COMMON STRUCTURAL CONSTITUENTS CONFER I-KAPPA-B ACTIVITY TO NF-KAPPA-B P105 AND I-KAPPA-B/MAD-3

The vertebrate NF-chiB/c-rel inhibitors MAD-3/IchiB alpha, IchiB gamma/pdI and bcl-3 all share a conserved ankyrin repeat domain (ARD) consisting of six complete repeats, a short acidic motif and/or an incomplete seventh repeat. We present here a detailed analysis of the domain in p105/pdI and MAD-3/IchiB involved in inhibition of DNA binding and in protein interaction with rel factors. We demonstrate that in both cases an acidic region and six ankyrin-like repeats are sufficient and required for protein interaction with the rel factors. However, for p105/pdI to achieve the high affinity needed to suppress DNA binding, an incomplete seventh repeat is required in addition. Both pdI and MAD-3 associate with rel proteins by forming heterotrimeric complexes, as shown by native gel analysis and by cross-linking. Furthermore, we demonstrate that deletion of only three amino acids in the first repeat converts the subunit specificity of the p105 ARD into that of MAD-3/IchiB. We conclude that functionally the ARD in these molecules has a modular structure, with different subregions determining the specificity for the NF-chiB subunits p50 and p65.