Structural and Functional Characterization of a Complex between the Acidic Transactivation Domain of EBNA2 and the Tfb1/p62 Subunit of TFIIH

Abstract

Infection with the Epstein-Barr virus (EBV) can lead to a number of human diseases including Hodgkin's and Burkitt's lymphomas. The development of these EBV-linked diseases is associated with the presence of nine viral latent proteins, including the nuclear antigen 2 (EBNA2). The EBNA2 protein plays a crucial role in EBV infection through its ability to activate transcription of both host and viral genes. As part of this function, EBNA2 associates with several host transcriptional regulatory proteins, including the Tfb1/p62 (yeast/human) subunit of the general transcription factor IIH (TFIIH) and the histone acetyltransferase CBP(CREB-binding protein)/p300, through interactions with its C-terminal transactivation domain (TAD). In this manuscript, we examine the interaction of the acidic TAD of EBNA2 (residues 431–487) with the Tfb1/p62 subunit of TFIIH and CBP/p300 using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimeter (ITC) and transactivation studies in yeast. NMR studies show that the TAD of EBNA2 binds to the pleckstrin homology (PH) domain of Tfb1 (Tfb1PH) and that residues 448–471 (EBNA2 _448–471) are necessary and sufficient for this interaction. NMR structural characterization of a Tfb1PH-EBNA2 _448–471 complex demonstrates that the intrinsically disordered TAD of EBNA2 forms a 9-residue α-helix in complex with Tfb1PH. Within this helix, three hydrophobic amino acids (Trp458, Ile461 and Phe462) make a series of important interactions with Tfb1PH and their importance is validated in ITC and transactivation studies using mutants of EBNA2. In addition, NMR studies indicate that the same region of EBNA2 is also required for binding to the KIX domain of CBP/p300. This study provides an atomic level description of interactions involving the TAD of EBNA2 with target host proteins. In addition, comparison of the Tfb1PH-EBNA2 _448–471 complex with structures of the TAD of p53 and VP16 bound to Tfb1PH highlights the versatility of intrinsically disordered acidic TADs in recognizing common target host proteins.

Author Summary

Infection with the Epstein-Barr virus (EBV) is linked to a number of human diseases and the nuclear antigen EBNA2 is one of nine viral latent proteins that plays a key role in EBV-linked diseases. EBNA2 activates expression of both viral and host gene in part through interaction between its C-terminal acidic transactivation domain (TAD) and a number of host transcriptional regulatory proteins including the general transcription factor IIH (TFIIH) and the histone acetyltransferase CBP/p300. In this manuscript, we demonstrate that the TAD of EBNA2 binds to the pleckstrin homology (PH) domain from the Tfb1/p62 subunit of TFIIH and determine a three-dimensional structure of a complex between EBNA2 and Tfb1/p62. The structure shows that three hydrophobic residues from the TAD of EBNA2 make key interactions at the complex interface and these same residues also play an important role in the binding to CBP/p300. Comparison of the structure of the EBNA2-Tfb1 complex with complexes containing acidic TADs from other proteins (p53 and VP16) bound to the same Tfb1/p62 target highlights the inherent versatility of these intrinsically disordered domains and how minor variations in positioning of key hydrophobic residues allows them to bind to common targets using different functional interfaces.