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pISSN 2288-6982 l eISSN 2288-7105 Biodesign MINI REVIEW P 143-153 Structure and function of Zalpha, a Z conformation-specific nucleic acid binding domain Xu Zheng, Chan Yang Park, So-Young Park, Jinhyuk Choi and Yang-Gyun Kim* Department of Chemistry, Sungkyunkwan University, Suwon 16419, Korea. *Correspondence: [email protected] Right-handed B-DNA is a predominant form of natural DNA and used for the structural support of genetic information. Nevertheless, DNA can assume many different shapes. Left-handed Z-DNA is one of well-known non-B-DNA structures. An increasing number of studies show direct connections between Z-DNA and its potent roles in biological processes. The discovery of the proteins with Z-DNA binding domain(s), Zα, provides new opportunities to elucidate the relevant biological function of Z-DNA in biological systems. Thus, a great deal of recent researches has focused on questions surrounding Z-DNA and its binding proteins in vivo. In this review, we compare structures of Zα domains from various Z-DNA binding proteins and discuss their conformational specificity adapted from the common fold that prevalently used by many B-DNA binding proteins. In addition, we summarize recent developments regarding the biological roles of Z-DNA and Z-DNA binding proteins. INTRODUCTION The most common conformation of double-stranded DNA (dsDNA) in biological systems is right-handed B-DNA with Watson and Crick base pairings. However, flexibility of the phosphodiester bond in DNA (and RNA) structure allows diverse non-B-DNA conformations of such as Z-DNA, four-stranded cruciform DNA and G-quadruplex, usually occurring in certain conditions (Choi and Majima, 2011). The first indication of the presence of left-handed DNA conformation came from the optical studies of poly d(CG) in high salt concentration (Pohl and Jovin, 1972). Before long, the first X-ray crystal structure of the left-handed dsDNA, named as Z-DNA for their zigzag phosphodiester backbone was reported by Rich group (Wang et al., 1979). Besides its handedness, Z-DNA has distinctive structural characteristics from those of B-DNA: in contrast to B-DNA, where all bases are in anti conformation, the bases in Z-DNA adopt alternate syn (purines) and anti (pyrimidines) conformations which produce the zigzag backbone pathway (Wang et al., 1979). Sequences consisting of alternating purines and pyrimidines (APPs) energetically favor Z-DNA conformation. Among them, the alternating dG and dC is the most favorable sequence to form stable Z conformation (Rich et al., 1984). Other sequences including alternating d(CA/TG) and even nonAPPs can also adopt Z conformation (Eichman et al., 1999). Positively charged molecules and cations such as spermidine and cobalt hexamine can greatly improve stability of dsDNA in Z conformation (Rich et al., 1984). In addition, chemical modifications on bases such as methylation or bromination of cytosines stabilize Z-DNA (Rich et al., 1984). bdjn.org Since its discovery, biochemical and biophysical properties of Z-DNA have been intensively studied in vitro as well as in vivo (Rich and Zhang, 2003). Although earlier studies on Z-DNA were not quite successful to pin down its role in vivo, those works clearly provide evidences for the formation of Z-DNA in vitro and in vivo. In addition to its unusual structural aspect, the existence of Z-DNA in vivo raises biologically intriguing questions. It was suggested that Z-DNA preferring sequences near promoter region could function as cis-elements in transcription (Schroth et al., 1992). Indeed, negative supercoiling produced by the movement of RNA polymerase during the transcription can stabilize Z-DNA conformation at transcription start sites (Liu and Wang, 1987; Singleton et al., 1982). Moreover, it has been shown that Z-DNA formation is strongly related to transcription and chromatin remodeling (Kim et al., 2010; Liu et al., 2001; Rothenburg et al., 2001; Wittig et al., 1992). In genomic DNA, Z-DNA has also been shown to function as a nucleosomal boundary element (Wong et al., 2007), and to induce genomic instability (Wang et al., 2006; Wang and Vasquez, 2006). Although having accepted the existence of left-handed Z-DNA, the biological function of Z-DNA has not been completely established yet. Since the discovery of Z-DNA, the presence of Z-DNA binding proteins (ZBPs) in cells had long been hypothesized and identification of such proteins could provide a pivotal lead in understanding of the biological function of Z-DNA. The specific antibody against Z-DNA was firstly found in human autoimmune disease since Z-DNA is antigenic unlike B-DNA (Nordheim et al., 1981). This antibody was used as a useful tool to probe the Z-DNA in vivo (Lancillotti et al., 1987; Biodesign l Vol.3 l No.4 l Dec 30, 2015 © 2015 Biodesign 143 Structure and function of Zalpha, a Z conformation-specific nucleic acid binding domain Nordheim et al., 1981). Ultimately, a family of true cellular ZBPs has been found and they subsequently facilitate Z-DNA study. Double-stranded RNA adenosine deaminase 1 (ADAR1) was the first identified ZBP that has Zα, a Z-DNA binding domain (ZBD) (Herbert et al., 1997). Several other cellular and viral proteins including DNA-dependent activator of interferon (IFN) regulatory factor (DAI, also known as DLM-1 or ZBP1) (Schwartz et al., 2001), protein kinase containing Z-DNA binding domain (PKZ) in fish (Rothenburg et al., 2005), viral Z-DNA binding protein E3L in poxvirus family (Herbert et al., 1997) and the recently added ORF112 from cyprinid herpesvirus 3 in fish (Tome et al., 2013) have been identified (Figure 1). Intriguingly, all identified proteins of this ZBP family, probably including ORF112 of fish virus, are somehow closely involved in the innate immune response. For example, ADAR1 belongs to an RNA editing enzyme that modifies dsRNA (cellular and viral RNA nucleotides) via deamination of adenosine to produce inosine, and it acts as a regulator of antiviral responses and is suggested to be involved in RNA interference (RNAi) pathway (Athanasiadis, 2012). However, the exact role of the Zα domain is yet to be clearly elucidated. Biochemical and biophysical studies on Zα domains have yielded crucial information for understanding their conformationspecific binding to Z-DNA. Most of all, all solved Zα domains share a common fold of the winged helix-turn-helix (wHTH) motif (Schwartz et al., 1999). Remarkably, this folding motif is also widely used by many B-DNA binding proteins. This finding implies that the same protein folding can be evolved to two classes of DNA binding proteins with completely opposite conformation specificities. To understand how ZBDs achieve conformational specificity, more research is needed on structure and function relationship of ZBDs. Lastly, competent ability of ZBDs for specific binding to Z conformation of nucleic acids could broaden its use in the fields of biology and biotechnology. Z CONFORMATION-SPECIFIC BINDING PROTEINS The first firmly-established cellular ZBP is ADAR1, which was initially extracted by a Z-DNA affinity column with a 5-bromodeoxycytosine-modified probe from the chicken blood nuclei (Herbert et al., 1995). The following work confirmed that a ZBD, referred as to Zα, is located in the N-terminal region of human ADAR1 (Herbert et al., 1997). Thus far, several cellular and viral proteins are identified to contain Zα domain(s), and certainly more proteins will be added in this list as genome sequence information from various organisms accumulates. Although homology of amino acid sequences is not high, critical residues involved in the Z-DNA binding and the Zα fold formation are highly conserved among Zα domains from various proteins and species (Figure 2). ADAR1 belongs to a family of enzymes that modify cellular and viral dsRNA by deamination of adenosine to yield inosine, which is decoded as guanosine instead by ribosomes (Polson and Bass, 1994). This base modification process can alter the genetic information of mRNA and ultimately the protein function. ADAR1 comprises of two tandem Zα domains at its N terminus denoted as Zα (ZαADAR1) and Zβ (ZβADAR1), respectively, three dsRNA binding domains (dsRBDs) in the middle and the C-terminal deaminase domain (Herbert et al., 1997). The first Zα domain has shown to bind left-handed Z-DNA with high affinity (Rich and Zhang, 2003). However, the second Zα domain (i.e., ZβADAR1) of human ADAR1 is not an effective ZBD (Kim et al., 2004). ADAR1 was suggested to be involved in host defense mechanisms, apoptosis and organogenesis, and in miRNA processing and RNAi mechanisms (Athanasiadis, 2012). Interestingly, as shown in Figure 1, ADAR1 exists in two protein isoforms in cells, the long form of ADAR1 (P150) and the short form of ADAR1 (P110), which differ by the extended N-terminal region that includes the first Zα domain, ZαADAR1 (Patterson and Samuel, 1995). The FIGURE 1 I Schematic representation of domain organization of Zα family proteins. Two protein isoforms of ADAR1 are shown as P150 (long) and P110 (short). Two of Z-DNA binding domains and IFN-inducible P150 shows both nuclear double-stranded RNA binding domains are shown as Zα, Zβ and dsRBDs, respectively. The third and cytoplasmic localization while the DNA-binding domain and the receptor interacting protein kinase homotypic interaction motifs in constitutively-expressed P110 exists DAI protein are represented by D3 and RHIMs, respectively. The TBK1/IRF3-binding domain of DAI is indicated as TID. A eukaryote initiation factor 2α kinase domain (eIF2α kinase) presents in the predominantly in nucleus (George et al., C-terminal of both PKZ/PKR while two tandem Zα domains for PKZ and two dsRBDs for PKR are 2011). located in their N terminus, respectively. Vaccinia virus protein E3L has an N-terminal Zα domain DNA-dependent activator of IFN and a C-terminal dsRBD. ORF112 from Cyprinid Herpesvirus 3 (CyHC3) has a single Zα domain. Its hypothetical N-terminal region (dotted box) does not contain any homology to known proteins and regulatory factor (DAI, also known as its expression is not also confirmed (h: human, ca: carassius auratus, dr: danio rerio, vv: vaccinia DLM-1 and ZBP1) was initially identified virus). 144 Biodesign l Vol.3 l No.4 l Dec 30, 2015 © 2015 Biodesign bdjn.org Xu Zheng, Chan Yang Park, So-Young Park, Jinhyuk Choi and Yang-Gyun Kim FIGURE 2 I Amino acid sequence alignment of Zα domains from Zα family proteins. The amino acid sequences of Zα domains are shown underneath the secondary structure diagram deduced from the co-crystal structures of human ZαADAR1 (Schwartz et al., 1999). Residues interacting with Z-DNA (blue triangles) and residues important for the protein fold (red dots) are indicated. Three residues (bold), Asn173, Tyr177, and Trp195 in hZαADAR1, central to interaction with Z-DNA are completely conserved within the Zα family except hZβADAR1, which is not a Z-DNA binder but has the Zα fold. The amino acid numbering at the beginning and the end of each sequence is indicated. The GenBank accession numbers for the various sequences are as follows: ADAR1, AAB06697.1 (Homo sapiens); DAI/DLM-1/ZBP1, NP_001153891 (Homo sapiens) and NP_067369 (Mus musculus); PKZ, AAP49830 (Carassious auratus) and CAH68533 (Danio rerio); E3L, AAA02759 (vaccinia virus) and NP_073419 (yaba-like disease virus); CyHC3 ORF112, (Cyprinid Herpesvirus 3). as an IFN- γ -inducible protein (Fu et al., 1999) and functions as a cytosolic foreign dsDNA sensor and activator of innate immune responses (Takaoka et al., 2007). DAI also contains two ZBDs (ZαDAI and ZβDAI) like ADAR1, a DNA binding domain (D3) in the middle and an additional C-terminal domain whose function has not been clearly known (Figure 1). It was proposed that DAI binds cytosolic viral DNA by use of the D3 domain for initial binding and then the ZBDs for a tighter DNA-DAI complex (Wang et al., 2008). In addition, it was shown that the receptorinteracting protein homotypic interaction motifs (RHIMs) present in the middle of DAI mediate DAI-induced NF-κB signals through interacting with two RHIM-containing kinases − RIP1 and RIP3 (Kaiser et al., 2008; Rebsamen et al., 2009). PKZ (protein kinases containing ZBDs) is described as a functional ortholog of protein kinase R (PKR) in fish (Rothenburg et al., 2005). Unlike PKR, PKZ has two ZBDs (ZαPKZ and ZβPKZ) instead of two dsRBDs at the N terminus but shares a eukaryote translation initiation factor 2α (eIF2α) kinase domain at the C terminus (Figure 1). As PKR plays an important role in the innate immune response by recognizing infecting viral dsRNA in cytosol (Garcia et al., 2007), it is suggested that the phosphorylation of eIF2α by PKZ is regulated via specific Z-DNA binding (Bergan et al., 2008). Vaccinia virus (VV) E3L protein is expressed early during infection and exists in both the nucleus and cytoplasm (Brandt and Jacobs, 2001). E3L possesses two domains; single Zα ZBD (vvZα E3L) at the N terminal, and a typical dsRBD at the C terminus. E3L is believed to play a key role in avoiding the IFN-mediated defense of host cells including inhibition of PKR activation (Brandt and Jacobs, 2001). The N-terminal Zα domain of E3L is also widely existed in many distantly related poxviruses such as variola virus, swinepox, Orf virus and yaba-like disease virus (Ha et al., 2004). VV E3L is needed for viral pathogenesis bdjn.org (Brandt and Jacobs, 2001). Notably, it was demonstrated that Z-DNA binding ability of vvZαE3L is the essential requirement for full viral pathogenesis (Kim et al., 2003). Despite of structural similarity among Zα domains existed in poxviruses, sequence identity is surprisingly low (Ha et al., 2004). Zα domains of E3L and its homologs in poxviruses show highly diverse array of amino acids especially in the N-terminal half, which may mirror subtly different functional roles (Ha et al., 2004). Lastly, a herpesvirus Zα-domain-containing protein (ORF112) from cyprinid herpesvirus 3 is the latest addition of Zα ZBD family. The X-ray crystal structure of free Zα domain of ORF112 (CyHCVZα revealed that it adopts a similar Zα fold found in other Zα domains from ADAR1, DAI, PKZ, and E3L, probably binding to Z-DNA in the same way as observed in other Zα/Z-DNA structures (Tome et al., 2013). It was suggested that this protein plays a role in the inhibition of IFN response by blocking antiviral host defense system, similarly to E3L of poxviruses (Tome et al., 2013). STRUCTURES OF Zα DOMAINS To date, several co-crystal structures of Zα ZBDs complexed with Z-DNA are available including Zα domain of human ADAR1 (hZα ADAR1) (Schwartz et al., 1999), Zα domain of mouse DAI (mZαDAI) (Schwartz et al., 2001), Zα domain of Yaba-like disease virus (yabZα E3L) (Ha et al., 2004), Zβ domain of human DAI (hZβDAI) (Ha et al., 2008), Zα domain of zebrafish PKZ (drZαPKZ) (de Rosa et al., 2013) and Zα domain of goldfish PKZ (caZαPKZ) (Kim et al., 2014) (Table 1). Structural study of Zα domains indisputably shows that they principally share a common fold, the wHTH DNA binding motif consisting of three α-helices and three β strands. Most of the observed interactions in Zα domains bound to Z-DNA occur through a common structurespecific recognition core within the binding domain which Biodesign l Vol.3 l No.4 l Dec 30, 2015 © 2015 Biodesign 145 Structure and function of Zalpha, a Z conformation-specific nucleic acid binding domain TABLE 1 I List of the solved Zα domain structures Protein Nucleic Acid Method* Notable Structural features Reference hZαADAR1 dsDNA Crystal (Schwartz et al., 1999) hZαADAR1 no Solution (Schade et al., 1999a) mZαDAI dsDNA Crystal (Schwartz et al., 2001) yabZαE3L dsDNA Crystal Some variation in hairpin region (Ha et al., 2004) vvZαE3L no Solution Y48 adopts two major rotamer positions in the unbound state (Kahmann et al., 2004) hZαADAR1 dsDNA Crystal Non-CG repeat DNAs (Ha et al., 2009) hZβADAR1 no Crystal The terminal helix α4 involved in metal binding and dimerization (Athanasiadis et al., 2005) hZαADAR1 dsDNA Crystal hZβDAI dsDNA Crystal hZαADAR1 dsDNA Solution hZαADAR1 dsDNA Crystal B-Z junction (Ha et al., 2005) hZαADAR1 dsDNA Crystal Z-Z junction (de Rosa et al., 2010) yabZαE3L dsDNA Solution hZβDAI dsDNA Solution Alterations in the helix α3, the β-wing, and Y145. (Kim et al., 2011) CyHCVZ no Crystal The terminal helix α4 involved in dimerization (Tome et al., 2013) drZαPKZ dsDNA Crystal (de Rosa et al., 2013) caZαPKZ dsDNA Crystal (Kim et al., 2014) hZαDAI no Solution (Yang et al., 2014) (Placido et al., 2007) The α3 recognition helix adopts a310 helix conformation. (Ha et al., 2008) (Kang et al., 2009) (Lee et al., 2010) *Solved either by X-ray crystallography (Crystal) or by NMR spectroscopy (Solution) is complementary to the backbone structure of Z-DNA. All observed structural characteristics point that Zα domains bind to Z-DNA in a conformation-specific manner. Possible deviation in DNA structure caused by different Z-forming sequences seems not interfering major interactions of Zα domain. Despite the close resemblance in the overall structure, a few distinctive variations are also observed in the Z-DNA binding modes and activities of some Zα domains as listed in Table 1. The structure of hZα ADAR1 was the firstly solved among Zα domains and its co-crystal structure was complexed with double-stranded dTd(CG)3 (Schwartz et al., 1999) (Figure 3A). This study showed the structural basis of how a Zα domain achieves specificity for the Z conformation. Two hZα ADAR1s bind symmetrically to each strand of the palindromic dsDNA substrate. However, they do not interact with each other. A hydrophobic core is stabilized by interdigitated interaction among aliphatic residues from the three α helices and strand β3 (Figure 2). The conserved hydrophobic residues are important to maintain this Zα fold. Direct and water-mediated contacts with phosphodiester backbones are made by helix α3 and β-wing (loop connecting β2 and β3). Interactions made by conserved amino acid residues from helix α3 and β-wing appear to be crucial for Z-DNA binding. Particularly, Lys169, Asn173, Tyr177 and Trp195 play key roles in recognizing Z-DNA backbone (Figure 3A). These complementary contacts between hZαADAR1 and the 146 Biodesign l Vol.3 l No.4 l Dec 30, 2015 © 2015 Biodesign zigzag phosphodiester backbone of Z-DNA are appropriate for delivering conformational specificity of hZαADAR1. In contrast, only a single base contact was observed between Tyr177 of helix α3 to guanine via CH-π interaction in the syn conformation, characteristic of Z-DNA (Figure 3A). This highly conserved tyrosine plays a pivotal role in binding and stabilizing Z-DNA. All these residues that participate Z-DNA recognition show a high degree of conservation throughout Zα domains and their importance for Z-DNA binding were confirmed by extensive mutational analysis (Ha et al., 2004; Kim et al., 2004; Kim et al., 2003; Quyen et al., 2007; Schade et al., 1999b). All vertebral proteins containing Zα domain have two tandem Zα domains (Figure 1). For the second Zα domain, generally referred as to Zβ, their structures are also available, either unbound or complexed form with Z-DNA (Table 1). Zβs also have very similar Z-DNA binding and protein folding, analogous to Zαs. One insightful example is hZβADAR1 lacking Z-DNA binding ability. In the crystal structure of free hZαADAR1, its overall fold closely resembles the co-crystal structure of hZαADAR1, with the exception of the missing tyrosine residue in helix α3 (Athanasiadis et al., 2005). The co-crystal structure of hZβDAI also demonstrates a highly conserved fold and Z-DNA interactions with minor differences (Ha et al., 2008). The solution structure of hZα ADAR1 complexed with Z-DNA confirmed that its global fold and conformational specific bdjn.org Xu Zheng, Chan Yang Park, So-Young Park, Jinhyuk Choi and Yang-Gyun Kim interaction are all consistent with those observed in co-crystal structure of hZα ADAR1 (Jeong et al., 2014; Kang et al., 2009). In addition, NMR study could provide uniquely informative observations about critical Z-DNA binding residues of hZαADAR1 from the crystal structure study (Jeong et al., 2014). Individual mutation of either Lys169, Asn173, or Tyr177 causes unusual A B conformational changes of hZαADAR1 resulting multiple loss of interactions with Z-DNA backbone and subsequently significant reduction of Z-DNA binding ability, while Trp195 only affects Z-DNA binding. As shown in Figure 3 and summarized in Table 1, there are small structural variations seen among the reported crystal and solution structures of Zα domains. One noticeable example is dimerization of some Zα domains. Unbound crystal structures of hZα ADAR1 (Athanasiadis et al., 2005) and the Zα domain of CyHV3 ORF112 (Tome et al., 2013) have the additional 4th α-helix at the C-terminal end, which are responsible for dimerization. This distinct structural feature has not been observed in other Zα domains. However, its biological relevance and functional significance have not been confirmed yet. Taken together, all structural and mutational studies indicate that Zα domain recognizes Z-DNA in a conformation-specific manner. Even free Zα domains have almost identical folding that is poised to bind Z-DNA. The most variable region of Z-DNA contacting amino acids is detected at β-wing. Mutational studies suggested that diversity of amino acid residues in the loop region of β-wing in poxvirus Zα domains influences on modulation of Z-DNA binding strength (Quyen et al., 2007). It was suggested that differences in the host cells of the various poxviruses may require differentiated Z-DNA binding ability. CONFORMATIONAL SPECIFICITY OF Zα BINDING Structures of various Zα domains complexed with Z-DNA provide much of information to significantly improve our understanding of their Z conformation specificity. However, even knowing these detailed interactions occurred at the Zα/Z-DNA interface may not TABLE 2 I Three-dimensional homology analysis of Zα domains comparing with hZαADAR1 (PDB ID: 1QBJ) FIGURE 3 I Co-crystal structures of Zα domains. (A) The basis of Zα/Z-DNA interactions is shown in the enlarged co-crystal structure of hZα ADAR1 protein and dTd(CG) 3 DNA duplex (PDB ID: 1QBJ). Helix α3 and β-wing regions of the hZα ADAR1 are shown as complexed to a fragment of Z-DNA. A number of backbone residues of hZα ADAR1 are shown to interact with left-handed Z-DNA by electrostatic and van der Waals interactions. Three residues, Asn173, Tyr177, and Trp195 in hZαADAR1, central to interaction with Z-DNA are emphasized by boxes. (B) Structural comparisons of co-crystal structures of other Zα domains by superimposing with helix α3. Protein structures of, mZαDAI (PDB ID: 1J75), yabZαE3L (PDB ID: 1SFU) and caZαPKZ (PDB ID: 4KMK) are drawn to show close resemblance of overall Zα/Z-DNA interactions among them. bdjn.org Protein PDB ID Z-SCORE RMSD (Å) Co-crystal hZαADAR1 2GXB 13.8 0.6 Z-RNA caZαPKZ 4KMK 12.9 0.9 Z-DNA yabZαE3L 1SFU 12.5 1.3 Z-DNA mZαDAI 1J75 10.8 1.1 Z-DNA hZβADAR1 1XMK 10.2 1.3 free hZβDAI 4KA4 9.9 1.6 Z-DNA CAP 1CGP 8.4 1.4 B-DNA H5 1HST 7.9 2.0 free vvZαE3L 1OYI 7.6 2.4 free HNF-3γ 1VTN 5.4 3.0 B-DNA The Z-score, calculated using the program DALI, describes the similarity between structures. Protein domains with Z-scores < 2.0 are structurally dissimilar. Full name or description of abbreviated names are as follows: catabolite activator protein (CAP); the globular domain of Chicken histon 5 (H5); hepatic nuclear factor 3gamma (HNF-3γ). Biodesign l Vol.3 l No.4 l Dec 30, 2015 © 2015 Biodesign 147 Structure and function of Zalpha, a Z conformation-specific nucleic acid binding domain A B FIGURE 4 I Schematic structures of DNA binding domains with the winged helix-turn-helix motif. (A) The structure superposition between hZαADAR1 (PDB ID: 1QBJ, in pink) and CAP (PDB ID: 1CGP, in yellow). (B) Schematics show the close structural similarity in the topology of Zα domains (Top) and right-handed B-DNA binding domains (Bottom) compared with hZαADAR1 (PDB ID: 1QBJ, boxed). The order of structures represents increasing structural deviations from left to right. All PDB IDs and RMSDs for the structures are listed in the Table 2. enough to clarify how ZBDs evolves as a Z conformation specific protein from a common wHTH folding motif that used by many B-DNA binding proteins (Figure 4). In structural comparison of proteins made of the wHTH folding motif, Z-score and RMSD show very close structural similarity even between Zα domains and B-DNA binding proteins (Table 2). For example, the DNA binding domain of catabolite gene activator protein (CAP), a typical B-DNA binder, is superimposed equally or better with the crystal structure of hZαADAR1 than those of hZβDAI or vvZαE3L, respectively (Figure 4A). This manifests that two different classes of DNA binding proteins use essentially the identical fold for binding to two radically different DNA conformations. Thus, small structural variation alone in wHTH folds observed in these structures is not sufficient to explain different conformational specificity of these proteins. What makes a Zα domain Z conformation specific while many other proteins use the same wHTH fold for B-DNA? In fact, mutational analysis can offer vital information for comprehending conformation-specific intermolecular interactions between ZBDs and Z-DNA. One clue came from the gain-of-function mutant of 148 Biodesign l Vol.3 l No.4 l Dec 30, 2015 © 2015 Biodesign hZβADAR1. From the amino acid sequence alignment and structure comparison, it is certain that hZβADAR1 belongs to Zα domain. Moreover, its overall fold is very similar to hZαADAR1 with a Z-score and RMSD of 10.2 and 1.3 Å (Table 3). hZβADAR1 has all critical amino acid residues for both Z-DNA binding and proper wHTH folding, except a key tyrosine residue in the α3 recognition helix (Athanasiadis et al., 2005). From the in vitro assays for Z-DNA binding, hZβ ADAR1 does not show Z-DNA binding and B-to-Z conversion activities (Kim et al., 2003). Since this key tyrosine residue in helix α3 is the only residue that mediates direct contacts to a base of the bound Z-DNA, it was postulated that no Z-DNA-binding activity of hZβADAR1 was attributed to the lack of the tyrosine. Remarkably, the single substitution of Ile335 to Tyr in helix α3 (the corresponding residue Tyr177 in the hZαADAR1) of hZβADAR1 is sufficient to obtain Z-DNA binding activity (Kim et al., 2003). The significance of this tyrosine residue in helix α3 was reemphasized by the charateriztion of vvZα E3L . The Tyr48 residue in helix α3 of vvZαE3L in its unbound protein structure is not favorably prepositioned for Z-DNA binding as seen in the bdjn.org Xu Zheng, Chan Yang Park, So-Young Park, Jinhyuk Choi and Yang-Gyun Kim TABLE 3 I Summary of the suggested cellular functions of Zα family proteins Z-DNA binding proteins Reference ADAR1 ADAR1 responses often are proviral and antiapoptotic. (George et al., 2011) Genomic DNA segments probed for hZαADAR1 binding reveal an enrichment in centromeric repeats. (Li et al., 2009) hZαADAR1 binds specific regions of ribosomal RNA of ribosomes to inhibit translation. (Feng et al., 2011) hZαADAR1 is required for its localization to stress granules. (Ng et al., 2013) hZαADAR1 interacts with the oncogenic c-Myc promoter G-quadruplex. (Kang et al., 2014) Z-RNA binding by hZαADAR1 alters efficiency and pattern of A-to-I editing. (Koeris et al., 2005) DAI DAI requires three DNA-binding domains for full activation in vivo. (Wang et al., 2008) The Zα domain of DAI is responsible for localization of DAI to stress granules. (Deigendesch et al., 2006) PKZ Activity of PKZ depends on the presence of Z-DNA forming CpG dsDNA. (Bergan et al., 2008) E3L Z-DNA binding of vvZαE3L is essential for vaccinia virus pathogenesis. (Kim et al., 2003) vvZαE3L blocks a part of the interferon signaling cascade. (Langland et al., 2006) Z-DNA binding of vvZαE3L is required for gene transactivation and antiapoptotic activity. (Kwon and Rich, 2005) vvZαE3L is required for PKR inhibition, but in a Z-DNA binding independent manner. (Thakur et al., 2014) hZαADAR1, which explains molecular basis for no B-to-Z conversion activity of vvZαE3L (Kahmann et al., 2004). The assay based on 1:1 B-Z equilibrium promoted by cobalt hexamine conclusively demonstrated that vvZαE3L indeed binds to preformed Z-DNA in vitro while hZβADAR1 does not (Kim et al., 2004). Further the in vivo Z-DNA binding assay using the modified yeast one-hybrid system confirmed that vvZαE3L binds Z-DNA in vivo (Kim et al., 2004). These results concluded that vvZαE3L can bind to Z-DNA conformation specifically despite of its no B-to-Z conversion activity while hZβADAR1 is truly not a Z-DNA binder. Comparison of structure and activity of vvZαE3L and hZβADAR1 with hZαADAR1 leads to important conclusion about the role of the properly positioned tyrosine that is decisive for the conformational specificity of Zα domains. In the crystal structures of the Zα domains bound to Z-DNA, it is clear that interactions of Zα domains with the left-handed phosphodiester backbone adopt essentially the same mode and no indication of base specific interaction (Figure 3). These observations suggested that their recognition of Z-DNA is sequence-independent. Other experiments also proved that Zα domains do not discriminate sequences of Z-forming DNA segment. hZαADAR1 is seemingly a powerful Z-DNA binder, which is capable of inducing B-to-Z conversion of non-APP sequences as well as various APP sequences (Kim, 2007). Ultimately, crystallization of representative non-CG-repeat Z-DNAs in complex with hZαADAR1 conclusively demonstrated that Z-DNA bdjn.org recognition by hZα ADAR1 is conformation-specific rather than sequence-specific, displaying very limited structural deviation irrespective of the DNA sequence (Ha et al., 2009). Molecular dynamic simulation also supports that hZαADAR1 uses the same binding mode irrespective of Z-DNA sequences (Wang et al., 2014). Sequence-independence of Z-DNA binding by Zα domains suggests considering new view on their biological roles and substrates. Alternating CG residues of dsRNA can form lefthanded conformation similar to that of Z-DNA in high-ionicstrength solutions (Popenda et al., 2004). In addition, hZαADAR1 can stabilize Z-RNA in a physiological condition (Brown et al., 2000), and the crystal structure of hZαADAR1 complexed to a dUr(CG)3 showed that the binding of Z-RNA by hZαADAR1 is almost identical with that of Z-DNA (Placido et al., 2007). Thus, it confirmed that Zα domains are able to bind both dsRNA and dsDNA of the same sequence in a conformation-specific way. It is plausible that both Z-DNA and Z-RNA are equally suitable substrates for hZαADAR1 and possibly other Zα domains as well. Further, hZαADAR1 also stabilizes the Z conformation of DNA-RNA hybrid duplex (Bae et al., 2013; Kim et al., 2009) and its kinetic formation by hZαADAR1 was shown to be more rapid than dsDNA and dsRNA (Bae et al., 2013). Taken together, it is reasonable to consider that efforts to find in vivo substrates of Zα domains should extend to all Z conformers of nucleic acids. Biodesign l Vol.3 l No.4 l Dec 30, 2015 © 2015 Biodesign 149 Structure and function of Zalpha, a Z conformation-specific nucleic acid binding domain BIOLOGICAL ROLES OF Zα DOMAINS AND Z-DNA BINDING PROTEINS The common function of Zα domains should interconnect with biological roles of ZBPs containing them. ZBPs have been known to be involved in cellar processes associated with innate immune response. Specifically, cellular proteins found in vertebrate, ADAR1, DAI and PKZ, play as a regulator in the antiviral responses while poxvirus E3L proteins and possibly CyHCV ORF112 act as a potent inhibitor of IFN response against host antiviral response. In regard to biological roles of Zα domains, Table 3 addresses reported biological functions of ZBPs associated with activity of Zα domain. Here, the possible roles of Zα domains that may include probable recognition of Z conformation of other nucleic acids will be briefly discussed. VV E3L is one of well-characterized dsRNA binding proteins and required for viral pathogenesis. Although only the C-terminal dsRBD domain is necessary for viral growth in cell culture, the whole protein containing both Zα and dsRBD domains are required for full pathogenesis in mice (Brandt and Jacobs, 2001). For viral pathogenesis in a mouse model, the Zα domain of E3L appears to be essential and modulating its Z-DNA binding activity greatly influences on efficacy of vaccinia virus (Kim et al., 2003). Mutations of vvZαE3L decreasing Z-DNA binding indeed correlate with decreases in viral pathogenicity. More importantly, Zα domains are functionally interchangeable for E3L protein. The replacement of vvZαE3L in E3L with other Zα domains or their mutants showed Z-DNA-binding dependent viral pathogenesis, as do analogous mutations in the wild-type E3L. It is possible that vvZαE3L may compete with host IFN-responsive proteins such as ADAR1 and DAI to inhibit its Z-DNA or Z conformer nucleic acids binding and subsequently the induction of immune response. Additionally, it was suggested that Z-DNA binding of vvZαE3L is crucial for the activity of VV E3L as a viral transactivator that inhibits apoptosis of host cells (Kwon and Rich, 2005). The Z-DNA or Z conformer nucleic acid binding by vvZαE3L for viral pathogenesis is seemingly persuasive based on domain swapping and mutations of key residues in the Zα domain of VV E3L. However, this explanation is not conclusive yet. The recent report showed that some key residues in vvZαE3L for viral pathogenesis may not be responsible for Z-DNA binding (Thakur et al., 2014). Further study will be necessary to resolve these somewhat conflicting results. It has not been clear that the nature of the target nucleic acid substrate(s) of Zα domains for their biological functions, which remains to be mostly elusive. Biophysical studies strongly suggest that any Z conformer of dsDNA, dsRNA, or DNARNA hybrid could be a target substrate for Zα domains in vivo. Since dsRNA is a natural substrate for ADAR1, it is possible that Z-RNA formed in Z-forming sequences embedded in cellular or viral RNAs may act as a cis-factor to recruit ADAR1 through its Zα domain and guide it correctly to a target site for deamination. In fact, the previous report demonstrated that Z-RNA recognition by ADAR1 using its Zα domain to improve 150 Biodesign l Vol.3 l No.4 l Dec 30, 2015 © 2015 Biodesign its target site-selectivity in vitro (Koeris et al., 2005). Possible functional association of Zα domain with Z-RNA was also stated by other studies that showed Zα-domain dependent subcellular localization of DAI (Ng et al., 2013) and ADAR1 (Deigendesch et al., 2006) to stress granules. Z-RNA bound by Zα domain could be used to direct ZBPs in cytoplasmic stress granule in stress situation such as viral infection. Similarly the cross-linking experiment of hZαADAR1 with cellular RNAs demonstrated that it binds specific regions of ribosomal RNA of ribosomes (Feng et al., 2011). ADAR1-bound mRNAs may be specifically inhibited by interference of translational activity caused by Zα associating to the ribosome. It is also plausible that the localization of DAI in stress granules may correlate with stress granule formation promoted by accumulation of stalled translation preinitiation complexes (Deigendesch et al., 2006). In addition, occurrence of DNA-RNA hybrid abundantly takes place in both the nucleus and cytosol during cellular processes like transcription or virus infection. Since short stretches of Z-forming sequences are prevalent in human and viral genomes, it is quite feasible that DNA-RNA hybrids may be also one of the natural substrates for ZBPs. FUTURE PERSPECTIVES OF Z-DNA AND ITS BINDING PROTEIN STUDY It is conceivable that Z-DNA could be an interesting example for how nature utilizes dynamic, elusive conformation of biomolecules for beneficial cellular activities. Since its discovery decades ago, there has been skeptical about biological roles of Z-DNA while not rejecting its existence in vivo. For researchers, Z-DNA is a difficult biological target to study for getting reliable and informative outcome. However, owing to slow but steady progress on Z-DNA study, the broad outline of biological roles of Z-DNA has finally begun to emerge. Z-DNA is a short-lived structure embedded in a massive amount of B-DNA matrix and occasionally formed and disappeared quickly during biological processes such as transcription. This is why the finding of ZBPs is particularly important and exciting. The Zα domain itself is both a subject of study and a tool for Z-DNA study at the same time. Most studies indicate that cellular ZBPs function in innate immune response and viral ZBPs play a role in blocking antiviral host response, possibly competing with cellular counterparts (Athanasiadis, 2012). However, the detailed functions of Zα domains present in various ZBPs are not clearly understood yet. Also biologically relevant targets (or substrates) of the Zα domains has not been determined yet. Recent results suggest that biological targets of Zα domains may be more diverse than considered previously (Ng et al., 2013). For example, hZαADAR1 recognizes non-Z-DNA conformer such as G-qudruplex (Kang et al., 2014) although it may share some structural characteristics with Z-DNA such as the exposed sugar-phosphate backbone. Besides its biological importance, Z conformation-specific binding ability of Zα domain makes it an interesting object of study. First of all, Zα domain is a promising tool to detect and bdjn.org Xu Zheng, Chan Yang Park, So-Young Park, Jinhyuk Choi and Yang-Gyun Kim capture Z-DNA in vivo, which has been successfully applied for the direct detection of Z-DNA formation during the chromatin remodeling (Liu et al., 2001) and probing Z-DNA segments in genomic DNA (Li et al., 2009). Another important use of Zα domains would be a probing tool to explore how B-to-Z conversion occurs. Recent studies from NMR and single molecule spectroscopy has added interesting clues for this question. However, conformational selection mechanism by Zα domain suggested by the single molecule study (Bae et al., 2010) is contradictory to the conclusion from the NMR study (Kang et al., 2009). To clarify these somewhat opposing results, it should be necessary to use a carefully designed experiment to examine both mechanisms in the same experimental condition. Finally, in protein engineering and synthetic biology, Zα domain could be valuable for understanding evolutionary strategy for creating conformation-specific DNA binding proteins. The intriguing structural adaptation of Zα domain for conformational specificity raises a question that how nature uses the same folding motif to recognize two opposite conformations of dsDNA. The conversion of a Z-DNA binding protein to a B-DNA binding protein or the other way round will provide new insight and venue for protein engineering. In addition, the dynamic and transient nature of Z-DNA formation would be suitable for a regulatory module to instruct momentary and timely controls over biological processes such as gene expression and genetic control networks. domains of the vaccinia virus interferon resistance gene, E3L, are required for pathogenesis in a mouse model. J Virol 75, 850-856. ACKNOWLEDGEMENTS Ha, S.C., Kim, D., Hwang, H.Y., Rich, A., Kim, Y.G., and Kim, K.K. (2008). The crystal structure of the second Z-DNA binding domain of human DAI (ZBP1) in complex with Z-DNA reveals an unusual binding mode to Z-DNA. Proc Natl Acad Sci USA 105, 20671-20676. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (2015R1A2A2A01008367). AUTHOR INFORMATION The Authors declare no potential conflicts of interest. Brown, B.A., 2nd, Lowenhaupt, K., Wilbert, C.M., Hanlon, E.B., and Rich, A. (2000). The Zα domain of the editing enzyme dsRNA adenosine deaminase binds left-handed Z-RNA as well as Z-DNA. Proc Natl Acad Sci USA 97, 13532-13536. Choi, J., and Majima, T. (2011). Conformational changes of non-B DNA. Chem Soc Rev 40, 5893-5909. de Rosa, M., de Sanctis, D., Rosario, A.L., Archer, M., Rich, A., Athanasiadis, A., and Carrondo, M.A. (2010). Crystal structure of a junction between two Z-DNA helices. 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