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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).
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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;
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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).
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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
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(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
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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
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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
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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.
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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).
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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
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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
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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
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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.
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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
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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.
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Original Submission: Nov 14, 2015
Revised Version Received: Dec 2, 2015
Accepted: Dec 4, 2015
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