Three conformational states of the p300 CH1 domain define its functional properties

Biochemistry. 2003 Aug 26;42(33):9937-45. doi: 10.1021/bi034989o.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Escherichia coli / metabolism
  • Humans
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Molecular Sequence Data
  • Nuclear Proteins / chemistry*
  • Nuclear Proteins / metabolism
  • Protein Binding
  • Protein Conformation
  • Protein Folding
  • Sequence Homology, Amino Acid
  • Spectrometry, Fluorescence
  • Trans-Activators / chemistry*
  • Trans-Activators / metabolism
  • Transcription Factors / chemistry
  • Transcription Factors / metabolism
  • Zinc / chemistry
  • Zinc / metabolism


  • HIF1A protein, human
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Nuclear Proteins
  • Trans-Activators
  • Transcription Factors
  • Zinc