Biochemical analysis of the Kruppel-associated box (KRAB) transcriptional repression domain - Spectral, kinetic, and stoichiometric properties of the KRAB.KAP-1 complex

The Kruppel-associated box CE(RAB) domain is a 75-amino acid transcriptional repressor module commonly found in eukaryotic zinc finger proteins. RRAB-mediated gene silencing requires binding to the RING-B box-coiled-coil domain of the corepressor KAP-1. Little is known about the biochemical properties of the KRAB domain or the KRAB.KAP-1 complex. Using purified components, a combination of biochemical and biophysical analyses has revealed that the KRAB domain from the KOX1 protein is predominantly a monomer and that the KAP-1 protein is predominantly a trimer in solution. The analyses of electrophoretic mobility shift assays, GST association assays, and plasmon resonance interaction data have indicated that the KRAB binding to KAP-1 is direct, highly specific, and high affinity The optical biosensor data for the complex was fitted to a model of a one-binding step interaction with fast association and slow dissociation rates, with a calculated bh of 142 nw. The fitted R-max indicated three molecules of KAP-1 binding to one molecule of the KRAB domain, a stoichiometry that is consistent with quantitative SDS-polyacrylsmide gel electrophoresis analysis of the complex. These structural and dynamic parameters of the KRAB/KAP-1 interaction have implications for identifying downstream effecters of RAP-I silencing and the de novo design of new repression domains.