Atypical bZIP Domain of Viral Transcription Factor Contributes to Stability of Dimer Formation and Transcriptional Function

Abstract

The Epstein-Barr virus transcription factor Zta (encoded by BZLF1) is a bZIP protein containing an ␣-helical coiled-coil homodimerization motif (zipper). The Zta zipper forms less-stable dimers than other bZIP proteins, and an adjacent region (CT) interacts with the zipper to form a novel structure that is proposed to strengthen the dimer. Here we question the role of the CT region for Zta function. Cross-linking experiments demonstrate that the entire CT region lies adjacent to the zipper. Detailed analyses of Zta truncation mutations identify an involvement of the proximal CT region (221 to 230) in dimer formation with a further contribution from the distal region (236 to 243). Biophysical analyses reveal that residues 221 to 230 enhance the stability of the coiled coil. The ability of the Zta truncation mutants to interact with three Zta-binding sites also requires the proximal CT region. Fine mapping of DNA-binding requirements highlighted the contribution of these amino acids for Zta function. Thus, the proximal part of the CT region is required to aid the dimerization of Zta and thereby its DNA-binding ability. In contrast, although the distal part of the CT region aids dimerization, it promotes only a modest increase in DNA binding. To probe this further, we defined the contribution from the CT region for Zta to transactivate a promoter embedded within the viral genome. From this we conclude that the proximal part of the CT region is absolutely required, whereas the distal part is dispensable.

Efficient Induction of Nuclear Aggresomes by Specific Single Missense Mutations in the DNA-binding Domain of a Viral AP-1 Homolog

“…This unique feature of ZEBRA lends itself to domain swapping, a well described structural mechanism for protein aggregation in proteins that harbor two independently folded domains connected by a loop (54 -56). This C-terminal region of ZEBRA has been reported to interact with the zipper domain nearly as far as the basic domain (57). Perturbations in the stability of the DNA-binding domain as a result of amino acid substitutions may be transmitted to other regions of the ZEBRA homodimer, including the C-terminal regions.…”

Interaction of Epstein-Barr Virus BZLF1 C-Terminal Tail Structure and Core Zipper Is Required for DNA Replication but Not for Promoter Transactivation

“…1A and B). Chemical cross-linking in the C-terminal region of a purified fragment of BZLF1 expressed in Escherichia coli and biophysical analyses of synthetic peptides were consistent with the X-ray structure (9). Results from transcription reporter assays indicated that the coiled-coil region is absolutely required for transactivation (presumably because of the requirement for dimerization) but that the C-terminal tail is not essential for this process (9).…”

Functional Interaction between Epstein-Barr Virus Replication Protein Zta and Host DNA Damage Response Protein 53BP1

“…Vectors used for the in vitro translation of Zta and expression in eukaryotic cells have been described previously (16,44,51).…”

“…Subsequent studies to refine the region(s) of Zta required for the interaction with 53BP1 used a series of carboxyl-terminal termination mutants of Zta (Fig. 4) (44). These 35 S-labeled truncated proteins were generated using an in vitro translation system and were applied to the in vitro association assay with GST-tagged 53BP1-BRCT.…”

“…Zta has homology to the bZIP family of transcription factors whose general structure includes a transactivation domain and a bZIP domain consisting of a basic DNA contact region and a coiled-coil dimerization motif, termed a leucine zipper (24,49,50). Zta has a more complex dimerization domain than other bZIP family members, consisting of a dimeric leucine zipper entwined with an adjacent carboxyl-terminal region (35,38,44,50). Zta is multifunctional; through its basic region, it interacts with specific sequence DNA motifs (ZREs) that occur in the promoters of several viral and cellular genes (49) and in the viral origin of lytic replication (Ori-lyt) (46,47).…”