Use of SAXS in ubiquitin conjugation research Ubiquitin conjugation - PDF document

Use of SAXS in ubiquitin conjugation research

Ubiquitin conjugation is a signalling system Like phosphorylation, ubiquitination changes the fate of the target protein Ubiquitin is a 76-amino acid protein Targeted to amino groups (Lys, N-term) Ubiquitin can make chains Ubiquitin

Ubiquitin conjugation is a signalling system Regulates many essential pathways Potentially interesting drug targets E1 E3 ligases Dubs Ubiquitin Figure from Hochstrasser Nature 2009

Ubiquitin conjugation and SAXS Three stories E3 ligase RNF8 Dub USP7 Ubiquitinated target PCNA Ubiquitin Figure from Hochstrasser Nature 2009

H2A ubiquitination H2A H2A UB UB UB UB UB • Polycomb repressive • Rnf8 and Rnf168 complex 1 (Ring1B/A, Bmi1 and Mel18) Transcriptional silencing DNA damage response

Crystal Structure of the RING domain of RNF8 Mattiroli et al, Cell 2012

Dimerization in Class 1 RING/Ubox dimers Members of Class 1 RING dimers Brca1/Bard1 Ring1b/Bmi1 Traf6 Traf2 Class 1 Trim37 Ubox dimer CHIP Class 1 Dimer contacts primarily through four-helix bundle from flanking helices, N- and C- terminal to RING. N-terminal C-terminal RING Helix helix Hibbert et al., DNA repair, 2009 Huang, Hibbert et al, J Mol Biol 2011

Dimerization in Brca1/Bard1 is important Brca1 is the breast cancer susceptibility gene 1 Brca1/Bard1 is a RING dimer Breast cancer-associated point mutations in Brca1 are found in the dimer interface with Bard1 Only BRCA1 is active as E3 ligase Klevit lab: Brzovic et al, NSB 2001/ JBC 2001

Asymmetry is common in class 1 RING dimers Mutation of E2/E3 interface in Ring1b/Bmi1 • in Ring1b causes loss of activity • in Bmi1 has no effect H2A-Ub H2A 15 30 60 120 15 30 60 120 15 30 60 120 mins TIME - E3 Ring1B Ring1B Ring1B I53A Bmi1 Bmi1 L20A Bmi1 Heterodimer Ring1b/Bmi1 • Part of Polycomb PRC1 complex • Interface extended by N-terminal embracing arm Buchwald et al, EMBO J, 2006 Flora Groothuizen, Francesca Mattiroli

Spectacular asymmetry in CHIP Ubox homodimer Full length CHIP structure shows Class 1 dimerization fold - Ubox has RING fold without Zinc ions Flanking TPR domains are highly asymmetrically arranged CHIP is a chaperone protein with E3 ligase CHIP function collaborating with e.g HSP90 UBox domain Pearl Lab: Zhang et al, Mol Cell 2006

CHIP asymmetry causes 2:1 stoichiometry with E2s Only one E2 binding site is exposed in the full length protein CHIP UBox domain Pearl Lab: Zhang et al, Mol Cell 2006

Asymmetry is conserved in Rad18 Full-length Rad18 is an asymmetric homodimer Strep-Rad6 pull-down: brings down His-Rad18 but not His-Rad6 His6 Tag Strep Tag Coomassie RING domains of Rad18 Strep Tag 2:1 Anti-His Huang, Hibbert et al, J. Mol Biol 2011

RNF8 is highly asymmetric in the crystal 348 Chain B 35 o Asymmetry could be Chain A due to crystal packing 483 Superposition of the RING

The point of divergence is conserved between RNF8 and CHIP CHIP and Rnf8 monomers RING superimposed

Small angle X-ray scattering of RNF8 RING domain 100 10 measured value (expected value in brakets) log(I) 1 0.1 scattering curve theoretical curve 0.01 0.0 0.1 0.2 0.3 0.4 s = 4 π sin( θ )/ λ Five different concentrations (from 0.4 to 7 mg/ml) in gel-filtration buffer. Data were collected at EMBL Hamburg, data analyzed using ATSAS Mattiroli et al, Cell 2012

Small angle X-ray scattering Envelope from DAMAVER where 10 DAMMIF ab initio models were used. All 10 models showed an elongated shape and their NSD (normalized spatial discrepancy) values were between 0.6 and 0.74

AB Generated models for symmetric dimers Two times molecule A Two times molecule B Tried to fit these separately. The asymmetric dimer seems to fit best, AA BB

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 All proteins critical in oncogenic pathways

USP7/HAUSP protein

The C-terminal domain of USP7/HAUSP is important for activity (Fernandez-Montalvan et al., 2007) USP7 In U2OS cells Against purified ubiquitinated p53 Faesen et al, Mol Cell 2011

The HUBL domain necessary for full USP7 activity on minimal substrate Ub-AMC + AMC USP7 AMC

Crystallization of HUBL domain Initial selenomethionine phased map

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

The di-Ubl units have similar relative arrangement

Faesen et al Mol Cell 2011 HUBL-13 HUBL-45 USP7CD

Faesen et al Mol Cell 2011 HUBL-13 HUBL-45 USP7CD

USP7CD-HUBL 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 binds the catalytic domain HUBL-45 activates in trans C-terminal 19 amino acid tail is important but not sufficien -requires HUBL-45 for binding

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Point mutations block activation by HUBL-45

Usp7 point mutations block activation by HUBL-45 Faesen et al, Mol Cell 2011 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 HUBL activation of USP7 Questions: - Is this equilibrium unique for USP7 - Most USPs don’t show inactive state for isolated catalytic domain - UBP8 in the SAGA complex is activated through Sgf11 interaction at switching loop - Does the equilibrium exist in cells ? - Are there proteins that regulate it ? - Analyze the activation by GMP synthase

GMP synthase (GMPS) is an activator of USP7 USP7 and GMPS form a stable complex USP7 can be activated by GMPS Activation does not require GMPS catalytic activity Van der Knaap et al (2005) Molecular Cell 17:695-707 van der Knaap (2010) Mol Cell Biol. 30:736-44 Sarkari et al, (2009), PloS Pathog. e1000624

GMP synthase (GMPS) is an activator of USP7 Drosophila USP7 and GMPS collaborate genetically Drosophila USP7 and GMPS form a stable complex GMPS activates USP7 against p53 and is absolutely required for H2b activity GMPS catalytic activity not involved Human USP7 and GMPS form complex and are active against H2b Van der Knaap et al (2005) Molecular Cell 17:695-707 van der Knaap (2010) Mol Cell Biol. 30:736-44 Sarkari et al, (2009), PloS Pathog. e1000624

GMPS activates USP7 - 5.5-fold increase in k cat , - No effect on K M - - No change in ubiquitin affinity Ubiquitin affinity Substrate dependent effects likely

GMPS binds HUBL-13 SPR analysis of GMPS binding reveals - HUBL-13 is required for binding of GMPS (K d ~ 35 nM) - Point mutants don’t interfere with binding - Neither catalytic domain nor HUBL-45 has affinity for GMPS

GMPS activation requires a functional HUBL Cys-223 Switching loop Activation peptide

USP7 switching model GMPS binds to HUBL-13 GMPS requires HUBL-45 activation If GMPS shifts the equilibrium, GMPS should stabilize the interaction between CD and HUBL-45

Binding of HUBL to USP7CD is enhanced by GMPS USP7 catalytic domain: K d = 50 mM for HUBL + K d = 2 mM for HUBL in presence of GMPS Conclusion: - GMPS stabilizes interaction between HUBL-45 and catalytic domain

Model for activation of USP7 Are there other regulators - Stabilizing the ‘on’ state - Stabilizing the ‘off’ state Conclusions: - GMPS binds to HUBL-13 - GMPS stabilizes interaction between HUBL-45 and catalytic domain - GMPS allosterically activates by stabilization of the active state

Analysis of a ubiquitinated target

PCNA promotes processivity of DNA polymerases in replication

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

Methods of produce ubiquitinated PCNA • 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 - No E3 required - Fully specific for K164 Hibbert & Sixma, JBC 2012

USP1/UAF1 deconjugation Facilitates the study of

Gel filtration / MALS analysis

SAXS analysis

SAXS analysis

SAXS analysis

SAXS analysis

The ubiquitin on PCNA-Ub is flexible