Deciphering the substrate specificity of ubiquitin conjugating enzymes

Fábio M. Marques Madeira

Supervisor: Professor Ronald T. Hay

24th July 2013

Protein ubiquitylation

1Hochstrasser, M. (2009) Nature 458, 422–9

The ubiquitin modification cascade

2Woelk, et al. (2007) Cell Division 2:11

STUbL having a key role in DNA damage response

RNF4

RNF4 RING bond to ubiquitin-loaded UbcH5a

3Plechanovová, et al. (2012) Nature 489, 115–20

RNF4 RING bond to ubiquitin-loaded UbcH5a

4Plechanovová, et al. (2012) Nature 489, 115–20

ε-amino groupof lysine

Tetrahedral transition state intermediary

pKa 10.5 ± 1.1

Ube2W conjugates ubiquitin to α-amino groups of protein N-termini

5Tatham, et al. (2013) The Biochemical Journal 453, 137–45

α-amino groupof the substrate N-terminus

pKa 7.7 ± 0.5

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

1. Sequence and structure-informed mutational analysis of key residues

2. Protein expression and purification of the mutant proteins

3. Biochemical characterization of the proteins and in vitro ubiquitin conjugation

assays

6

*

Helix 1

Helix 2

D117

N77

Ube2W model (I-TASSER)Ube2W (2A7L:A)UbcH5a (4AP4:E) Ubiquitin (4AP4:F)

Ube2W model (Phyre2)

N

Structural analysis of UbcH5a and Ube2W

7

Helix 1 Helix 2

8

Multiple alignment analysis of UbcH5a and Ube2W

UbcH5a~Ubiquitin (4AP4:E~F)

Model of Ube2W~Ubiquitin(4AP4:F)

UbcH5aM1 – N77H M2 – P115K/D116R/D117RM3 – M1/M2M4 – D112S/insC/P113K/N114E/P115K/D116R/D117RM5 – M1/M4

Helix 1 Helix 2

Ube2WM1 – H94NM2 – K133P/R134D/R135DM3 – M1/M2M4 – S129D/delC130/K131P/E132N/K133P/R134D/R135DM5 – M1/M4

8

Multiple alignment analysis of UbcH5a and Ube2W

kDa

75 -

50 -

37 -

25 -

15 -

10 -

20 -

E. coli BL21 (DE3)

wt M1 M2 M3 M4 M5

- + - + - + - + - + - + IPTG

His6-UbcH5a

Rosetta (DE3)

M2 M3 M4 M5

- + - + - + - + IPTG

M2 M3 M4 M5

- + - + - + - +

ArcticExpress (DE3)

75 -

50 -

37 -

25 -

15 -

10 -

20 -

kDa

Cpn60

His6-UbcH5a

Cpn10

Protein expression and purification

9

Site-directed mutagenesis

DpnI digestionMutant strand synthesis

18

DNA sequencing

E. coli Rosetta (DE3)

wt M1 M2 M3 M4 M5

kDa

75 -

50 -

37 -

25 -

15 -

10 -

20 - His6-Ube2W

- + - + - + - + - + - + IPTG

9

Protein expression and purification

kDa

75 -

50 -

37 -

25 -

15 -

10 -

20 -

E. coli BL21 (DE3)

wt M1 M2 M3 M4 M5

- + - + - + - + - + - + IPTG

His6-UbcH5a

Site-directed mutagenesis

DpnI digestionMutant strand synthesis

18

DNA sequencing

10

Protein expression and purification

kDa

75 -

50 -

37 -

25 -

15 -

10 -

20 -

S F B W E T

His6-UbcH5a UbcH5a

His6-tag

Ube2W wt and M1

S F B W E TCkDa

75 -

50 -

37 -

25 -

15 -

10 -

20 - His6-Ube2W Ube2W

His6-tag

S F B W E TCkDa

75 -

50 -

37 -

25 -

15 -

10 -

20 - His6-Ube2W Ube2W

His6-tag

Ube2W M2-5

C – Cell suspensionS – Supernatant

F – Flow-through B – First wash

W – Second washE – Elution

T – After His6-tag cleavage with TEV

Expression vector

Lyse cells

Ni-NTA resin

Wash

Elute

His6-tagged proteins Cleavage with TEV protease

Purified proteins

UbcH5a wt and M1-3

Protein expression and purification

11

50 55 60 65 70 75 80 850

50

100

150

200

250

Elution volume (ml)

Abs

orva

nce

at 2

80 n

m (m

AU

)1 6 7 13

1 6 7 132 3 4 5 8 9 10 11 1220 -

15 -

kDa S F B W E TCkDa

75 -

50 -

37 -

25 -

15 -

10 -

20 - His6-Ube2W Ube2W

His6-tag

Ube2W M2-5

Gel filtration on a HiLoad 16/60 Superdex 75 pg

Equilibrium of moners and dimers

Vittal, et al. (2013) Cell Biochemistry and Biophysics 13, 9633-5

The ability of mutant proteins to form E2~Ub thioester bonds

12

kDa25 -

15 -

10 -

20 -

wt M1 M2 M3

Time

UbcH5a

UbcH5a

Ubiquitin

UbcH5a~Ub

25 -

15 -

10 -

20 -

Ubiquitin

UbcH5a

kDa25 -

15 -

10 -

20 -

25 -

15 -

10 -

20 -

Ubiquitin

Ube2W

Ube2W~Ub

Ube2W

wt M1 M2 M3 M4 M5

Time

Ubiquitin

Ube2W

Non-reducingSDS-PAGE

ReducingSDS-PAGE

M1 – N77H M2 – P115K/D116R/D117RM3 – M1/M2

E1 E2 UbReaction mix: + + + ATP

His6

N SUMO-2 SUMO-2 SUMO-2 SUMO-2

pH titration analysis of UbcH5a and Ube2W

13

6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 pH

Time

His6-SUMO-2x4

kDa

75 -

50 -

His6-SUMO-2x4~Ub

His6-SUMO-2x4

His6-SUMO-2x4~Ub75 -

50 -

UbcH5a

UbcH5a N77H

6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 pH

Time

His6-SUMO-2x4

kDa

75 -

50 -

His6-SUMO-2x4~Ub

His6-SUMO-2x4

His6-SUMO-2x4~Ub75 -

50 -

Ube2W

Ube2W M3

Peptide-His6-SUMO-2x4

E3E1 E2 UbReaction mix: + + + ATP + +

M3 – H94N/ K133P/R134D/R135D

Conclusions

1. Key residues in the active site of Ube2W are different from most of

the conserved E2s

2. Ube2W shows an equilibrium of monomers and dimers that does not

rely on the C-terminus

3. Most of the mutant proteins can still form a thioester bond with

ubiquitin, although their ability to modify a poly-SUMO2 substrate is

affected

4. Ube2W shows pH-dependent activity at pH below 9.0

14

Future work

1. Try to overcome low expression of UbcH5a mutants by DNA synthesis

of the constructs with codon optimization

2. Investigate what are the key features of N-terminal amino groups

modified by Ube2W, using N-terminal modified substrates (myc-tag,

in vitro carbamylation, etc.)

3. Try solving the structure of RNF4-Ube2W~Ubiquitin mutating the

catalytic Cys to Lys to form an isopeptide linkage

15

Acknowledgements

Professor Ronald T. Hay

Anna Plechanovová

Ellis Jaffray

Linnan Shen

Mike Tatham

… all members of the Hay group!