The structurally disordered KRAB repression domain is incorporated into a protease resistant core upon binding to KAP-1-RBCC domain Journal Article
| Authors: | Peng, H.; Gibson, L. C.; Capili, A. D.; Borden, K. L. B.; Osborne, M. J.; Harper, S. L.; Speicher, D. W.; Zhao, K.; Marmorstein, R.; Rock, T. A.; Rauscher, F. J. 3rd |
| Article Title: | The structurally disordered KRAB repression domain is incorporated into a protease resistant core upon binding to KAP-1-RBCC domain |
| Abstract: | 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. © 2007 Elsevier Ltd. All rights reserved. |
| Keywords: | protein expression; unclassified drug; sequence analysis; dna-binding proteins; protein domain; protein function; mass spectrometry; protein analysis; complex formation; protein degradation; protein protein interaction; protein binding; transcription factor; tetrapoda; amino acid sequence; molecular sequence data; amino terminal sequence; protein purification; sequence alignment; nucleotide sequence; recombinant proteins; nuclear magnetic resonance spectroscopy; protein structure, tertiary; gene silencing; proteinase; protein structure; steroid receptor coactivator 1; zinc finger protein; repressor proteins; stoichiometry; zinc fingers; nucleic acid binding protein; transcriptional repression; gel filtration; protein-protein interaction; kap-1; krab domain; structurally disordered protein; kruppel associated box protein; protein rbcc; transcription factor kap 1 |
| Journal Title: | Journal of Molecular Biology |
| Volume: | 370 |
| Issue: | 2 |
| ISSN: | 0022-2836 |
| Publisher: | Academic Press Inc., Elsevier Science |
| Date Published: | 2007-07-06 |
| Start Page: | 269 |
| End Page: | 289 |
| Language: | English |
| DOI: | 10.1016/j.jmb.2007.03.047 |
| PUBMED: | 17512541 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 4" - "Export Date: 17 November 2011" - "CODEN: JMOBA" - "Molecular Sequence Numbers: GENBANK: AF242376, L28167, NM_003455, NM_015394, NM_016325, NM_023988;" - "Source: Scopus" |
