Significance: Infection with the Rift Valley fever virus (RVFV) has the capacity to cause fatal hemorrhagic fever in humans. A unique characteristic of RVFV infection is the presence of nuclear filaments whose formation is linked to synthesis of the viral NSs protein. We identify a crucial interaction between a ΩX a V motif present in the NSs protein and the p62 subunit of the host TFIIH. This interaction is required for nuclear filament formation, NSs-dependent degradation of p62 and for virulence. This ΩX a V motif is also found in human proteins that bind p62 and our results are an example of how viruses incorporate simple motifs into their protein sequences to mimic human proteins and enhance their functional capabilities in host cells during infections.

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 TAF_II31. Like other acidic TADs, the p65 TAD contains two subdomains (p65_TA1 and p65_TA2) 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 p65_TA1 bound to a target transcriptional regulatory factor. In this work, we characterize the interaction of p65_TA1 with two factors, the Tfb1/p62 subunit of TFIIH and the KIX domain of CBP. In these complexes, p65_TA1 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 p65_TA1 is able to bind different transcriptional regulatory factors needed to activate NF-κB target genes.

Erythroid Krüppel-like factor (EKLF) plays an important role in erythroid development by stimulating β-globin gene expression. We have examined the details by which the minimal transactivation domain (TAD) of EKLF (EKLFTAD) interacts with several transcriptional regulatory factors. We report that EKLFTAD displays homology to the p53TAD and, like the p53TAD, can be divided into two functional subdomains (EKLFTAD1 and EKLFTAD2). Based on sequence analysis, we found that EKLFTAD2 is conserved in KLF2, KLF4, KLF5, and KLF15. In addition, we demonstrate that EKLFTAD2 binds the amino-terminal PH domain of the Tfb1/p62 subunit of TFIIH (Tfb1PH/p62PH) and four domains of CREB-binding protein/p300. The solution structure of the EKLFTAD2/Tfb1PH complex indicates that EKLFTAD2 binds Tfb1PH in an extended conformation, which is in contrast to the α-helical conformation seen for p53TAD2 in complex with Tfb1PH. These studies provide detailed mechanistic information into EKLFTAD functions as well as insights into potential interactions of the TADs of other KLF proteins. In addition, they suggest that not only have acidic TADs evolved so that they bind using different conformations on a common target, but that transitioning from a disordered to a more ordered state is not a requirement for their ability to bind multiple partners.

The general transcription factor IIH is recruited to the transcription preinitiation complex through an interaction between its p62/Tfb1 subunit and the α-subunit of the general transcription factor IIE (TFIIEα). We have determined that the acidic carboxyl terminus of TFIIEα (TFIIEα _336–439 ) directly binds the amino-terminal PH domain of p62/Tfb1 with nanomolar affinity. NMR mapping and mutagenesis studies demonstrate that the TFIIEα binding site on p62/Tfb1 is identical to the binding site for the second transactivation domain of p53 (p53 TAD2). In addition, we demonstrate that TFIIEα _336–439 is capable of competing with p53 for a common binding site on p62/Tfb1 and that TFIIEα _336–439 and the diphosphorylated form (pS46/pT55) of p53 TAD2 have similar binding constants. NMR structural studies reveal that TFIIEα _336–439 contains a small domain (residues 395–433) folded in a novel ββααα topology. NMR mapping studies demonstrate that two unstructured regions (residues 377–393 and residues 433–439) located on either side of the folded domain appear to be required for TFIIEα _336–439 binding to p62/Tfb1 and that these two unstructured regions are held close to each other in three-dimensional space by the novel structured domain. We also demonstrate that, like p53, TFIIEα _336–439 can activate transcription in vivo . These results point to an important interplay between the general transcription factor TFIIEα and the tumor suppressor protein p53 in regulating transcriptional activation that may be modulated by the phosphorylation status of p53.

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. 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.