title: Three conformational states of the p300 CH1 domain define its functional properties.
abstract: Numerous transcription factors interact with the basal transcriptional machinery through the transcriptional co-activators p300 and CREB-binding protein (CBP). The Zn(2+)-binding cysteine/histidine-rich 1 (CH1) domain of p300/CBP binds many of these transcription factors, including hypoxia-inducible factor (HIF). We studied the structural and biophysical properties of the p300 CH1 domain alone and bound to the HIF-1 alpha C-terminal transactivation domain (TAD) to understand the diverse binding properties of CH1. The Zn(2+)-bound CH1 domain (CH1-Zn(2+)) and the HIF-1 alpha TAD-CH1 complex (CH1-Zn(2+)-HIF-1 alpha) are similarly helical, whereas metal-free CH1 is mostly random coil. CH1-Zn(2+) undergoes noncooperative thermal denaturation, does not have a near-UV elliptical signal, and binds the hydrophobic fluorophore ANS. In contrast, the CH1-Zn(2+)-HIF-1 alpha complex undergoes cooperative thermal denaturation, does produce a near-UV signal, and does not bind ANS. Addition of Zn(2+) ions to metal-free CH1 produced one conformational change, and subsequent addition of a HIF-1 alpha TAD peptide induced a second conformational change as detected by intrinsic tryptophan fluorescence spectroscopy. The NMR (1)H-(15)N HSQC spectrum of CH1-Zn(2+) exhibits few poorly dispersed peaks with broad line widths. Removal of metal ions produces more poorly dispersed peaks with sharper line widths. Addition of a HIF-1 alpha TAD peptide to CH1-Zn(2+) produces many well-dispersed peaks with sharp line widths. Taken together, these data support three conformational states for CH1, including an unstructured metal-free domain, a partially structured Zn(2+)-bound domain with molten globule characteristics, and a stable, well-ordered HIF-1 alpha TAD-CH1 complex.
authors.affiliation: Division of Hemostasis and Thrombosis and Division of Hematology-Oncology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, Massachusetts 02215, USA.
authors.forename: Ravina
authors.initials: R
authors.lastname: Dial
authors.forename: Zhen-Yu J
authors.initials: ZY
authors.lastname: Sun
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Freedman
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D015032:Zinc