A biophysical characterisation of factors controlling dimerisation and selectivity in the NF-kappaB and NFAT families.
The Rel/NF-kappaB 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-kappaB 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-kappaB 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-kappaB-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-kappaB dimers and have determined, for the first time, the intrinsic homodimerisation constant for NF-kappaB p50. These data support a model in which NF-kappaB 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.
de Lumley
Marie
M
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.
Hart
Darren J
DJ
Cooper
Matthew A
MA
Symeonides
Stefan
S
Blackburn
Jonathan M
JM
D000595:Amino Acid Sequence; D000818:Animals; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D019143:Evolution, Molecular; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011485:Protein Binding; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D050778:NFATC Transcription Factors; C494005:Nfatc1 protein, mouse; D009687:Nuclear Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA