A biophysical characterisation of factors controlling dimerisation and selectivity in the NF-kappa B and NFAT families

de Lumley, Marie, Hart, Darren J., Cooper, Matthew A., Symeonides, Stefan and Blackburn, Jonathon M. (2004) A biophysical characterisation of factors controlling dimerisation and selectivity in the NF-kappa B and NFAT families. Journal of Molecular Biology, 339 5: 1059-1075.


Author de Lumley, Marie
Hart, Darren J.
Cooper, Matthew A.
Symeonides, Stefan
Blackburn, Jonathon M.
Title A biophysical characterisation of factors controlling dimerisation and selectivity in the NF-kappa B and NFAT families
Journal name Journal of Molecular Biology    (ERA 2010 Rank B)   Check publisher's open access policy
Publication date 2004-06-18
Sub-type Article
DOI 10.1016/j.jmb.2004.03.083
Volume number 339
Issue number 5
ISSN 0004-9425; 1448-0038
Start page 1059
End page 1075
Total pages 17
Place of publication Cambridge , U.K.
Publisher Elseiver B.V.
Language eng
Subject 03 Chemical Sciences
0304 Medicinal and Biomolecular Chemistry
Abstract The Rel/NF-kB family of eukaryotic transcription factors bind DNA with high specificity and affinity as homo- or heterodimers to mediate a diverse range of biological processes. By comparison, the nuclear factor of activated T-cells (NFAT) family has been recognised as Rel homologues due to structural similarities between the DNA-binding domains, yet they bind DNA as lower-affinity monomers. The structural and functional overlap between the NF-kB and NFAT families suggests that they may be evolutionarily divergent from a common, monomeric ancestor but have evolved different mechanisms to achieve high-affinity binding to their target DNA sequences. In order to understand the origin of these mechanistic differences, we constructed two chimeric proteins, based on molecular modelling, comprising the DNA-binding domain of NFAT and the dimerisation domain of NF-kB p50, differing only in the position of the splice site. Biophysical characterisation of the wild-type and chimeric proteins revealed that one of the chimeras bound DNA as a high-affinity, NF-kB-like cooperative dimer, whilst the other bound as a lower-affinity, NFAT-like monomer, demonstrating the importance of the interdomain linker in controlling the intrinsic ability of NFATc to form dimers. In addition, we have studied the rate of exchange of monomers between preformed NF-kB dimers and have determined, for the first time, the intrinsic homodimerisation constant for NF-kB p50. These data support a model in which NF-kB proteins bind DNA both in vitro and in vivo as high-affinity preformed homo- or heterodimers, which in an unbound form can still exchange monomer units on a physiologically relevant timescale in vivo. c 2004 Elsevier Ltd. All rights reserved.
Keyword Transcription Factors
Rel family
NFAT family
Evolution
Dimerisation
Transcription Factor NFATC
P50 Homodimer
Dimer interface
DNA
Proteins
Site
Technology
Affinity
Complex
Target
Q-Index Code C1

 
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