Insights on HIV-1 Tat: P/CAF bromodomain molecular recognition from in vivo experiments and molecular dynamics simulations

ABSTRACT Structural and functional studies indicate that, through its bromodomain, the cellular acetyltransferase P/CAF binds the acetylated Tat protein of human immunodeficiency virus type 1 (HIV-1) and promotes transcriptional activation of the integrated provirus. Based on the NMR struc-ture of P/CAF complexed with an acetylated Tat peptide, here we use molecular dynamics simula-tions to construct a model describing the interac-tion between full lengthTat and theP/CAFbromodo-main. Our calculations show that the protein– protein interface involves hydrophobic interactions between theP/CAFZA loop and the Tat core domain. In particular, tyrosines 760 and 761 of P/CAF, two residues that are highly conserved in most known bromodomains, play an essential role for the bind-ing. Fluorescence resonance energy transfer (FRET) experiments performed in this work demonstrate that P/CAF proteins in which these tyrosines are mutated into hydrophilic residues neither bind to Tat inside the cells nor mediate Tat transactivation. The combination of theoretical and in vivo studies provides new insights into the specificity of bromo-domain recognition. Proteins 2006;62:1062–1073