Use of SAXS to study DNA regulation Titia Sixma t.sixma@nki.nl - - PowerPoint PPT Presentation

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Use of SAXS to study DNA regulation Titia Sixma t.sixma@nki.nl - - PowerPoint PPT Presentation

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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

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Use of SAXS to study DNA regulation

Titia Sixma t.sixma@nki.nl

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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

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SLIDE 3

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

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

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DNA mismatch repair

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Fitting of WT MutS DNA Binding Surface plasmon resonance to DNA with a mismatch

 

kd2 ka2 ka1 kd2 kd1 ka2 kd2 ka1 kd1 ka kd ka kd

AB* KD AB B A AB KD B A

  

      

21GT

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SLIDE 6

Dimer-tetramer equilibrium of MutS

Lamers et al. 2000 Nature

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Dimer-tetramer equilibrium of MutS

324 190 kDa 381 kDa

Multi-angle laser light scattering MALLS analysis of wild type MutS

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SLIDE 8

Preventing tetramerization

Mendillo et al. 2007 JBC

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Preventing tetramerization

Mendillo et al. 2007 JBC 324 189 198

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Fitting of D835R MutS DNA Binding

21GT 21AT

Heteroduplex Homoduplex

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Fitting kinetics for the dimer

Wild type Dimer (D835R)

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Crystallization of D835R MutS

Crystallographic table

Spacegroup P21 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

25 mM Tris pH 8/8,5 750 mM NaCl 12% PEG 6K 10 mM MgCl2

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SLIDE 13

Structure of full-length MutS dimer (D835R)

Resolution 3.1 Å Rwork/Rfree 20.7/25.0

Groothuizen, Fish et al, NAR 2013

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Structure of full-length MutS dimer (D835R)

Tetramerization domain is located in a crystal contact

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Tetramer MutS modeling

Tight structure Loose structure

Extrapolation based on Full length and tetramerization domain structures

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Use SAXS to asses shape of full-length MutS dimer (D835R)

Groothuizen, Fish et al, NAR 2013

DAMMIF 10 independent models Averaged in DAMAVER

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Comparison to crystal structure and extended state

Conclusion: Tetramerization domain is flexible with respect to the the main body of MutS

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Single-cysteine MutS R848C

Capturing the MutS tetramer single cysteine crosslinking

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410 324 189 198

Single-cysteine MutS R848C

Capturing the MutS tetramer single cysteine crosslinking

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Guinier plots for the SAXS data of the dimer and the tetramer

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Shape of the cross-linked tetramer

Groothuizen, Fish et al, NAR 2013

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SLIDE 22
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How does the tetramer bind to DNA?

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Differences in DNA binding kinetics between tetramer and dimer

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Slow off-rate of the MutS tetramer

  • nly on longer DNA

Fitting of kinetic data for extended tetramer

  • n DNA not possible
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Slow off-rate of the MutS tetramer

Mixture of straight and bend-over MutS

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Bending over of the tetramer is possible

M4M  8 Å M17M  22 Å ~100 Å

Crosslinking experiment

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Conformations representative of the major peaks from the EOM (red: selected conformations) 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 Fit of EOM selected conformations to SAXS data Maxim Petoukhov

Bending over of the tetramer is possible

Groothuizen, Fish et al, NAR 2013

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SLIDE 29

Groothuizen, Fish et al, NAR 2013

  • 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

Generation of MutS dimers and tetramers

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SLIDE 30

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

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SLIDE 31

Figure from Hochstrasser Nature 2009

Regulates many essential pathways Potentially interesting drug targets E1 E3 ligases Dubs

Ubiquitin conjugation is a signalling system

Ubiquitin

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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

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USP7/HAUSP protein

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AMC USP7 AMC

+

The HUBL domain necessary for full USP7 activity

  • n minimal substrate Ub-AMC

(Novartis: Fernandez-Montalvan et al., 2007)

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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

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USP7CD HUBL-13 HUBL-45 SAXS data ID14-3

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HUBL USP7CD-HUBL

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14 nm 5.5 nm 8 nm 5 nm

HUBL domain

  • elongated,
  • long atom-atom distances

HUBL + catalytic domain

  • long distances lost
  • HUBL domain folds back onto CD
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SLIDE 39

HUBL-45 indeed binds the catalytic domain HUBL-45 activates in trans

Understanding the activation process

C-terminal 19 amino acid tail is important but not sufficient

  • requires HUBL-45 for binding

HUBL-45 is sufficient for full activity

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SLIDE 40
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Shi lab: Hu et al, Cell 2002

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Zoom

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Zoom

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Point mutations block activation by HUBL-45

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Over-expression in U2OS

Usp7 point mutations block activation by HUBL-45

Annette Dirac

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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
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SLIDE 48

Model for activation of USP7

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.
  • Are there other regulators
  • Stabilizing the ‘on’ state
  • Stabilizing the ‘off’ state
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Activation of USP4

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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
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SLIDE 51

Analysis of a ubiquitinated target

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During DNA replication PCNA promotes processivity of DNA polymerases

PCNA

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Mono-ubiquitination of PCNA causes a switch from replicative to TLS polymerase

K164

Exquisitely specific for Lysine 164 (K164)

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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)

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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.

+

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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

```````````` ````

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Gel filtration / MALS analysis

Hibbert & Sixma, JBC 2012

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Small angle X-ray Scattering

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SAXS analysis

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SAXS analysis

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SAXS analysis

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The ubiquitin on PCNA-Ub is flexible

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Observed radius of gyration(40-44) from X-ray, light scattering indicates flexible conformations

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NMR analysis

Hibbert & Sixma, JBC 2012 Red: 15N labelled Ubiquitin Blue 15N laeblled Ubiquitin on unlabelled PCNA

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NMR analysis

10 20 30 40

Ub PCNA-Ub Residue number Linewidth (Hz) Hibbert & Sixma JBC 2012

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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

–

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SLIDE 68

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

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