Abstract
The Kruppel-associated box (KRAB) domain is a 75-amino acid transcriptional repressor module commonly found in eukaryotic zinc finger proteins. KRAB-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 K(d) of 142 nm. 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-polyacrylamide gel electrophoresis analysis of the complex. These structural and dynamic parameters of the KRAB/KAP-1 interaction have implications for identifying downstream effectors of KAP-1 silencing and the de novo design of new repression domains.
Highlights
The Kruppel-associated box (KRAB) domain is a 75amino acid transcriptional repressor module commonly found in eukaryotic zinc finger proteins
The fitted Rmax indicated three molecules of KAP-1 binding to one molecule of the KRAB domain, a stoichiometry that is consistent with quantitative SDSpolyacrylamide gel electrophoresis analysis of the complex
These helices have an amphipathic nature as defined by helical wheel analysis. These predictions suggest that the KRAB domain provides an interface for protein/protein interactions and that it may bind to the KAP-1 corepressor via a helix/helix interaction
Summary
Preparation of Plasmids—The plasmids pQE30 GAL4-KRAB-(1–90), pQE30 KOX1-KRAB, GST-KRAB, GST-KRAB(DV), pQE30 KAP-1RBCC, and pVL1392 KAP-1-RBCC have been described previously. The eluted KOX1-(1–161) and KAP-1-RBCC proteins were mixed at a 1:1 molar ratio in a volume of 20 ml of buffer containing 8 M urea, 0.1 M sodium phosphate, 0.01 M Tris-HCl, pH 8.0, 10% glycerol, 20 M ZnSO4, and 0.5 mM dithiothreitol. The KAP-1-(22– 618) protein and the KOX1-(1–161)1⁄7KAP-1-RBCC complex were purified using a Superose 6 HR 10/30 column equilibrated with the P300 buffer plus 10% glycerol. The KOX1-KRAB protein in P300 buffer plus 10% glycerol was incubated with 20 mM C8E5 for 1 h at 4 °C by rotation to attempt to dissociate aggregates before gel filtration. The KAP-1-(22– 618) protein was purified by gel filtration in P300 buffer plus 10% glycerol without detergent. Sedimentation equilibrium experiments were performed in an Optima XL-I ultracentrifuge, using either the absorbance (280 nm) optics (for KOX1-KRAB) or the interference optics
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