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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

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

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

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

Biochemically validated structural model of the 15-subunit intraflagellar transport complex IFT-B

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2022-12-02 | PXD037049 | Pride

Mass Spectrometry Uncovers Protein Interactions and Stoichiometry of Intermediates and Sub-complexes for SNARE Complex Assembly

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2023-01-25 | PXD030619 | Pride

Quantitative cross-linking analysis of chemoresistant HeLa cells

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