Structural characterization of interactions between transactivation domain 1 of the p65 subunit of NF-κB and transcription regulatory factors.

p65 is a member of the NF-κB family of transcriptional regulatory proteins that functions as the activating component of the p65-p50 heterodimer. Through its acidic transactivation domain (TAD), p65 has the capacity to form interactions with several different transcriptional regulatory proteins, including TFIIB, TFIIH, CREB-binding protein (CBP)/p300 and TAFII31. Like other acidic TADs, the p65 TAD contains two subdomains (p65TA1 and p65TA2) that interact with different regulatory factors depending on the target gene. Despite its role in controlling numerous NF-κB target genes, there are no high-resolution structures of p65TA1 bound to a target transcriptional regulatory factor. In this work, we characterize the interaction of p65TA1 with two factors, the Tfb1/p62 subunit of TFIIH and the KIX domain of CBP. In these complexes, p65TA1 transitions into a helical conformation that includes its characteristic ΦXXΦΦ motif (Φ = hydrophobic amino acid). Structural and functional studies demonstrate that the two binding interfaces are primarily stabilized by three hydrophobic amino acids within the ΦXXΦΦ motif and these residues are also crucial to its ability to activate transcription. Taken together, the results provide an atomic level description of how p65TA1 is able to bind different transcriptional regulatory factors needed to activate NF-κB target genes.

A ΩXaV motif in the Rift Valley fever virus NSs protein is essential for degrading p62, forming nuclear filaments and virulence.

Rift Valley fever virus (RVFV) is a single-stranded RNA virus capable of inducing fatal hemorrhagic fever in humans. A key component of RVFV virulence is its ability to form nuclear filaments through interactions between the viral nonstructural protein NSs and the host general transcription factor TFIIH. Here, we identify an interaction between a ΩXaV motif in NSs and the p62 subunit of TFIIH. This motif in NSs is similar to ΩXaV motifs found in nucleotide excision repair (NER) factors and transcription factors known to interact with p62. Structural and biophysical studies demonstrate that NSs binds to p62 in a similar manner as these other factors. Functional studies in RVFV-infected cells show that the ΩXaV motif is required for both nuclear filament formation and degradation of p62. Consistent with the fact that the RVFV can be distinguished from other Bunyaviridae-family viruses due to its ability to form nuclear filaments in infected cells, the motif is absent in the NSs proteins of other Bunyaviridae-family viruses. Taken together, our studies demonstrate that p62 binding to NSs through the ΩXaV motif is essential for degrading p62, forming nuclear filaments and enhancing RVFV virulence. In addition, these results show how the RVFV incorporates a simple motif into the NSs protein that enables it to functionally mimic host cell proteins that bind the p62 subunit of TFIIH.

Structural and functional characterization of a complex between the acidic transactivation domain of EBNA2 and the Tfb1/p62 subunit of TFIIH.

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 (EBNA2448₋471) are necessary and sufficient for this interaction. NMR structural characterization of a Tfb1PH-EBNA2448₋471 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-EBNA2448₋471 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.