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

Yujun Zheng, Fatemah Mamdani, Dimitri Toptygin, Ludwig Brand, James T. Stivers, Philip A. Cole

Research output: Contribution to journalArticle

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 α5-β6 loop was investigated in solution using tryptophan fluorescence. A mutant human PCAF HAT domain (PCAFWloop) was created in which the natural tryptophan (Trp-514) remote from the α5-β6 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 PCAFWloop, binding of the high-affinity bisubstrate analogue H3-CoA-20 led to a 2-fold fluorescence increase. These different effects correlate with the different α5-β6 loop conformations seen previously in X-ray structures. On the basis of stopped-flow fluorescence studies, binding of H3-CoA-20 to PCAFWloop 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 α5- β6 loop in the H3-CoA-20-PCAFWloop complex exists in a narrower ensemble of conformations compared to free PCAFWloop. The relevance of loop dynamics to PCAF/GCN5 catalysis and substrate specificity are discussed.

Original languageEnglish (US)
Pages (from-to)10501-10509
Number of pages9
JournalBiochemistry
Volume44
Issue number31
DOIs
StatePublished - Aug 9 2005

ASJC Scopus subject areas

  • Biochemistry

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    Zheng, Y., Mamdani, F., Toptygin, D., Brand, L., Stivers, J. T., & Cole, P. A. (2005). Fluorescence analysis of a dynamic loop in the PCAF/GCN5 histone acetyltransferase. Biochemistry, 44(31), 10501-10509. https://doi.org/10.1021/bi050776i