Use of SAXS to study DNA regulation Titia Sixma t.sixma@nki.nl
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. Groothuizen 1# , Alexander Fish 1# , Maxim V. Petoukhov 2 , Annet Reumer 1 , Laura Manelyte 3 , Herrie H.K. Winterwerp 1 , Martin G. Marinus 4 , Joyce H.G. Lebbink 5 , Dmitri I. Svergun 2 , Peter Friedhoff 3 and Titia K. Sixma 1 1 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
DNA mismatch repair
Fitting of WT MutS DNA Binding Surface plasmon resonance to DNA with a mismatch 21GT ka kd A B AB KD ka kd ka1 ka2 kd1 kd2 A B AB AB* KD ka1 ka2 kd2 kd1 kd2
Dimer-tetramer equilibrium of MutS Lamers et al . 2000 Nature
Dimer-tetramer equilibrium of MutS 324 190 kDa 381 kDa Multi-angle laser light scattering MALLS analysis of wild type MutS
Preventing tetramerization Mendillo et al . 2007 JBC
Preventing tetramerization 324 189 198 Mendillo et al . 2007 JBC
Fitting of D835R MutS DNA Binding Heteroduplex Homoduplex 21GT 21AT
Fitting kinetics for the dimer Wild type Dimer (D835R)
Crystallization of D835R MutS Crystallographic table 25 mM Tris pH 8/8,5 750 mM NaCl Spacegroup P2 1 12% PEG 6K 10 mM MgCl 2 Cell parameters Resolution 3Å Completeness 99,48% R value 28,1% Free R value 34,9% Mean B value 70,83 100 uM MutS 50 uM 21GT9 DNA 100uM ADP
Structure of full-length MutS dimer (D835R) Resolution 3.1 Å R work /R free 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 Fit of EOM selected conformations to from the EOM (red: selected conformations) 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 indeed binds the catalytic domain HUBL-45 is sufficient for full activity 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
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Point mutations block activation by HUBL-45
Usp7 point mutations block activation by HUBL-45 Annette Dirac Over-expression in U2OS
Model for HUBL activation of USP7 - Inactive state : HUBL-45 interaction - Active state : Interaction with HUBL-45 - Low ubiquitin affinity - High affinity for ubiquitin - Disorganized active site - Catalytically competent active site - ‘inactive’ switching loop - ‘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 ```````````` ````
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 indicates flexible conformations
NMR analysis Red: 15N labelled Ubiquitin Blue 15N laeblled Ubiquitin on unlabelled PCNA Hibbert & Sixma, JBC 2012
NMR analysis Ub PCNA-Ub 40 Linewidth (Hz) 30 20 10 0 Residue number Hibbert & Sixma JBC 2012
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