Biochemical analysis of the Kruppel-associated box (KRAB) transcriptional repression domain.
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.
Peng
H
H
Wistar Institute, Philadelphia, Pennsylvania 19104, USA.
Begg
G E
GE
Harper
S L
SL
Friedman
J R
JR
Speicher
D W
DW
Rauscher
F J
FJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000302:isolation & purification; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C117566:ZNF589 protein, human