Fluorescence analysis of a dynamic loop in the PCAF/GCN5 histone acetyltransferase

Biochemistry. 2005 Aug 9;44(31):10501-9. doi: 10.1021/bi050776i.

Abstract

PCAF and GCN5 are histone acetyltransferase (HAT) paralogs which play roles in the remodeling of chromatin in health and disease. Previously, a conformationally flexible loop in the catalytic domain had been observed in the X-ray structures of GCN5 in different liganded states. Here, the conformation and dynamics of this PCAF/GCN5 alpha5-beta6 loop was investigated in solution using tryptophan fluorescence. A mutant human PCAF HAT domain (PCAF(Wloop)) was created in which the natural tryptophan (Trp-514) remote from the alpha5-beta6 loop was replaced with tyrosine and a glutamate within the loop (Glu-641) was substituted with tryptophan. This PCAF(Wloop) protein exhibited catalytic parameters within 3-fold of those of the wild-type PCAF catalytic domain, suggesting that the loop mutation was not deleterious for HAT activity. While saturating CoASH induced a 30% quenching of Trp fluorescence in PCAF(Wloop), binding of the high-affinity bisubstrate analogue H3-CoA-20 led to a 2-fold fluorescence increase. These different effects correlate with the different alpha5-beta6 loop conformations seen previously in X-ray structures. On the basis of stopped-flow fluorescence studies, binding of H3-CoA-20 to PCAF(Wloop) proceeds via a rapid association step followed by a slower conformational change involving loop movement. Time-resolved fluorescence measurements support a model in which the alpha5-beta6 loop in the H3-CoA-20-PCAF(Wloop) complex exists in a narrower ensemble of conformations compared to free PCAF(Wloop). The relevance of loop dynamics to PCAF/GCN5 catalysis and substrate specificity are discussed.

Publication types

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

MeSH terms

  • Animals
  • Catalytic Domain* / genetics
  • Cell Cycle Proteins / chemistry*
  • Cell Cycle Proteins / genetics
  • Coenzyme A / chemistry
  • Histone Acetyltransferases / chemistry*
  • Histone Acetyltransferases / genetics
  • Humans
  • Kinetics
  • Mutagenesis, Site-Directed
  • Nanotechnology
  • Protein Conformation
  • Sequence Homology, Amino Acid
  • Spectrometry, Fluorescence
  • Substrate Specificity / genetics
  • Tetrahymena
  • Transcription Factors / chemistry*
  • Transcription Factors / genetics
  • Tryptophan / genetics
  • p300-CBP Transcription Factors

Substances

  • Cell Cycle Proteins
  • Transcription Factors
  • Tryptophan
  • Histone Acetyltransferases
  • p300-CBP Transcription Factors
  • p300-CBP-associated factor
  • tGCN5 histone acetyltransferase
  • Coenzyme A