Molecular recognition of human CBP by retroviral transcriptional activators

Abstract

HIV-1 Tat is required for the expression of the viral genome. The coactivator and acetyltransferase CREB binding protein (CBP), and the paralog p300, are recruited to the HIV-1 promoter by Tat to aid viral expression. Here we identify the interacting domains of Tat and CBP. Circular dichroism and pulldown assays show that full-length Tat binds to the KIX domain of CBP, but not to the C/H1 or CR2 domains of CBP. Circular dichroism and NMR studies of Tat deletion mutants localize the KlX-binding domain of Tat to the N-terminal 24 residues of Tat. Transient cotransfections demonstrate that exogenous KIX behaves as a dominant negative to Tat-mediated transcription in human T-cells, suggesting that Tat and KIX interact in vivo. These findings indicate that Tat targets the KIX domain of CBP and provide insight into the molecular interactions involved in regulating HIV-1 gene expression. Chemical-shift perturbation mapping with heteronuclear nuclear magnetic resonance spectroscopy was used to identify the surface of human KIX that interacts with Tat. It was found that that Tat binds to the c-Jun/MLL binding surface of KIX, as opposed to the CREB binding site. The results provide new insight into the molecular basis of the assembly of protein complexes involving p300/CBP and Tat during HIV gene expression. The HTLV-1 transcription activator Tax is required for viral replication and pathogenesis. In concert with human CREB, Tax recruits the human transcriptional coactivator and histone acetyltransferase p300/CBP to the HTLV-1 promoter. Here we investigate the structural features of the interaction between Tax and the KIX domain of human p300/CBP. Circular dichroism spectroscopy, nuclear magnetic resonance chemical-shift perturbation mapping and sedimentation equilibrium show that a subdomain of Tax (residues 59-98) binds KIX. Chemical-shift perturbation mapping reveals that the Tax-binding surface of KIX is distinct from that utilized by CREB, and corresponds to the site of KIX that interacts with MLL, c-Jun, and HIV-1 Tat. Sedimentation equilibrium shows that Tax and the phosphorylated KID domain of CREB can simultaneously bind KIX to form a ternary 1:1:1 complex. The results provide a molecular description of the concerted recruitment of p300/CBP via the KIX domain by Tax and phosphorylated CREB during Tax-mediated gene expression.