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DNA Binding and Phosphorylation Regulate the Core Structure of the NF-?B p50 Transcription Factor.

ABSTRACT: The NF-?B transcription factors are known to be extensively phosphorylated, with dynamic site-specific modification regulating their ability to dimerize and interact with DNA. p50, the proteolytic product of p105 (NF-?B1), forms homodimers that bind DNA but lack intrinsic transactivation function, functioning as repressors of transcription from ?B promoters. Here, we examine the roles of specific phosphorylation events catalysed by either protein kinase A (PKAc) or Chk1, in regulating the functions of p50 homodimers. LC-MS/MS analysis of proteolysed p50 following in vitro phosphorylation allows us to define Ser328 and Ser337 as PKAc- and Chk1-mediated modifications, and pinpoint an additional four Chk1 phosphosites: Ser65, Thr152, Ser242 and Ser248. Native mass spectrometry (MS) reveals Chk1- and PKAc-regulated disruption of p50 homodimer formation through Ser337. Additionally, we characterise the Chk1-mediated phosphosite, Ser242, as a regulator of DNA binding, with a S242D p50 phosphomimetic exhibiting a >?10-fold reduction in DNA binding affinity. Conformational dynamics of phosphomimetic p50 variants, including S242D, are further explored using ion-mobility MS (IM-MS). Finally, comparative theoretical modelling with experimentally observed p50 conformers, in the absence and presence of DNA, reveals that the p50 homodimer undergoes conformational contraction during electrospray ionisation that is stabilised by complex formation with ?B DNA. Graphical Abstract ?.

SUBMITTER: Vonderach M

PROVIDER: S-EPMC6318249 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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DNA Binding and Phosphorylation Regulate the Core Structure of the NF-κB p50 Transcription Factor.

Vonderach Matthias M Byrne Dominic P DP Barran Perdita E PE Eyers Patrick A PA Eyers Claire E CE

Journal of the American Society for Mass Spectrometry 20180605 1

The NF-κB transcription factors are known to be extensively phosphorylated, with dynamic site-specific modification regulating their ability to dimerize and interact with DNA. p50, the proteolytic product of p105 (NF-κB1), forms homodimers that bind DNA but lack intrinsic transactivation function, functioning as repressors of transcription from κB promoters. Here, we examine the roles of specific phosphorylation events catalysed by either protein kinase A (PKA<sub>c</sub>) or Chk1, in regulating ...[more]

PMID: 29873020

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Project description:The family of NF-κB transcriptional activators controls the expression of many genes, including those involved in cell survival and development. The family consists of homo- and heterodimers constituted by combinations of five subunits. Subunit p50 includes 13 tyrosine residues, but the relationship between specific tyrosine phosphorylations and p50 function is not well understood. Subunits of p50 and p65 prepared in vitro formed a heterodimer, but this NF-κB would not bind to the interleukin-2 (IL-2) promoter DNA. Treatment of p50 with guanosine triphosphate (GTP) and a lysate from activated Jurkat cells, effected rapid p50 phosphorylation, and, in the presence of wild-type subunit p65, was accompanied on the same time scale by IL-2 promoter DNA binding. Modified p50s containing one of seven stoichiometrically phosphorylated tyrosines in NF-κB p50/p65 heterodimers, included three that facilitated binding to the IL-2 DNA promoter region to a greater extent than the wild type. One of these three stoichiometrically phosphorylated p50/p65 heterodimers of NF-κB, containing pTyr60 in the p50 subunit, was treated with a lysate from activated Jurkat cells + GTP and shown to be phosphorylated on the same time scale as wild-type p50. This modified NF-κB also developed IL-2 promoter DNA binding activity on the same time scale as the wild type but exhibited greater binding to the IL-2 DNA promoters than the wild type. The nature of this enhanced binding was studied in greater detail using a metabolically stable pTyr derivative at position 60 of p50 and cellular phosphatases. We suggest that enhanced DNA binding of modified NF-κB containing pTyr60 in the p50 subunit may reflect stoichiometric NF-κB phosphorylation at a site that is not normally fully phosphorylated, or not phosphorylated at all, and is relatively resistant to the effects of Jurkat cell tyrosine phosphatase activity. This conclusion was reinforced by demonstrating that modification of Tyr60 of p50 with a metabolically stable methylenephosphonate moiety further increased the stability of the formed NF-κB p50/p65 heterodimer against the action of activated Jurkat cell phosphatases.

Project description:The unregulated activation of nuclear factor-κB (NF-κB) is a critical event in the progression of various inflammatory diseases such as ulcerative colitis, asthma, rheumatoid arthritis, bacterial induced gastritis, etc. Hence, blocking the transcriptional activity of NF-κB is a promising strategy towards the development of an anti-inflammatory agent. In this study, an integrated molecular and quantum mechanical approach was carried out to find a new potent andrographolide (AGP)-based analog that can inhibit DNA binding to NF-κB p50 and manifest anti-inflammatory activity. Our approach includes multiple sequence alignment, virtual screening, molecular docking (protein-ligand and protein-DNA), in silico site-directed mutagenesis, ADMET prediction, DFT (HOMO, LUMO, HLG, and EPM energy) analysis, MD simulation, and MM/GBSA rescoring. The virtual screening analysis of 237 AGP analogs yielded the five lead compounds based on the binding affinity. Further, molecular interactive docking and ADMET prediction of hit analogs revealed that Ana2 ((3Z,4S)-3-[2-[(4aR,6aS,7R,10aS,10bR)-3,3,6a,10b-tetramethyl-8-methylidene-1,4a,5,6,7,9,10,10a-octahydronaphtho[2,1-d][1,3]dioxin-7-yl]ethylidene]-4-hydroxyoxolan-2-one) is the most potent moiety as it displays the strongest binding affinity and better molecular/pharmacokinetic features. Moreover, DFT, MD simulation, and MM/GBSA studies corroborated the docking results and demonstrated better chemical and dynamic stability with the least binding free energy (- 29.99 kcal/mol) for the Ana2. Site-directed mutagenesis investigation (Cys62Ala) establishes the importance of the Cys62 amino acid residue towards the binding interaction and stability of Ana2 with NF-κB p50. Overall, the identified NF-κB p50 inhibitor opens up a new research horizon towards the development of plant-based anti-inflammatory drugs to combat progressive inflammatory diseases.Supplementary informationThe online version contains supplementary material available at 10.1007/s13205-022-03431-9.

Dataset Information

DNA Binding and Phosphorylation Regulate the Core Structure of the NF-?B p50 Transcription Factor.

Publications

DNA Binding and Phosphorylation Regulate the Core Structure of the NF-κB p50 Transcription Factor.

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