The transactivation domain within cysteine/histidine-rich region 1 of CBP comprises two novel zinc-binding modules.
cAMP-response element-binding protein-binding protein (CBP) is a transcriptional coactivator that interacts with a number of DNA-binding proteins and cofactor proteins involved in the regulation of transcription. Relatively little is known about the structure of CBP, but it has been noted that it contains three domains that are rich in cysteine and histidine (CH1, CH2, and CH3). The sequence of CH2 conforms to that of a leukemia-associated protein domain (PHD finger), and it has been postulated that this and both CH1 and CH3 may be zinc finger domains. This has not, however, been demonstrated experimentally. We have studied CH1 and show that it is composed of two novel zinc-binding modules, which we term "zinc bundles." Each bundle contains the sequence Cys-X(4)-Cys-X(8)-His-X(3)-Cys, and we show that a synthetic peptide comprising one zinc bundle from CH1 can fold in a zinc-dependent manner. CH3 also appears to contain two zinc bundles, one with the variant sequence Cys-X(2)-Cys-X(9)-His-X(3)-Cys, and we demonstrate that this variant motif also undergoes Zn(II)-induced folding. CH1 acts as a transcriptional activation domain in cellular assays. We show that mutations in any of the four zinc-chelating residues in either zinc bundle of CH1 significantly impair this activity and that these mutations also interfere with certain protein-protein interactions mediated by CH1. Our results indicate that CBP is a genuine zinc-binding protein and introduce zinc bundles as novel protein interaction domains.
Newton
A L
AL
Department of Biochemistry, G08, University of Sydney, Sydney, New South Wales 2006, Australia.
Sharpe
B K
BK
Kwan
A
A
Mackay
J P
JP
Crossley
M
M
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis; D001665:Binding Sites; D050882:CREB-Binding Protein; D017173:Caenorhabditis elegans; D002942:Circular Dichroism; D003545:Cysteine; D004331:Drosophila melanogaster; D006639:Histidine; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D010947:Plants, Toxic; D017510:Protein Folding; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D015533:Transcriptional Activation; D015032:Zinc / Q000378:metabolism; D016335:Zinc Fingers
D009687:Nuclear Proteins; D015534:Trans-Activators; D006639:Histidine; D050882:CREB-Binding Protein; C494363:CREBBP protein, human; C494364:Crebbp protein, mouse; D015032:Zinc; D003545:Cysteine