Role of the conserved lysine 80 in stabilisation of NF-kappaB p50 DNA binding.
The transcriptional rate of a variety of genes involved in acute-phase response, inflammation, lymphocytic activation, and cell growth or differentiation, is regulated by the DNA binding activity of the inducible transcription factor NF-kappaB. NF-kappaB p50 homodimers bind specifically to DNA, via base and backbone contacts mediated by residues in the flexible loops which link secondary structure elements in both of its two distinct domains. However, it has been suggested that additional contacts which stabilise DNA binding are made by lysine residues located in the C-terminus of the flexible loop which connects A and B beta-sheets of the N-terminal domain of p50. To determine the importance of each of the lysine residues in this region (K77, K79, K80), a series of mutated p50 proteins were generated in which the lysines were changed to alanines. The DNA binding properties of these mutants were analysed by gel electrophoresis DNA binding assays and surface plasmon resonance. This study revealed that the C-terminus of AB loop interacts with DNA through an additional lysine-phosphate backbone ionic bond which makes a significant contribution to the binding energy, thus stabilising the complex. The lysine residue responsible for this interaction is K80 which is conserved in all NF-kappaB/Rel/Dorsal molecules.
Michalopoulos
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School of Biomedical Sciences, Institute of Biomolecular Sciences, University of St Andrews, The North Haugh, St Andrews, Fife KY16 9ST, UK.
Hay
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D000595:Amino Acid Sequence; D000727:Androgen-Binding Protein; D001665:Binding Sites; D017124:Conserved Sequence; D008239:Lysine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
D000727:Androgen-Binding Protein; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; C027519:probasin; D008239:Lysine