Epstein-Barr Virus ZEBRA Protein DNA Binding Domain

Alexander Oles '16 and Stephanie Penix '16


I. Introduction

The Epstein-Barr virus (EBV), which primarily infects B-lymphocyte and epithelial cells, can be found in the majority of the world's population. Although only a small percentage of an infected individual's cells contain the virus, EBV is linked to the development of cancerous tumors of Burkitt's, Hodgkin's, and post-transplantation lymphomas, and nasopharyngeal and gastric carcinomas, as well as infectious mononucleosis. Infection by EBV begins with a short replication phase. The virus remains in a latent phase, only entering the lytic phase in response to a cascade of transcriptional signals. These signals are triggered by the ZEBRA protein (BZFL1)along with Rta (BRLF1), two immediate-early transcription factors of the EBV, that activate eachother's promoters for lytic cycle intitiation, as well as their own. Click Here for ZEBRA and Zta model. The key structural feature of ZEBRA that allows this initial binding to occur is a b-ZIP domain, or basic leucine zipper. This domain forms a common DNA binding protein motif which enables the two protein subunits to stabilize each other via cooperative binding by forming van der Waals interactions between the hydrophobic side chains of the proteins. This gives rise to a very common structural feature of two coiled coils, which are held together by several relatively weak interactions, allowing for dissociation between the two side chains if necessary, hence the "zipper". However, unlike a typical leucine zipper, there are several stabilizing interactions that are formed with a C-terminal tail of the domain. The C-terminal tails of the ZEBRA DNA binding region give the possibility of inhibiting the EBV lytic cycle activation by blocking the dimerization and preventing the activation cascade entirely.  

II. General Structure

The ZEBRA protein homodimer consists of two 245-residue subunits. The activating region , the region which binds to DNA, of this homodimer is   ~ 60 residue located between residues 175-236 . The ZEBRA protein, unlike many b-ZIP motifs, lacks the heptad repeats of leucine which typically assist with the formation of a leucine zipper dimer. The b-ZIP region is also unstable with decreased DNA binding affinity at bodily conditions. To compensate for the this unfavorable binding, the proteins are partially stabilized by the cooperative binding of the b-ZIP helices that forms the coiled-coil structure of the b-ZIP motif via alternative interactions to the normal leucine van der Waals interactions .   This region of ZEBRA is further stabilized by the binding of the basic region of each subunit, from residues 175-200, to the DNA via interactions with the major groove and the basic side chains . The missing leucine heptads of the b-ZIP region is also accounted for with a C-terminal tail on the activating regions of the ZEBRA protein . This critical section consists of an antiparallel one-turn helix and a continuing C-terminal tail which is connected to the b-ZIP region by a hairpin turn of residues. This C-Terminal tail results in the formation of multiple stablizing interactions within the ZEBRA protein's structure. These four helices result in the structure which comprisies the activating region of the ZEBRA protein.

III. Protein-Protein Interactions

In many proteins with a conical b-ZIP motif, amino acid heptad repeats form the coiled-coil region. These heptads usually consist of a hydrophobic residue in the first residue position and a Leu in the fourth residue position, allowing for extensive van der Waals contacts. The ZEBRA protein has four of these heptad regions: 1, 2, 3, and 4 . However, the ZEBRA protein does not contain residues that follow this common motif in four consecutive 1st and 4th positions. Instead, it contains Tyr-200 (4th position), Ala-204 (1st position), Lys-207 (4th position), and Asn-211 (1st position) . The first heptad of this coiled-coil motif ranges from residues Leu-197 to Val 203. The Tyr-200 hydroxyl group forms a single H-bond with an amine of Arg-201, and there are cation-pi interactions between Arg-201 and the aromatic ring of Tyr-200 . This close contact is mediated by the hydrophobic interactions between the Ala-204 on each helix, reducing the distance between the helices to about 4.2 .  The second heptad, ranging from residues 204- 210, not only contains Ala-204, but also a critical salt bridge between the amino group of Lys-207 and the carboxylate of Asp-236 on the C-terminal tail, as well as some stabilizing contacts with Thre-234 and Ser-208 . The third heptad, reaching from Asn-211 to Leu-217, contains the highly conserved H-bond between the Arg-211 residues. This heptad of the ZEBRA binding domain also forms the previously mentioned hydrogen bond network which further connects the helices to the C-terminal tail, thus, increasing stabilization of the dimer. The network consists of helical residues Glu-210 (of heptad 2), Asn-211, Asp-212, and Arg-215, and tail residues Asp-228, Arg-233, and Thr-234 . The fourth heptad ranges from residues Leu-218 to Ser-224. It has the sulfur-containing residues Met-221 and Cys-222. However, the Cys-222 residues, at 3.86 apart, are just out of reach to form a disulfide bond . The coiled coil dimer is further stabilized by its tail when it forms a hydrophobic pocket with five residues: Leu-218 (heptad 4), Leu-225, Val-227, Ile-230, and Ile-231 for each of the subunits . The Leu-217 on the third heptad plays a critical role in this hydrophobic pocket in that it inserts itself inside of the pocket of the adjacent subunit, which results in stabilizing hydrophobic interactions . The residues Leu-214 of heptad 3 and Leu-218 and Met-221 of heptad four also interact with the rim of the adjacent subunit's hydrophobic pocket . The Leu-218, not only forms part of the hydrophobic pocket, but it is also involved in making contacts with the neighboring pocket. Even though the ZEBRA b-ZIP diverges from the generic b-ZIP binding patterns, it generates stable interactions between the primary alpha-helices and the C terminal tail.

IV. DNA Binding

The ZEBRA protein, which binds a variety of viral and cellular genes, is less sequence-specific in its DNA binding than other common bZIP family proteins, such as the Fos/Jun or C/EBP proteins. The ZEBRA protein can to bind to either the Fos/Jun or the C/EBP promoter site. This promiscuity is attributed in part to its alpha-helical fork formed by the junction of the helices in the DNA binding region at the dimerization surface. This promoter flexibility is further enhanced by Phe-193, which is a structural feature unique to ZEBRA, and it is capable of being in the trans or the gauche + conformation, with each Phe-193 in the dimer assuming the opposite position. In the trans conformation, the Phe-193 points to the adjacent helix, but in the gauche + conformation, the Phe-193 makes cation-pi interactions with the adjacent Lys-194 . The ZEBRA molecule in this tutorial is binding to an AP-1 site which consists of the conserved heptamer of TGA(G or C)TCA. The ZEBRA protein recognizes the binding site with some protein-base specific contacts and by tightly binding to the DNA backbone through various protein-DNA hydrogen bonds, water mediated hydrogen bonds and van der Waals contacts. Each subunit of the ZEBRA protein binds to five consecutive phosphates of DNA through Lys and Arg residues separated by one turn of the alpha helix. The hydrogen bonds to the phosphate back bone involve several residues: Arg-179, Lys-181, Arg-183, Arg-187, Lys-188, Ser-189, Arg-190, Lys-192, and Lys-194 . However, the amine group of Lys-190 makes a direct hydrogen bond with only one of the alpha helices. The ZEBRA protein also makes several water-mediated H-bonds with the DNA backbone as well as the DNA bases, but they have not been depicted in this model. For both subunits, residue Asn-182 binds to the 4th position N of the cytosine base (+2 of the central CG base pair) and the 4th position O in the thymine base (-3 of the central CG base pair) . This crystal structure shows a ZEBRA binding domain with mutations S186A and C189S in order to obtain a better crystal. However, the contacts that would have been illustrated in this model would be that Ser-186 makes a hydrogen bond to the O4 of thymine (+1), in contrast to this model that shows a hydrophobic interaction between the point mutation insertion of Ala-186 and the 5th position carbon of thymine (+1) . On one helix, Arg-190 forms a water mediated hydrogen bond to the 4th position N on cytosine (0), but on the other helix, Arg-190 forms hydrogen bonds to the 6th position O and the 7th position N on the Guanine of the promoter (0) . Along with these interactions, Ala-185 on both helices also makes van der Waals contacts with the 5th position C. Together, these few specific protein-base interactions and protein-backbone interactions allow for the less sequence specific b-ZIP ZEBRA protein to bind to DNA promoter sites and initiate the lytic cycle.

V. Pharmacological Applications

The EBV lytic cycle initiation begins a large cascade of events which can result in a detrimental disease. Since the ZEBRA protein is an immediate-early transcription factor, the cascade of events can be terminated by manipulating this protein. The structure of the ZEBRA protein is very unusual, but also offers some areas for pharmacological interference. The hydrophobic loop on the C-terminal tail can be exploited by insertion of a large hydrophobic drug which prevents homodimerization and, therefore, lytic cycle initiation. This drug could also have an increased binding affinity by altering the adjacent functional groups of the ZEBRA protein. The hydrophobic loop is large enough to accommodate the common antiviral drugs, so antiviral drugs can attack this b-ZIP but not cellular ones. This protein is an exception to the common b-ZIP motif, and, therefore, provides insight for other ways that proteins can bind to DNA. Research on these proteins provides insight for the development of cures and treatments towards such diseases.

VI. References

Chang, Y., Chang, S.S., Lee, H.H., Doong, S.H., Takada K., Tsai, C.H. 2004. Inhibition of the Epstein-Barr virus lytic cycle by Zta-targeted RNA interference. Journal of General Virology. 85:1371-1379. Web.

Miller, G., El-Guindy, A., Countryman, J., Ye, J., Gradoville, L. 2007. Lytic Cycle Switches of Oncogenic Human Gammaherpesviruses. Advances in Cancer Research. 97:81?109. Web.

Petosa, C., Morand, P., Baudin, F., Moulin, M., Artero, J.B., Muller, C.W. 2006. Structural basis of lytic cycle activation by the Epstein-Barr virus ZEBRA protein. Molecular Cell. 21: 565-572. Web.

Wolfgang, A., Farrell, P.J. 2005. Reactivation of Epstein-Barr virus from latency. Review in Medical Virology. 5: 149-156. Web.

Young, L.S., Rickinson, A.B. 2004. Epstein-Barr virus: 40 years on. Nature Reviews. 4:757-768. Web.

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