The structurally disordered KRAB repression domain is incorporated into a protease resistant core upon binding to KAP-1-RBCC domain.
The KRAB domain is a 75 amino acid transcriptional repression module that is encoded by more than 400 zinc finger protein genes, making it the most abundant repression domain in the human proteome. KRAB-mediated gene silencing requires a direct high affinity interaction with the RBCC domain of KAP-1 co-repressor. The structures of the free KRAB domain or the KRAB-RBCC complex are unknown. To address this, we have performed a systematic biophysical analysis of all KRAB isoforms using purified recombinant proteins. All KRAB domains are predominantly monomeric either alone or in a complex with KAP-1-RBCC protein, while a KRAB-SCAN isoform exists as a stable dimer. The KRAB:KAP-1-RBCC interaction requires only the A box in the context of the KRAB(Ab) or KRAB(AC) but both A and B boxes in the context of KRAB(AB). All isoforms bind the KAP-1-RBCC in a stable, zinc dependent fashion with a stoichiometry of KRAB1:3 RBCC with a zinc content of four atoms per RBCC monomer. Limited proteolysis, mass spectrometry and N-terminal sequence analyses suggest that a core complex comprises the entire RBCC domain of KAP-1 and the AB box of the KRAB domain rendering it resistant to proteolysis. NMR spectroscopy showed that unbound KRAB domain does not exist as a well-folded globular protein in solution but may fold into an ordered structure upon binding to the KAP-1-RBCC protein. This is the first example of a structurally disordered repressor domain that is the most widely conserved silencing domain in tetrapods.
Peng
Hongzhuang
H
The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA.
Gibson
Lisa C
LC
Capili
Allan D
AD
Borden
Katherine L B
KL
Osborne
Michael J
MJ
Harper
Sandra L
SL
Speicher
David W
DW
Zhao
Kehao
K
Marmorstein
Ronen
R
Rock
Thomas A
TA
Rauscher
Frank J
FJ
D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D020868:Gene Silencing; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D000076183:Tripartite Motif-Containing Protein 28; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C101116:TRIM28 protein, human; D000076183:Tripartite Motif-Containing Protein 28