DNA binding alters the protease susceptibility of the p50 subunit of NF-kappa B.
The subdomain structure of the p50 subunit of NF-kappa B (amino acids 35-381) was investigated by partial proteolysis of the native protein. Trypsin cleaves p50 at a limited number of sites with an initial cleavage at low trypsin concentration occurring after R362 and a second cleavage taking place at higher trypsin concentration after K77. The cleavage after R362 does not alter the DNA binding characteristics of p50 but removes the nuclear localisation signal indicating that this region occupies a highly exposed position on the surface of the protein. The second cleavage after K77 generates a protein that although dimeric is incapable of binding DNA, thus emphasising the importance of residues 35-77 in DNA recognition. However p50 dimers containing one molecule cleaved after K77 and one molecule with this region intact are capable of binding DNA. When very high concentrations of trypsin are employed p50 is completely degraded. However if p50 is bound tightly to DNA containing its specific recognition site prior to trypsin addition the cleavage after K77 is almost completely blocked and the protein becomes highly resistant to proteolysis. These data suggest that bound DNA may mask critical trypsin cleavage sites or that DNA binding is accompanied by a conformational change in protein structure that renders the protein resistant to proteolysis.
Hay
R T
RT
School of Biological and Medical Sciences, University of St Andrews, Fife, UK.
Nicholson
J
J
D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000648:ultrastructure; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D011994:Recombinant Proteins; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D016328:NF-kappa B; D011994:Recombinant Proteins; D004247:DNA; D014357:Trypsin