Deciphering the substrate specificity of ubiquitin
conjugating enzymes
Fábio M. Marques Madeira
Supervisor: Professor Ronald T. Hay
24th July 2013
Protein ubiquitylation
Hochstrasser, M. (2009) Nature 458, 422–9
1
The ubiquitin modification cascade
RNF4
STUbL having a key role in DNA damage response
Woelk, et al. (2007) Cell Division 2:11
2
RNF4 RING bond to ubiquitin-loaded UbcH5a
Plechanovová, et al. (2012) Nature 489, 115–20
3
RNF4 RING bond to ubiquitin-loaded UbcH5a
ε-amino group
of lysine
Tetrahedral transition state intermediary
pKa 10.5 ± 1.1
Plechanovová, et al. (2012) Nature 489, 115–20
4
Ube2W conjugates ubiquitin to α-amino groups of protein N-termini
α-amino group
of the substrate
N-terminus
pKa 7.7 ± 0.5
Tatham, et al. (2013) The Biochemical Journal 453, 137–45
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 εamino 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
Structural analysis of UbcH5a and Ube2W
Helix 1
D117
N
*
N77
Helix 2
UbcH5a (4AP4:E)
Ube2W (2A7L:A)
Ube2W model (I-TASSER)
Ube2W model (Phyre2)
Ubiquitin (4AP4:F)
7
Multiple alignment analysis of UbcH5a and Ube2W
Helix 1
UbcH5a~Ubiquitin
(4AP4:E~F)
Helix 2
Model of Ube2W~Ubiquitin
(4AP4:F)
8
Multiple alignment analysis of UbcH5a and Ube2W
Helix 1
Helix 2
UbcH5a
Ube2W
M1 – N77H
M2 – P115K/D116R/D117R
M3 – M1/M2
M4 – D112S/insC/P113K/N114E/P115K/D116R/D117R
M5 – M1/M4
M1 – H94N
M2 – K133P/R134D/R135D
M3 – M1/M2
M4 – S129D/delC130/K131P/E132N/K133P/R134D/R135D
M5 – M1/M4
8
Protein expression and purification
Site-directed
mutagenesis
18
Mutant strand synthesis
DpnI digestion
ArcticExpress (DE3)
Rosetta (DE3)
E. coli BL21 (DE3)
kDa
wt
M1
M2
M3
M4
M5
- +
- +
- +
- +
- +
- +
IPTG
M2
M3
M4
M5
M2
M3
M4
M5
- +
- +
- +
- +
- +
- +
- +
- +
IPTG
75 -
75 -

Cpn60

His6-UbcH5a

Cpn10
50 -
50 -
37 -
37 25 20 -
kDa
DNA sequencing

His6-UbcH5a
25 20 -
15 -
15 -
10 -
10 -
9
Protein expression and purification
Site-directed
mutagenesis
18
Mutant strand synthesis
DpnI digestion
E. coli Rosetta (DE3)
E. coli BL21 (DE3)
kDa
wt
M1
M2
M3
M4
M5
- +
- +
- +
- +
- +
- +
IPTG
M2
M3
M4
M5
- +
- +
- +
- +
- +
- +
IPTG
37 -
37 -
10 -
M1
50 -
50 -
15 -
kDa
wt
75 -
75 -
25 20 -
DNA sequencing

His6-UbcH5a
25 20 -


His6-Ube2W
15 10 -
9
Protein expression and purification
Lyse cells
Wash
Elute
Expression
vector
Ni-NTA resin
UbcH5a wt and M1-3
kDa
S F B W
His6-tagged proteins
kDa
75 50 37 -
25 -
C S F B W
E T
His6-UbcH5a
UbcH5a
15 -
kDa
75 -
75 -
50 -
50 -
37 -
37 -
25 



20 -
His6-Ube2W
Ube2W
15 -
10 
C – Cell suspension
S – Supernatant
His6-tag
F – Flow-through
B – First wash
C S F B W
 His6-tag
W – Second wash
E – Elution
E T
25 20 15 -
10 -
Purified proteins
Ube2W M2-5
Ube2W wt and M1
E T
20 -
Cleavage with
TEV protease
10 -
His6-Ube2W
 Ube2W

 His6-tag
T – After His6-tag cleavage with TEV
10
Protein expression and purification
Gel filtration on a HiLoad 16/60 Superdex 75 pg
Ube2W M2-5
C S F B W
E T
kDa
250
75 50 37 -
25 20 15 10 -
His6-Ube2W
 Ube2W

Absorvance at 280 nm (mAU)
kDa
1
2
3
4 5 6 7
8
9 10 11 12 13
20 15 -
200
150
100
50
7
6
1
13
 His6-tag
0
50
55
60
65
70
75
80
85
Elution volume (ml)
Equilibrium of moners and dimers
Vittal, et al. (2013) Cell Biochemistry and Biophysics 13, 9633-5
11
The ability of mutant proteins to form E2~Ub thioester bonds
Reaction mix:
E1
+
E2
+ Ub + ATP
Ube2W
UbcH5a
wt
kDa
25 -
20 -
Non-reducing
SDS-PAGE
M1
M2
wt
M3
Time
kDa
25 -
UbcH5a~Ub
 UbcH5a
20 -

15 -

Ubiquitin
M3
M4
M5
Time

Ube2W~Ub


Ube2W

Ubiquitin


Ube2W

Ubiquitin
25 -
25 -
20 -
20 
UbcH5a
15 -
15 10 -
M2
10 -
10 -
Reducing
SDS-PAGE
15 -
M1

Ubiquitin
10 -
M1 – N77H
M2 – P115K/D116R/D117R
M3 – M1/M2
12
pH titration analysis of UbcH5a and Ube2W
Reaction mix:
+
E1
Peptide-His6-SUMO-2x4
6.5
7.0
7.5
8.0
E3
+ Ub + ATP +
E2
8.5
9.0
9.5
N
SUMO-2 SUMO-2 SUMO-2 SUMO-2
His6
10.0
10.5
11.0
pH
Time
kDa
UbcH5a
UbcH5a N77H
75 50 -
His6-SUMO-2x4~Ub
 His6-SUMO-2x4
75 -

50 -


6.5
Ube2W
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
His6-SUMO-2x4~Ub
His6-SUMO-2x4
pH
kDa
Time
75 -

50 -
75 -
Ube2W M3
+
50 -



His6-SUMO-2x4~Ub
His6-SUMO-2x4
His6-SUMO-2x4~Ub
His6-SUMO-2x4
M3 – H94N/ K133P/R134D/R135D
13
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!