Mutational analysis of the kinetics and thermodynamics of transcription factor NF-kappaB homodimerisation.
Dimeric transcription factors of the NF-kappaB/Rel family are sequence-specific DNA-binding proteins that mediate the inducible expression of immunologically important eukaryotic genes by competing for kappaB sites. The kinetic and thermodynamic components of these interactions were probed by mutation of the subunit interface of the p50 homodimer, a paradigm for other family members. Guided by the crystal structure, we selected the side chains of five key residues (R255, Y270, L272, A311 and V313) for individual and combinatorial truncation, with the aim of generating a mutant panel. Homodimerisation was assessed indirectly by measurement of DNA binding with an optical biosensor in order to unmask the relative contributions of each residue. Surface plasmon resonance revealed that a unanimous bias for a palindromic kappaB site over an asymmetric one was mainly the result of a slower dissociation rate for the DNA/homodimer complex in the case of the palindromic kappaB site. Y270 and L272 were individually the most critical residues in homodimerisation. DNA binding was abolished when all five residues were substituted, which reinforces the notion that only a subset of residues contributes crucial dimer-forming contacts. The role of Y270 was unique, since its mutation to glycine dramatically slowed both the association and dissociation rates for DNA binding. Surprisingly, R255 was shown to be of little importance in the stability of the p50 homodimer, despite its apparent participation in a salt bridge at the dimer interface. Our results suggest that binding modes inferred from structural data should be treated cautiously.
Day
Y S N
YS
Department of Chemistry, The University of Manchester, Oxford Road, Manchester M13 9PL, UK.
Bacon
S L
SL
Hughes-Thomas
Z
Z
Blackburn
J M
JM
Sutherland
J D
JD
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016328:NF-kappa B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D052002:NF-kappa B p50 Subunit; D011485:Protein Binding / Q000235:genetics; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D014157:Transcription Factors; D004247:DNA