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 structure of P/CAF complexed with an acetylated Tat peptide, here we use molecular dynamics simulations to construct a model describing the interaction between full length Tat and the P/CAF bromodomain. Our calculations show that the protein-protein interface involves hydrophobic interactions between the P/CAF ZA 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 binding. 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 bromodomain recognition.
2005 Wiley-Liss, Inc.