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!