Fluorescence analysis of a dynamic loop in the PCAF/GCN5 histone acetyltransferase.
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.
Zheng
Yujun
Y
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Mamdani
Fatemah
F
Toptygin
Dimitri
D
Brand
Ludwig
L
Stivers
James T
JT
Cole
Philip A
PA
D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics; D003065:Coenzyme A / Q000737:chemistry; D051548:Histone Acetyltransferases / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D036103:Nanotechnology; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity / Q000235:genetics; D013768:Tetrahymena; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics; D014364:Tryptophan / Q000235:genetics; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor
D018797:Cell Cycle Proteins; D003065:Coenzyme A; D051548:Histone Acetyltransferases; D014157:Transcription Factors; D014364:Tryptophan; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor; C120128:tGCN5 histone acetyltransferase