DNA binding reorganizes the intrinsically disordered C-terminal region of PSC in Drosophila PRC1

AbstractPolycomb Group (PcG) proteins regulate gene expression by modifying chromatin. A key PcG complex, Polycomb Repressive Complex 1 (PRC1), has two activities: a ubiquitin ligase activity for histone H2A, and a chromatin compacting activity. In Drosophila, the Posterior Sex Combs (PSC) subunit of PRC1 is central to both activities. The N-terminal homology region (HR) of PSC assembles into PRC1, including partnering with dRING to form the ubiquitin ligase for H2A. The intrinsically disordered C-terminal region of PSC (PSC-CTR) compacts chromatin, and inhibits chromatin remodeling and transcription in vitro. Both the PSC-HR and the PSC-CTR are essential in vivo. To understand how these two activities may be coordinated in PRC1, we used cross-linking mass spectrometry (XL-MS) to analyze the conformations of the PSC-CTR in PRC1 and how they change on binding DNA. XL-MS identifies interactions between the PSC-CTR and the core of PRC1, including between the PSC-CTR and PSC-HR. New contacts and overall more compacted PSC-CTR conformations are induced by DNA binding. Protein footprinting of accessible lysine residues in the PSC-CTR reveals an extended, bipartite candidate DNA/chromatin binding surface. Our data suggest a model in which DNA (or chromatin) follows a long path on the flexible PSC-CTR. Intramolecular interactions of the PSC-CTR detected by XL-MS can bring the high affinity DNA/chromatin binding region close to the core of PRC1 without disrupting the interface between the ubiquitin ligase and the nucleosome. Our approach may be applicable to understanding the global organization of other large IDRs that bind nucleic acids.HighlightsAn intrinsically disordered region (IDR) in Polycomb protein PSC compacts chromatinCross-linking mass spectrometry (XL-MS) was used to analyze topology of the PSC IDRProtein footprinting suggests a bipartite DNA binding surface in the PSC IDRA model for the DNA-driven organization of the PSC IDRCombining XL-MS and protein footprinting is a strategy to understand nucleic acid binding IDRsAbstract Figure

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A DNA-binding domain in the C-terminal region of Cdt2 enhances the DNA synthesis-coupled CRL4Cdt2 ubiquitin ligase activity for Cdt1

The Journal of Biochemistry ◽

10.1093/jb/mvz001 ◽

2019 ◽

Vol 165 (6) ◽

pp. 505-516 ◽

Cited By ~ 3

Author(s):

Muadz Ahmad Mazian ◽

Naohiro Suenaga ◽

Takashi Ishii ◽

Akiyo Hayashi ◽

Yasushi Shiomi ◽

...

Keyword(s):

Dna Binding ◽

Dna Synthesis ◽

Ubiquitin Ligase ◽

Binding Domain ◽

Terminal Region ◽

Dna Binding Domain ◽

Ubiquitin Ligase Activity

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The intrinsically disordered amino-terminal region of human RecQL4: multiple DNA-binding domains confer annealing, strand exchange and G4 DNA binding

Nucleic Acids Research ◽

10.1093/nar/gku993 ◽

2014 ◽

Vol 42 (20) ◽

pp. 12614-12627 ◽

Cited By ~ 38

Author(s):

Heidi Keller ◽

Kristin Kiosze ◽

Juliane Sachsenweger ◽

Sebastian Haumann ◽

Oliver Ohlenschläger ◽

...

Keyword(s):

Dna Binding ◽

Terminal Region ◽

Strand Exchange ◽

Dna Binding Domains ◽

Amino Terminal ◽

Intrinsically Disordered ◽

Binding Domains ◽

G4 Dna

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Handcuffing intrinsically disordered regions in Mlh1-Pms1 disrupts mismatch repair

10.1101/2021.03.02.433678 ◽

2021 ◽

Author(s):

Christopher M. Furman ◽

Ting-Yi Wang ◽

Qiuye Zhao ◽

Kumar Yugandhar ◽

Haiyuan Yu ◽

...

Keyword(s):

Mass Spectrometry ◽

Mismatch Repair ◽

Cross Linking ◽

Dna Mismatch ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

On Demand ◽

Transient Loss ◽

Disordered Regions

AbstractThe DNA mismatch repair (MMR) factor Mlh1-Pms1 contains long intrinsically disordered regions (IDRs). While essential for MMR, their exact functions remain elusive. We performed cross-linking mass spectrometry to identify the major interactions within the Mlh1-Pms1 heterodimer and used this information to insert FRB and FKBP dimerization domains into the IDRs of Mlh1 and Pms1. Yeast bearing these constructs were grown with rapamycin to induce dimerization. Strains containing FRB and FKBP domains in the Mlh1 IDR displayed complete MMR defects when grown with rapamycin, but removing rapamycin restored MMR functions. Furthermore, linking the Mlh1 and Pms1 IDRs through FRB-FKBP dimerization disrupted Mlh1-Pms1 binding to DNA, inappropriately activated Mlh1-Pms1, and caused MMR defects in vivo. We conclude that dynamic and coordinated rearrangements of the MLH IDRs regulate how the complex clamps DNA to catalyze MMR. The application of the FRB-FKBP dimerization system to interrogate in vivo functions of a critical repair complex will be useful for probing IDRs in diverse enzymes and to probe transient loss of MMR on demand.

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The linear ubiquitin chain assembly complex LUBAC generates heterotypic ubiquitin chains

10.1101/2020.05.27.117952 ◽

2020 ◽

Author(s):

Alan Rodriguez Carvajal ◽

Carlos Gomez Diaz ◽

Antonia Vogel ◽

Adar Sonn-Segev ◽

Katrin Schodl ◽

...

Keyword(s):

Electron Microscopy ◽

Mass Spectrometry ◽

Catalytic Activity ◽

Ubiquitin Ligase ◽

Bond Formation ◽

Mass Spectrometry Data ◽

Cross Linking ◽

Concerted Action ◽

Ubiquitin Chain ◽

C Terminus

AbstractThe linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase that generates linear/Met1-linked ubiquitin chains. One of the LUBAC components, HOIL-1L, was recently shown to catalyse oxyester bond formation between the C-terminus of ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. We present the first 3D reconstruction of LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that addition of the oxyester-bound branches depends on HOIL-1L catalytic activity. We suggest a coordinated ubiquitin relay mechanism between the HOIP and HOIL-1L ligases supported by cross-linking mass spectrometry data, which show proximity between the catalytic RBR domains. Mutations in the linear ubiquitin chain-binding NZF domain of HOIL-1L reduces chain branching confirming its role in the process. In cells, these heterotypic chains were induced by TNF. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains with linear and oxyester-linked branches by the concerted action of HOIP and HOIL-1L.

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DNA Binding Reorganizes the Intrinsically Disordered C-Terminal Region of PSC in Drosophila PRC1

Journal of Molecular Biology ◽

10.1016/j.jmb.2020.07.002 ◽

2020 ◽

Vol 432 (17) ◽

pp. 4856-4871 ◽

Cited By ~ 2

Author(s):

Jin Joo Kang ◽

Denis Faubert ◽

Jonathan Boulais ◽

Nicole J. Francis

Keyword(s):

Dna Binding ◽

Terminal Region ◽

Intrinsically Disordered

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DNA-Binding Surface of RecA Protein. Photochemical Cross-Linking of the First DNA Binding Site on RecA Filament

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1995.695_a.x ◽

1995 ◽

Vol 234 (3) ◽

pp. 695-705 ◽

Cited By ~ 16

Author(s):

Katsumi Morimatsu ◽

Toshihiro Horii

Keyword(s):

Dna Binding ◽

Binding Site ◽

Reca Protein ◽

Cross Linking ◽

Binding Surface ◽

Dna Binding Site

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Probing the Binding Region of the Single-Stranded DNA-Binding Domain of Rat DNA Polymerase β Using Nanosecond-Pulse Laser-Induced Cross-Linking and Mass Spectrometry

Photochemistry and Photobiology ◽

10.1111/j.1751-1097.1998.tb09685.x ◽

1998 ◽

Vol 68 (3) ◽

pp. 299-308 ◽

Cited By ~ 13

Author(s):

Dallas A. Connor ◽

Arnold M. Falick ◽

Mark C. Young ◽

Martin D. Shetlar

Keyword(s):

Mass Spectrometry ◽

Dna Binding ◽

Dna Polymerase ◽

Pulse Laser ◽

Binding Domain ◽

Cross Linking ◽

Dna Binding Domain ◽

Binding Region ◽

Dna Polymerase Β ◽

Nanosecond Pulse Laser

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Native Mass Spectrometry Reveals the Conformational Diversity of the UVR8 Photoreceptor

10.1101/371658 ◽

2018 ◽

Cited By ~ 1

Author(s):

Inês S. Camacho ◽

Alina Theisen ◽

Linus O. Johannissen ◽

L. Aranzazú Díaz-Ramos ◽

John M. Christie ◽

...

Keyword(s):

Mass Spectrometry ◽

Uv Light ◽

Native Mass Spectrometry ◽

Full Length ◽

Structural Investigations ◽

Core Domain ◽

Intrinsically Disordered ◽

The Core ◽

C Terminus ◽

Conformational Diversity

AbstractUVR8 is a plant photoreceptor protein that regulates photomorphogenic and protective responses to UV light. The inactive, homodimeric state absorbs UV-B light resulting in dissociation into monomers, which are considered to be the active state and comprise a β-propeller core domain and intrinsically disordered N- and C-terminal tails. The C-terminus is required for functional binding to signalling partner COP1. To date, however, structural studies have only been conducted with the core domain where the terminal tails have been truncated. Here, we report structural investigations of full-length UVR8 using native ion mobility mass spectrometry adapted for photo-activation. We show that, whilst truncated UVR8 photo-converts from a single conformation of dimers to a single monomer conformation, the full-length protein exist in numerous conformational families. The full-length dimer adopts both a compact state and an extended state where the C-terminus is primed for activation. In the monomer the extended C-terminus destabilises the core domain to produce highly extended yet stable conformations, which we propose are the fully active states that bind COP1. Our results reveal the conformational diversity of full-length UVR8. We also demonstrate the potential power of native mass spectrometry to probe functionally important structural dynamics of photoreceptor proteins throughout nature.TOC Graphic

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Native mass spectrometry reveals the conformational diversity of the UVR8 photoreceptor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1813254116 ◽

2019 ◽

Vol 116 (4) ◽

pp. 1116-1125 ◽

Cited By ~ 11

Author(s):

Inês S. Camacho ◽

Alina Theisen ◽

Linus O. Johannissen ◽

L. Aranzazú Díaz-Ramos ◽

John M. Christie ◽

...

Keyword(s):

Mass Spectrometry ◽

Uv Light ◽

Native Mass Spectrometry ◽

Full Length ◽

Structural Investigations ◽

Core Domain ◽

Intrinsically Disordered ◽

The Core ◽

C Terminus ◽

Conformational Diversity

UVR8 is a plant photoreceptor protein that regulates photomorphogenic and protective responses to UV light. The inactive, homodimeric state absorbs UV-B light, resulting in dissociation into monomers, which are considered to be the active state and comprise a β-propeller core domain and intrinsically disordered N- and C-terminal tails. The C terminus is required for functional binding to signaling partner COP1. To date, however, structural studies have only been conducted with the core domain where the terminal tails have been truncated. Here, we report structural investigations of full-length UVR8 using native ion mobility mass spectrometry adapted for photoactivation. We show that, while truncated UVR8 photoconverts from a single conformation of dimers to a single monomer conformation, the full-length protein exists in numerous conformational families. The full-length dimer adopts both a compact state and an extended state where the C terminus is primed for activation. In the monomer the extended C terminus destabilizes the core domain to produce highly extended yet stable conformations, which we propose are the fully active states that bind COP1. Our results reveal the conformational diversity of full-length UVR8. We also demonstrate the potential power of native mass spectrometry to probe functionally important structural dynamics of photoreceptor proteins throughout nature.

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