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, 41 (7), 4295-306

The Crystal Structure of the DNA-binding Domain of vIRF-1 From the Oncogenic KSHV Reveals a Conserved Fold for DNA Binding and Reinforces Its Role as a Transcription Factor

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The Crystal Structure of the DNA-binding Domain of vIRF-1 From the Oncogenic KSHV Reveals a Conserved Fold for DNA Binding and Reinforces Its Role as a Transcription Factor

Kelly Hew et al. Nucleic Acids Res.

Abstract

Kaposi's sarcoma-associated herpesvirus encodes four viral homologues to cellular interferon regulatory factors (IRFs), where the most studied is vIRF-1. Even though vIRF-1 shows sequence homology to the N-terminal DNA-binding domain (DBD) of human IRFs, a specific role for this domain in vIRF-1's function has remained uncertain. To provide insights into the function of the vIRF-1 DBD, we have determined the crystal structure of it in complex with DNA and in its apo-form. Using a thermal stability shift assay (TSSA), we show that the vIRF-1 DBD binds DNA, whereas full-length vIRF-1 does not, suggesting a cis-acting regulatory mechanism in similarity to human IRFs. The complex structure of vIRF-1 DBD reveals interactions with the DNA backbone and the positioning of two arginines for specific recognition in the major grove. A superimposition with human IRF-3 reveals a similar positioning of the two specificity-determining arginines, and additional TSSAs indicate binding of vIRF-1 to an IRF-3 operator consensus sequence. The results from this study, therefore, provide support that vIRF-1 has evolved to bind DNA and plays a role in DNA binding in the context of transcriptional regulation and might act on some of the many operator sequences controlled by human IRF-3.

Figures

Figure 1.
Figure 1.
(a) The predicted domain boundaries of vIRF-1: DBD and the IAD (30). Below are the different domain boundary truncations of our four vIRF-1 protein constructs. (b) A TSSA was performed for all the four vIRF-1 protein constructs with several dsDNA, including the PRDIII-I sequence from the human IRF operator (PRDIII-I), a two times repeat of S0 (S0_x2), two sequences from the viral operator region that contains sequences that are highly similar to S0 (v1 and v2) and a random DNA sequence that contains 25 bp (N25). The TSSA shows that only vIRF-188–196 is stabilized by the various dsDNA. (c) A dose–response experiment results of vIRF-188–196 with varying concentrations of the aforementioned dsDNA. A dose–responsive stabilization of vIRF-188–196 by the different dsDNA was observed.
Figure 2.
Figure 2.
The crystal structure of vIRF-1 DBD in complex with DNA. (a) The asymmetric unit of the complex structure is made up of two vIRF-1 DBDs (vIRF-188–196), namely, vIRF-1A (orange) and vIRF-1B (green), and two dsS0mm stacked on top of each other. Each dsS0mm is made up of two molecules of ssS0 (strands C and D) that form a distorted double-stranded DNA with two mismatching G–A base pairs in the center. Both vIRF-1A and vIRF-1B bind to the major grooves on the opposite sides of the DNA duplex (gray). vIRF-1A binds to the major groove of one DNA duplex, whereas vIRF-1B binds to the major groove that is created by the imperfect stacking of two DNA duplexes. The individual DNA bases that interact with vIRF-1A and vIRF-1B are colored light orange and light green, respectively. (b) Both vIRF-1A and vIRF-1B are essentially the same. The DBD is made up of three α-helices (α1–α3), four β-sheets (β1–β4) and three long loops (L1–L3). It forms the typical winged HTH motif.
Figure 3.
Figure 3.
An illustration of the interactions between the vIRF-1 DBD and the DNA duplex. (a) The DNA bases are labeled (guanine = G; adenine = A; cytosine = C; and thymine = T) and numbered according to their sequence from the 5′- to 3′-end. The DNA bases that are labeled with an asterisk are bases from the neighboring asymmetrical unit that are included in the complex structure. The pentose sugars of these bases are colored orange. The black and the red dotted lines represent the interactions between the respective amino acids with the DNA phosphate backbone and the DNA bases. The interacting amino acids from vIRF-1A and vIRF-1B are colored orange and green, respectively. The DNA nucleotides that interact with vIRF-1A and vIRF-1B are also colored orange and green, respectively. (b) Arg 163 and Arg 164 from vIRF-1A and (c) Arg 163 and Arg 171 from vIRF-1B are the only amino acids that were found to interact with the DNA bases through hydrogen bonds (blue dotted lines). (d) A superimposition of vIRF-1A and vIRF-1B is shown to illustrate the difference in the arrangement of Arg 163 and Arg 171 in both molecules.
Figure 4.
Figure 4.
The superimpositions of the vIRF-1 DBDs. (a) The superimpositions of the four molecules of the apo-vIRF-1 DBD found in an asymmetrical unit of the crystal structure. (b) The superimposition of the DNA bound vIRF-1 (orange) and the apo vIRF-1 DBD (red). Both the DNA bound form and the apo-vIRF-1 DBD are similar, except at the L2.
Figure 5.
Figure 5.
Comparison of the vIRF-1 DBD with the human IRFs DBD. (a) The sequence alignment of the DBDs of vIRF-1 and the human IRF-1, -2, -3, -4 and -7. The completely conserved amino acids are highlighted in red, and the similar amino acids are red. The light blue boxes indicate the amino acids that take part in the direct and water-mediated DNA interactions. (b) The DBD of the human IRF-1 (green), IRF-2 (yellow), IRF-3 (blue), IRF-4 (cyan), IRF-7 (orange) and vIRF-1 (red) was superimposed. The amino acid residues that were found to interact with the DNA base(s) as marked in the sequence alignment are shown as sticks and are colored accordingly. Most of the protein residues that participate in the protein–DNA interactions are located on the recognition helix (α3). (c) The superimposition of IRF-3 and vIRF-1B in complex with DNA. The specificity determining arginines of both IRF-3 (blue) and vIRF-1B (red) are shown in sticks and are colored accordingly.

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