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Structural proteomics based investigation of DNA binding domain (DBD) of FOXO4 transcription factor in complex with DNA binding element (DNA)

ABSTRACT: The limited information available on the structure of complexes involving transcription factors and cognate DNA response elements represents a major obstacle in the quest to understand their mechanism of action at the molecular level. We implemented a concerted structural proteomics approach, which combined hydrogen-deuterium exchange (HDX), quantitative protein-protein and protein-nucleic acid cross-linking (XL), and homology analysis, to model the structure of the complex between the full-length DNA binding domain (DBD) of FOXO4 and its DNA binding element (DBE).

INSTRUMENT(S): solariX

ORGANISM(S): Homo Sapiens (human) Escherichia Coli

SUBMITTER: Lukáš Slavata

LAB HEAD: Petr Novak

PROVIDER: PXD013969 | Pride | 2020-05-26

REPOSITORIES: Pride

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			Action	DRS
	F1D_10m_11677.mzML	Mzml
	F1D_20s_11669.mzML	Mzml
	F1D_2h_11685.mzML	Mzml
	F1D_2m_11673.mzML	Mzml
	F1D_30m_11681.mzML	Mzml

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Publications

MS-Based Approaches Enable the Structural Characterization of Transcription Factor/DNA Response Element Complex.

Slavata Lukáš L Chmelík Josef J Kavan Daniel D Filandrová Růžena R Fiala Jan J Rosůlek Michal M Mrázek Hynek H Kukačka Zdeněk Z Vališ Karel K Man Petr P Miller Michael M McIntyre William W Fabris Daniele D Novák Petr P

Biomolecules 20190926 10

The limited information available on the structure of complexes involving transcription factors and cognate DNA response elements represents a major obstacle in the quest to understand their mechanism of action at the molecular level. We implemented a concerted structural proteomics approach, which combined hydrogen-deuterium exchange (HDX), quantitative protein-protein and protein-nucleic acid cross-linking (XL), and homology analysis, to model the structure of the complex between the full-leng ...[more]

PMID: 31561554

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TEAD1-DBD-DNA interaction studied by structural MS

Project description:TEAD transcription factors mediate gene expression regulation through interactions with their DNA response M-CAT motif. They are active mainly during growth and development and induce gene expression of proteins involved in cell proliferation, differentiation or apoptosis prevention. TEADs and many of their target proteins are known to be upregulated in several types of cancers. To study the interaction of DNA binding domain of TEAD1 with M-CAT motifs of different orientations originating from regulatory regions of human genes (CTGF, SRF, C-MYC and GLUT1), dissociation constant of each complex was determined using fluorescence anisotropy assay. Structural mass spectrometry techniques (H/D exchange and quantitative chemical cross-linking) and molecular docking were utilized for structural characterization of the binding interface. In-vitro results were correlated with situation in living cells studied by ChIP and Q-PCR.

2020-12-08 | PXD012127 | Pride

Proteome-wide identifying site non-specific cross-linking peptides with SpotLink

Project description:We developed SpotLink to identify site non-specific cross-links from the proteome scale. Here described the Abeta dataset and the HeLa dataset to evaluate SpotLink.

2022-08-25 | PXD030274 | Pride

The linear ubiquitin chain assembly complex LUBAC generates heterotypic ubiquitin chains

Project description:The 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.

2021-06-28 | PXD019771 | Pride

How Myosin VI Traps its Off-State, is Activated and Dimerizes

Project description:Myosin VI (Myo6) is the only minus-end directed nanomotor on actin, allowing it to uniquely contribute to numerous cellular functions. As for other nanomotors, proper functioning of Myo6 relies on precise spatio-temporal control of motor activity via a poorly defined off-state and interactions with partners. Our structural, functional, and cellular studies reveal key features of myosin regulation and indicate that not all partners can activate Myo6. TOM1 and Dab2 cannot bind the off-state while, GIPC1 binds Myo6, releases its auto-inhibition and triggers proximal dimerization. Myo6 partners thus differentially recruit Myo6. We solved a crystal structure of the proximal dimerization domain, and show that its disruption compromises endocytosis in HeLa cells, emphasizing the importance of Myo6 dimerization. Finally, we show that the L926Q deafness mutation disrupts Myo6 auto-inhibition and indirectly impairs proximal dimerization. Our study thus demonstrates the importance of partners in the control of Myo6 auto-inhibition, localization, and activation.

2024-01-26 | PXD044767 | Pride

A2M-3K DSSO cross-linking to identify the bait region position

Project description:An A2M mutant, A2M-3K, with the bait region arginines replaced with lysines, was cross-linked with DSSO. Its monomeric gel band was analyzed by XL-MS to identify the bait region position within a subunit.

2021-05-21 | PXD019048 | Pride

An inhibitory segment within G-patch activators tunes Prp43-ATPase activity during ribosome assembly

Project description:Chemical cross-linking coupled to mass spectrometry using the amine-reactive DSS reagent was used to study the interactions between the RNA helicase Prp43 and its interactors, Pxr1 and Tma23, in Saccharomyces cerevisiae.

2024-10-02 | PXD047173 | Pride

Effects of a phosphomimetic mutation and nucleotide binding on the interaction of Hsp90a with co-chaperone Aha1 analyzed by chemical cross-linking with mass spectrometry

Project description:Complex conformational dynamics are essential for the chaperone function of heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimerization establishing ATPase competence. Biochemical data demonstrate that the intrinsic, but weak, ATP hydrolyzing activity of Hsp90 is markedly enhanced by the co-chaperone Aha1. However, cellular concentration of Aha1 is substoichiometric relative to Hsp90. In cells, interaction of this important co-chaperone with Hsp90 is up-regulated by posttranslational modifications (PTMs), including phosphorylation of a highly conserved tyrosine (Y313 in Hsp90a). Here we use chemical cross-linking with mass spectrometry to explore the the impacts of a phosphomimetic mutation (Y313E) and binding of a non-hydrolyzable ATP analog (AMP-PNP),on the structure Hsp90a and its interaction with Aha1.

2019-06-17 | PXD013476 | Pride

Reconstitution of mammalian Cleavage Factor II

Project description:Cleavage factor II (CF II) is a poorly characterized component of the multi-protein complex catalyzing 3' cleavage and polyadenylation of mammalian mRNA precursors. We have reconstituted CF II as a heterodimer of hPcf11 and hClp1. The heterodimer is active in partially reconstituted cleavage reactions, whereas hClp1 by itself is not. Pcf11 moderately stimulates the RNA 5' kinase activity of hClp1; the kinase activity is dispensable for RNA cleavage. CF II binds RNA with nanomolar affinity. Binding is mediated mostly by the two zinc fingers in the C-terminal region of hPcf11. RNA is bound without pronounced sequence-specificity, but extended G-rich sequences appear to be preferred. We discuss the possibility that CF II contributes to the recognition of cleavage/polyadenylation substrates through interaction with G-rich far-downstream sequence elements.

2020-05-26 | PXD010177 | Pride

Structural Assessment of the Full-Length Tumor Suppressor Protein p53 by Mass Spectrometry-Guided Computational Modeling

Project description:The tetrameric tumor suppressor p53 represents a great challenge for 3D structural analysis due to its high degree of intrinsic disorder (ca. 40%). We developed and applied an integrative structural biology approach combining complementary techniques of structural mass spectrometry (MS), namely cross-linking mass spectrometry (XL-MS), protein footprinting, and hydrogen/deuterium exchange mass spectrometry (HDX-MS), with advanced protein structure prediction approaches to gain insights into the disordered C-terminal region of p53. Additionally, we evaluate possible differences in p53 regarding solvent accessibility and topology upon DNA binding. Our quantitative XL-MS and lysine labeling data show no major conformational differences in p53 between DNA-bound and DNA-free states. Integration of experimental data generate p53 models for p53’s intrinsically disordered regions (IDRs) that reflect substantial compaction of the molecule. Our models provide the most detailed description of the relationship between p53’s folded regions and IDRs that is available to date. The synergies between complementary structural MS techniques and computational modeling as pursued in our integrative approach is envisioned to serve as general strategy for studying intrinsically disordered proteins (IDPs) and IDRs.

2023-06-26 | PXD037030 | Pride

Integrative structure models of the α-Synuclein dimer conformations

Project description:α-Synuclein is an intrinsically disordered protein known for its contributions to functions in neurons but mostly in its contributions to the development of Parkinson's disease via formation of self-associated forms, fibrils and Lewy bodies. Among the multiple targets it interacts with and gains a folded structure within the bound state, are homo-oligomers. Logically, the shortest homo-oligomer a protein can form is a dimer. However, until today the existence of the α-Synuclein dimer has been a subject of much debate. Using an array of measurements, we show that in vitro α-Synuclein exhibits primarily a monomer-dimer equilibrium within a concentration range of hundreds of nanomolars and up to a few micromolars. Furthermore, we performed hetero-isotopic cross-linking mass spectrometry experiments, from which we gained spatial information on the dimer that was then used in constructing restraints for discrete molecular dynamics simulations. This allowed us to retrieve integrative structure models of α-Synuclein dimer structural subpopulations, having different degrees of compactness and binding energies. Interestingly, one of dimers structures is compact, stable, abundant and exhibits partially-exposed β-sheet structures in the N-terminal and non-amyloid component segments. This compact dimer form is also the only one that exhibits interaction distances between hydroxyl groups of tyrosine 39 in the two subunits. We suggest that the high abundance compact dimer form serves as an important α-Synuclein dimeric species and propose different hypotheses as to its function, whether on pathway to aggregation or not

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