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

ABSTRACT: Polycomb Group proteins regulate gene expression by modifying chromatin. 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 of PSC assembles into PRC1, including partnering with dRING to form the ubiquitin ligase. The intrinsically disordered C-terminal region of PSC compacts chromatin and inhibits chromatin remodeling and transcription in vitro. Both regions of PSC are essential in vivo. To understand how these two activities may be coordinated in PRC1, we used crosslinking mass spectrometry to analyze the conformations of the C-terminal region of PSC in PRC1 and how they change on binding DNA. Crosslinking identifies interactions between the C-terminal region of PSC and the core of PRC1, including between N and C-terminal regions of PSC. New contacts and overall more compacted PSC C-terminal region conformations are induced by DNA binding. Protein footprinting of accessible lysine residues reveals an extended, bipartite candidate DNA/chromatin binding surface in the C-terminal region of PSC. Our data suggest a model in which DNA (or chromatin) follows a long path on the flexible disordered region of PSC. Intramolecular interactions of PSC detected by crosslinking 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 intrinsically disordered regions that bind nucleic acids.

SUBMITTER: Kang JJ

PROVIDER: S-EPMC7415685 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description:Polycomb Group (PcG) proteins regulate gene expression by modifying chromatin. A key PcG complex, PRC1, has two activities: a ubiquitin ligase activity for histone H2A, and a chromatin compacting/nucleosome bridging activity that can inhibit transcription and chromatin remodeling. In Drosophila, the Posterior Sex Combs (PSC) subunit of PRC1 is central to both activities. The N-terminal homology region (HR) of PSC partners with dRING to form the ubiquitin ligase, while its intrinsically disordered C-terminal region (PSC-CTR) compacts chromatin, and inhibits chromatin remodeling and transcription in vitro. Both the 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 the PSC HR. New contacts and overall more compacted PSC-CTR conformations are induced by DNA binding. We used chemical acetylation of accessible lysines for MS-based protein footprinting of the PSC-CTR. Protein footprinting 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.

2020-05-15 | MSV000085418 | MassIVE

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

Project description:Human RecQL4 belongs to the ubiquitous RecQ helicase family. Its N-terminal region represents the only homologue of the essential DNA replication initiation factor Sld2 of Saccharomyces cerevisiae, and also participates in the vertebrate initiation of DNA replication. Here, we utilized a random screen to identify N-terminal fragments of human RecQL4 that could be stably expressed in and purified from Escherichia coli. Biophysical characterization of these fragments revealed that the Sld2 homologous RecQL4 N-terminal domain carries large intrinsically disordered regions. The N-terminal fragments were sufficient for the strong annealing activity of RecQL4. Moreover, this activity appeared to be the basis for an ATP-independent strand exchange activity. Both activities relied on multiple DNA-binding sites with affinities to single-stranded, double-stranded and Y-structured DNA. Finally, we found a remarkable affinity of the N-terminus for guanine quadruplex (G4) DNA, exceeding the affinities for other DNA structures by at least 60-fold. Together, these findings suggest that the DNA interactions mediated by the N-terminal region of human RecQL4 represent a central function at the replication fork. The presented data may also provide a mechanistic explanation for the role of elements with a G4-forming propensity identified in the vicinity of vertebrate origins of DNA replication.

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Phosphorylated intrinsically disordered region of FACT masks its nucleosomal DNA binding elements.

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Long-range regulation of p53 DNA binding by its intrinsically disordered N-terminal transactivation domain.

Project description:Atomic resolution characterization of the full-length p53 tetramer has been hampered by its size and the presence of extensive intrinsically disordered regions at both the N and C termini. As a consequence, the structural characteristics and dynamics of the disordered regions are poorly understood within the context of the intact p53 tetramer. Here we apply trans-intein splicing to generate segmentally 15N-labeled full-length p53 constructs in which only the resonances of the N-terminal transactivation domain (NTAD) are visible in NMR spectra, allowing us to observe this region of p53 with unprecedented detail within the tetramer. The N-terminal region is dynamically disordered in the full-length p53 tetramer, fluctuating between states in which it is free and fully exposed to solvent and states in which it makes transient contacts with the DNA-binding domain (DBD). Chemical-shift changes and paramagnetic spin-labeling experiments reveal that the amphipathic AD1 and AD2 motifs of the NTAD interact with the DNA-binding surface of the DBD through primarily electrostatic interactions. Importantly, this interaction inhibits binding of nonspecific DNA to the DBD while having no effect on binding to a specific p53 recognition element. We conclude that the NTAD:DBD interaction functions to enhance selectivity toward target genes by inhibiting binding to nonspecific sites in genomic DNA. This work provides some of the highest-resolution data on the disordered N terminus of the nearly 180-kDa full-length p53 tetramer and demonstrates a regulatory mechanism by which the N terminus of p53 transiently interacts with the DBD to enhance target site discrimination.

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Intrinsically disordered C-terminal tails of E. coli single-stranded DNA binding protein regulate cooperative binding to single-stranded DNA.

Project description:The homotetrameric Escherichia coli single-stranded DNA binding protein (SSB) plays a central role in DNA replication, repair and recombination. E. coli SSB can bind to long single-stranded DNA (ssDNA) in multiple binding modes using all four subunits [(SSB)65 mode] or only two subunits [(SSB)35 binding mode], with the binding mode preference regulated by salt concentration and SSB binding density. These binding modes display very different ssDNA binding properties with the (SSB)35 mode displaying highly cooperative binding to ssDNA. SSB tetramers also bind an array of partner proteins, recruiting them to their sites of action. This is achieved through interactions with the last 9 amino acids (acidic tip) of the intrinsically disordered linkers (IDLs) within the four C-terminal tails connected to the ssDNA binding domains. Here, we show that the amino acid composition and length of the IDL affects the ssDNA binding mode preferences of SSB protein. Surprisingly, the number of IDLs and the lengths of individual IDLs together with the acidic tip contribute to highly cooperative binding in the (SSB)35 binding mode. Hydrodynamic studies and atomistic simulations suggest that the E. coli SSB IDLs show a preference for forming an ensemble of globular conformations, whereas the IDL from Plasmodium falciparum SSB forms an ensemble of more extended random coils. The more globular conformations correlate with cooperative binding.

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A Dimerization Function in the Intrinsically Disordered N-Terminal Region of Src.

Project description:The mode of regulation of Src kinases has been elucidated by crystallographic studies identifying conserved structured protein modules involved in an orderly set of intramolecular associations and ligand interactions. Despite these detailed insights, much of the complex behavior and diversity in the Src family remains unexplained. A key missing piece is the function of the unstructured N-terminal region. We report here the function of the N-terminal region in binding within a hydrophobic pocket in the kinase domain of a dimerization partner. Dimerization substantially enhances autophosphorylation and phosphorylation of selected substrates, and interfering with dimerization is disruptive to these functions. Dimerization and Y419 phosphorylation are codependent events creating a bistable switch. Given the versatility inherent in this intrinsically disordered region, its multisite phosphorylations, and its divergence within the family, the unique domain likely functions as a central signaling hub overseeing much of the activities and unique functions of Src family kinases.

| S-EPMC6226010 | biostudies-literature

Regulation of Src tumor activity by its N-terminal intrinsically disordered region.

Project description:The membrane-anchored Src tyrosine kinase is involved in numerous pathways and its deregulation is involved in human cancer. Our knowledge on Src regulation relies on crystallography, which revealed intramolecular interactions to control active Src conformations. However, Src contains a N-terminal intrinsically disordered unique domain (UD) whose function remains unclear. Using NMR, we reported that UD forms an intramolecular fuzzy complex involving a conserved region with lipid-binding capacity named Unique Lipid-Binding Region (ULBR), which could modulate Src membrane anchoring. Here we show that the ULBR is essential for Src's oncogenic capacity. ULBR inactive mutations inhibited Src transforming activity in NIH3T3 cells and in human colon cancer cells. It also reduced Src-induced tumor development in nude mice. An intact ULBR was required for MAPK signaling without affecting Src kinase activity nor sub-cellular localization. Phospho-proteomic analyses revealed that, while not impacting on the global tyrosine phospho-proteome in colon cancer cells, this region modulates phosphorylation of specific membrane-localized tyrosine kinases needed for Src oncogenic signaling, including EPHA2 and Fyn. Collectively, this study reveals an important role of this intrinsically disordered region in malignant cell transformation and suggests a novel layer of Src regulation by this unique region via membrane substrate phosphorylation.

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Multiple intrinsically disordered sequences alter DNA binding by the homeodomain of the Drosophila hox protein ultrabithorax.

Project description:During animal development, distinct tissues, organs, and appendages are specified through differential gene transcription by Hox transcription factors. However, the conserved Hox homeodomains bind DNA with high affinity yet low specificity. We have therefore explored the structure of the Drosophila melanogaster Hox protein Ultrabithorax and the impact of its nonhomeodomain regions on DNA binding properties. Computational and experimental approaches identified several conserved, intrinsically disordered regions outside the homeodomain of Ultrabithorax that impact DNA binding by the homeodomain. Full-length Ultrabithorax bound to target DNA 2.5-fold weaker than its isolated homeodomain. Using N-terminal and C-terminal deletion mutants, we demonstrate that the YPWM region and the disordered microexons (termed the I1 region) inhibit DNA binding approximately 2-fold, whereas the disordered I2 region inhibits homeodomain-DNA interaction a further approximately 40-fold. Binding is restored almost to homeodomain affinity by the mostly disordered N-terminal 174 amino acids (R region) in a length-dependent manner. Both the I2 and R regions contain portions of the activation domain, functionally linking DNA binding and transcription regulation. Given that (i) the I1 region and a portion of the R region alter homeodomain-DNA binding as a function of pH and (ii) an internal deletion within I1 increases Ultrabithorax-DNA affinity, I1 must directly impact homeodomain-DNA interaction energetics. However, I2 appears to indirectly affect DNA binding in a manner countered by the N terminus. The amino acid sequences of I2 and much of the I1 and R regions vary significantly among Ultrabithorax orthologues, potentially diversifying Hox-DNA interactions.

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NeProc predicts binding segments in intrinsically disordered regions without learning binding region sequences.

Project description:Intrinsically disordered proteins are those proteins with intrinsically disordered regions. One of the unique characteristics of intrinsically disordered proteins is the existence of functional segments in intrinsically dis-ordered regions. These segments are involved in binding to partner molecules, such as protein and DNA, and play important roles in signaling pathways and/or transcriptional regulation. Although there are databases that gather information on such disordered binding regions, data remain limited. Therefore, it is desirable to develop programs to predict the disordered binding regions without using data for the binding regions. We developed a program, NeProc, to predict the disordered binding regions, which can be regarded as intrinsically disordered regions with a structural propensity. We only used data for the structural domains and intrinsically disordered regions to detect such regions. NeProc accepts a query amino acid sequence converted into a position specific score matrix, and uses two neural networks that employ different window sizes, a neural network of short windows, and a neural network of long windows. The performance of NeProc was comparable to that of existing programs of the disordered binding region prediction. This result presents the possibility to overcome the shortage of the disordered binding region data in the development of the prediction programs for these binding regions. NeProc is available at http://flab.neproc.org/neproc/index.html.

| S-EPMC7692026 | biostudies-literature

Intrinsically disordered chromatin protein NUPR1 binds to the C-terminal region of Polycomb RING1B.

Project description:Intrinsically disordered proteins (IDPs) are ubiquitous in eukaryotes, and they are often associated with diseases in humans. The protein NUPR1 is a multifunctional IDP involved in chromatin remodeling and in the development and progression of pancreatic cancer; however, the details of such functions are unknown. Polycomb proteins are involved in specific transcriptional cascades and gene silencing. One of the proteins of the Polycomb complex is the Ring finger protein 1 (RING1). RING1 is related to aggressive tumor features in multiple cancer types. In this work we characterized the interaction between NUPR1 and the paralogue RING1B in vitro, in silico, and in cellulo. The interaction occurred through the C-terminal region of RING1B (C-RING1B), with an affinity in the low micromolar range (?10 ?M). The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch at the 30s region of its sequence, as pinpointed by computational results and site-directed mutagenesis at Ala33. The association between C-RING1B and wild-type NUPR1 also occurred in cellulo as tested by protein ligation assays; this interaction is inhibited by trifluoperazine, a drug known to hamper binding of wild-type NUPR1 with other proteins. Furthermore, the Thr68Gln and Ala33Gln/Thr68Gln mutants had a reduction in the binding toward C-RING1B as shown by in vitro, in silico, and in cellulo studies. This is an example of a well-folded partner of NUPR1, because its other interacting proteins are also unfolded. We hypothesize that NUPR1 plays an active role in chromatin remodeling and carcinogenesis, together with Polycomb proteins.

| S-EPMC5547589 | biostudies-literature

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