M3 - + M5 - + - + M5 - + M4 M5 - + IPTG Site-directed mutagenesis kDa 18 75 - 50 - His6-UbcH5a 37 - 25 - 20 - Mutant strand synthesis DpnI digestion DNA sequencing 15 - 10 - ArcticExpress (DE3) Rosetta (DE3) IPTG kDa 75 -  Cpn60 50 - 37 - 25 - 20 - His6-UbcH5a 15 -  Cpn10 10 - 9

  12. Protein expression and purification E. coli Rosetta (DE3) E. coli BL21 (DE3) - + wt wt - + M1 M1 - + - + - + M2 M2 - + - + M3 M3 - + - + M4 M4 - + - + - + M5 M5 IPTG IPTG Site-directed mutagenesis kDa kDa 18 75 - 75 - 50 - 50 -  His6-Ube2W  His6-UbcH5a 37 - 37 - 25 - 25 - 20 - 20 - Mutant strand synthesis DpnI digestion DNA sequencing 15 - 15 - 10 - 10 - 9

  13. Protein expression and purification S F B W E T Ube2W M2-5 Ube2W wtand M1 C C S F S F B W B W E T E T kDa Lyse cells kDa kDa Wash 75 - 75 - 75 - 50 - 50 - 50 - Elute 37 - 37 - 37 - Expression vector Cleavage with TEV protease Purified proteins Ni-NTA resin His6-tagged proteins  His6-Ube2W His6-UbcH5a 25 - 25 - Ube2W 25 - UbcH5a  His6-Ube2W 20 - 20 - 20 - Ube2W 15 - 15 - 15 - UbcH5a wtand M1-3 His6-tag His6-tag  His6-tag 10 - 10 - 10 - F – Flow-through B – First wash C – Cell suspension S – Supernatant W – Second wash E – Elution T – After His6-tag cleavage with TEV 10

  14. Protein expression and purification Ube2W M2-5 kDa 13 2 3 4 5 7 8 9 10 11 12 6 1 20 - C S F B W E T 15 - kDa Gel filtration on a HiLoad 16/60 Superdex 75 pg 75 - 50 - 37 - 25 -  His6-Ube2W 20 - Ube2W 15 - His6-tag 10 - 7 13 6 1 Equilibriumofmonersanddimers Vittal, et al. (2013) Cell Biochemistry and Biophysics 13, 9633-5 11

  15. The ability of mutant proteins to form E2~Ub thioester bonds wt wt M1 M1 M2 M2 M3 M3 M4 M5 Reaction mix: + + + ATP E1 E2 Ub Ube2W UbcH5a Time Time Ube2W~Ub kDa kDa UbcH5a~Ub 25 - 25 - 25 - 25 - Ube2W  UbcH5a 20 - 20 - 20 - 20 - 15 - 15 - 15 - 15 - Ubiquitin Ubiquitin Non-reducing SDS-PAGE 10 - 10 - 10 - 10 - Ube2W  UbcH5a Ubiquitin Ubiquitin Reducing SDS-PAGE M1 – N77H M2 – P115K/D116R/D117R M3 – M1/M2 12

  16. pH titration analysis of UbcH5a and Ube2W 6.5 6.5 7.0 7.0 7.5 7.5 8.0 8.0 8.5 8.5 9.0 9.0 9.5 9.5 10.0 10.0 10.5 10.5 11.0 11.0 pH Time Reaction mix: + + + ATP + + E1 E2 Ub E3 kDa  His6-SUMO-2x4~Ub kDa UbcH5a  His6-SUMO-2x4 75 - 75 - 75 - 75 - 50 - 50 - 50 - 50 -  His6-SUMO-2x4~Ub UbcH5a N77H Peptide-His6-SUMO-2x4  His6-SUMO-2x4 SUMO-2 SUMO-2SUMO-2 SUMO-2 N His6 pH Time  His6-SUMO-2x4~Ub Ube2W  His6-SUMO-2x4  His6-SUMO-2x4~Ub Ube2W M3  His6-SUMO-2x4 M3 – H94N/ K133P/R134D/R135D 13

  17. Conclusions • Key residues in the active site of Ube2W are different from most of the conserved E2s • Ube2W shows an equilibrium of monomers and dimers that does not rely on the C-terminus • Most of the mutant proteins can still form a thioester bond with ubiquitin, although their ability to modify a poly-SUMO2 substrate is affected • Ube2W shows pH-dependent activity at pH below 9.0 14

  18. Future work • Try to overcome low expression of UbcH5a mutants by DNA synthesis of the constructs with codon optimization • Investigate what are the key features of N-terminal amino groups modified by Ube2W, using N-terminal modified substrates (myc-tag, in vitrocarbamylation, etc.) • Try solving the structure of RNF4-Ube2W~Ubiquitin mutating the catalytic Cys to Lys to form an isopeptide linkage 15

  19. Acknowledgements • Professor Ronald T. Hay • Anna Plechanovová • Ellis Jaffray • Linnan Shen • Mike Tatham • … all members of the Hay group!

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