Use of SAXS to study DNA regulation Titia Sixma

Use of SAXS to study DNA regulation
Titia Sixma
[email protected]
Integrating SAXS analysis into
functional analysis
DNA mismatch repair
- MutS dimers/tetramers
Resolving conformational states
Ubiquitin conjugation
- Activation of deubiquitinating enzymes
- USP7
Binding between domains
- USP4
Shape of catalytic domain
- PCNA ubiquitination
- Effect of ubiquitination
Flexibility of modulator
Uncoupling MutS dimer and tetramer
Flora S. Groothuizen1#, Alexander Fish1#, Maxim V. Petoukhov2,
Annet Reumer1, Laura Manelyte3, Herrie H.K. Winterwerp1,
Martin G. Marinus4, Joyce H.G. Lebbink5, Dmitri I. Svergun2, Peter
Friedhoff3 and Titia K. Sixma1
Division of Biochemistry, Netherlands Cancer Institute, Amsterdam, the Netherlands
2 European Molecular Biology Laboratory, Hamburg Outstation, Hamburg, Germany
3 Institute for Biochemistry, Justus-Liebig-University, Giessen, Germany
4 Department of Biochemistry University of Massachusetts Medical School, Worcester, USA
5 Department of Cell Biology and Genetics, Erasmus Medical Center, Rotterdam, the Netherlands
1
DNA mismatch repair
Fitting of WT MutS DNA Binding
Surface plasmon resonance to DNA with a mismatch
21GT
ka
A  B  AB KD 
kd
ka1
ka2
kd1
kd2
A  B  AB  AB* KD 
kd
ka
kd1kd2
ka1 ka2 kd2 
Dimer-tetramer equilibrium of MutS
Lamers et al. 2000 Nature
Dimer-tetramer equilibrium of MutS
324
190 kDa
Multi-angle laser light scattering
MALLS analysis of wild type MutS
381 kDa
Preventing tetramerization
Mendillo et al. 2007 JBC
Preventing tetramerization
324
189
198
Mendillo et al. 2007 JBC
Fitting of D835R MutS DNA Binding
Heteroduplex
21GT
Homoduplex
21AT
Fitting kinetics for the dimer
Wild type
Dimer (D835R)
Crystallization of D835R MutS
25 mM Tris pH 8/8,5
750 mM NaCl
12% PEG 6K
10 mM MgCl2
100 uM MutS
50 uM 21GT9 DNA
100uM ADP
Crystallographic table
Spacegroup
Cell parameters
Resolution
Completeness
R value
Free R value
Mean B value
P21
3Å
99,48%
28,1%
34,9%
70,83
Structure of full-length MutS dimer (D835R)
Resolution
Rwork/Rfree
3.1 Å
20.7/25.0
Groothuizen, Fish et al, NAR 2013
Structure of full-length MutS dimer (D835R)
Tetramerization domain is
located in a crystal contact
Tetramer MutS modeling
Extrapolation based on Full length and tetramerization domain structures
Loose structure
Tight structure
Use SAXS to asses shape of
full-length MutS dimer (D835R)
DAMMIF 10 independent models
Averaged in DAMAVER
Groothuizen, Fish et al, NAR 2013
Comparison to crystal structure and extended state
Conclusion:
Tetramerization domain is
flexible with respect to the
the main body of MutS
Capturing the MutS tetramer
single cysteine crosslinking
Single-cysteine MutS R848C
Capturing the MutS tetramer
single cysteine crosslinking
Single-cysteine MutS R848C
410
324
189 198
Guinier plots for the SAXS data of the dimer and
the tetramer
Shape of the cross-linked tetramer
Groothuizen, Fish et al, NAR 2013
How does the tetramer bind to DNA?
Differences in DNA binding kinetics
between tetramer and dimer
Slow off-rate of the MutS tetramer
only on longer DNA
Fitting of kinetic data for extended tetramer
on DNA not possible
Slow off-rate of the MutS tetramer
Mixture of straight and bend-over MutS
Bending over of the tetramer is possible
M4M  8 Å
M17M  22 Å
~100 Å
Crosslinking experiment
Bending over of the tetramer is possible
Conformations representative of the major peaks
from the EOM (red: selected conformations)
Fit of EOM selected conformations to
SAXS data
Ensemble optimization (EOM) of pool of 10000 conformations using model of D835R (1-800) linked via 22
dummy atoms to tetramerization domain structure (2OK2): those conformations that describe the scattering
curve best are selected, representative examples are shown
Maxim Petoukhov
Groothuizen, Fish et al, NAR 2013
Generation of MutS dimers and tetramers
- Allowed fitting of kinetic data
- Allowed structure solution of full length MutS
- Allowed SAXS analysis of dimers and tetramers
- SAXS shows that dimer is predominantly
extended
- Biochemical experiments and EOM show that
tetramer bends over occasionally
Groothuizen, Fish et al, NAR 2013
Conclusions
• The MutS tetramer can bend over and in that way dissociates
slowly from DNA
• Careful analysis of SAXS data required to analyse this
• The MutS dimer mutant is a single DNA-binding unit and
simplifies the system:quantitative analysis of mismatch
binding and sliding clamp formation
• Sliding clamp formation is impaired when binding a C.C
mismatch; may explain why this mismatch is not repaired
efficiently
Ubiquitin conjugation is a signalling system
Regulates many essential pathways
Potentially interesting drug targets
E1
E3 ligases
Dubs
Ubiquitin
Figure from Hochstrasser Nature 2009
Usp7/HAUSP
• DUB for MDM2 and p53
- Regulates stability
- Decision making for apoptosis, cell cycle and senescence
• DUB for PTEN and FOXO4
- Regulates cellular localization
• Interaction with DNMT1
All proteins in critical pathways
USP7/HAUSP protein
The HUBL domain necessary for full USP7 activity
on minimal substrate Ub-AMC
USP7
AMC
+
AMC
(Novartis: Fernandez-Montalvan et al., 2007)
The HAUSP C-terminal domain has 5 Ubl domains
5 Ubl domains
2+1+2 structure
USp7/Hausp Ubl domain: HUBL
Faesen et al, Mol Cell, 2011
USP7CD
HUBL-13
HUBL-45
SAXS data ID14-3
HUBL
USP7CD-HUBL
8 nm
5 nm
14 nm
5.5 nm
HUBL domain
- elongated,
- long atom-atom distances
HUBL + catalytic domain
- long distances lost
- HUBL domain folds back onto CD
Understanding the activation process
HUBL-45 is sufficient for full activity
HUBL-45 indeed binds the catalytic domain
HUBL-45 activates in trans
C-terminal 19 amino acid tail is important but not sufficient
-requires HUBL-45 for binding
Shi lab: Hu et al, Cell 2002
Zoom
Zoom
Point mutations block activation by HUBL-45
Usp7 point mutations block activation by HUBL-45
Over-expression in U2OS
Annette Dirac
Model for HUBL activation of USP7
- Inactive state: HUBL-45 interaction
- Low ubiquitin affinity
- Disorganized active site
- ‘inactive’ switching loop
-Active state: Interaction with HUBL-45
- High affinity for ubiquitin
- Catalytically competent active site
- ‘Active’ switching loop
Model for activation of USP7
Are there other regulators
- Stabilizing the ‘on’ state
- Stabilizing the ‘off’ state
GMPS is an allosteric activator
- Binds to HUBL-13 exclusively
- Promotes the interaction between HUBL-45 and catalytic domain (20-fold )
- Shifts the equlibrium to the active state.
-
Activation of USP4
USP4/USP7 regulation
USP4
-
Full length USP4 much more active than CD
DUSP-UBL binds to insert to promote Ub release
Switching loop serves as relay
USP7
-
Full length USP7 much more active than CD
-
UBL domains HUBL45 bind to CD allow ubiquitin binding
-
Switching loop serves as relay
-
GMPS can allosterically promote this type of activation
Analysis of a ubiquitinated target
During DNA replication PCNA promotes
processivity of DNA polymerases
PCNA
Mono-ubiquitination of PCNA causes a switch from
replicative to TLS polymerase
K164
Exquisitely specific for Lysine 164 (K164)
Attempts to produce
ubiquitinated PCNA for biophysical studies
• Native PCNA-Ub
– PNAS 2005, 2006
• ‘Split’ PCNA
– Co-expression of PCNA (1-163) with Ubiquitin fused in-line with PCNA
(164-261)
– Nature SMB (2010)
• Intein PCNA
– Nature Chem Biol (2010)
• Click PCNA
– Incorporating unnatural amino acids
– Chembiochem (2011)
How does PCNA change upon
ubiquitination
• Crystal structure of ‘split’ PCNA
– Co-expression of PCNA (1-163) with
Ubiquitin fused in-line with PCNA (164-261)
– Ubiquitin buries its hydrophobic patch
• Saxs analysis of ‘split’ PCNA and intein-based link
– 70% ordered structure, 2 major states.
+
in vitro ubiquitination of PCNA with E2
enzyme UbcH5c
````````````
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In vitro ubiquitination of PCNA with E2
enzyme UbcH5c
````````````
````
- Optimized conditions
Hibbert & Sixma, JBC 2012
Gel filtration / MALS analysis
Hibbert & Sixma, JBC 2012
Small angle X-ray Scattering
SAXS analysis
SAXS analysis
SAXS analysis
The ubiquitin on PCNA-Ub
is flexible
Observed radius of gyration(40-44) from X-ray, light scattering
NMR analysis
Red: 15N labelled Ubiquitin
Blue 15N laeblled Ubiquitin on unlabelled PCNA
Hibbert & Sixma, JBC 2012
Linewidth (Hz)
NMR analysis
Ub
PCNA-Ub
40
30
20
10
0
Residue number
Hibbert & Sixma JBC 2012
SAXS to make specific points
- Mismatch repair
- Analyse location of
– Ubiquitin E3 ligase RNF8
• Confirm extended helix
• Could not distinguish between symmetric or asymmetric states
– Deubiquitinating enzyme USP7
• In solution less extended HUBL domain
• HUBL domain folds back on catalytic domain
– Ubiquitin target PCNA
• Ubiquitin is flexible on the target
–
Beamline scientists
ESRF & SLS
For MutS:
Maxim V. Petoukhov,
Dmitri I. Svergun,
Martin G. Marinus,
Joyce H.G. Lebbink,
Peter Friedhoff
NKI
Annette Dirac
Farid El Oualid
Huib Ovaa
Tassos Perrakis
Netherlands Cancer Institute
Division of Biochemistry
Mismatch repair
Flora Groothuizen
Alexander Fish
Annet Reumer
Herrie Winterwerp
PCNA
Rick Hibbert
USP7/HAUSP
Alex Faesen
USP4
Marcello Clerici
Mark Luna-Vargas
Funding: EU Rubicon, SPINE2complexes, Ubiregulators, KWF, ERC, NWO-CW