Download Structural Analyses of Zinc Finger Domains for Specific Interactions

J. Microbiol. Biotechnol. (2016), 26(12), 2019–2029
http://dx.doi.org/10.4014/jmb.1609.09021
Review
Research Article
jmb
Structural Analyses of Zinc Finger Domains for Specific Interactions
with DNA
Ki Seong Eom1, Jin Sung Cheong2,3, and Seung Jae Lee3*
1
Department of Neurosurgery, School of Medicine and Hospital, Wonkwang University, Iksan 54538, Republic of Korea
Department of Neurology, School of Medicine and Hospital, Wonkwang University, Iksan 54538, Republic of Korea
3
Department of Chemistry and Research Institute of Physic and Chemistry, Chonbuk National University, Jeonju 54896, Republic of Korea
2
Received: September 19, 2016
Revised: September 26, 2016
Accepted: September 28, 2016
First published online
October 6, 2016
*Corresponding author
Phone: +82-63-270-3412;
Fax: +82-63-260-3407;
E-mail: [email protected]
pISSN 1017-7825, eISSN 1738-8872
Copyright © 2016 by
The Korean Society for Microbiology
and Biotechnology
Zinc finger proteins are among the most extensively applied metalloproteins in the field of
biotechnology owing to their unique structural and functional aspects as transcriptional and
translational regulators. The classical zinc fingers are the largest family of zinc proteins and
they provide critical roles in physiological systems from prokaryotes to eukaryotes. Two
cysteine and two histidine residues (Cys2His2) coordinate to the zinc ion for the structural
functions to generate a ββα fold, and this secondary structure supports specific interactions
with their binding partners, including DNA, RNA, lipids, proteins, and small molecules. In
this account, the structural similarity and differences of well-known Cys 2His2-type zinc fingers
such as zinc interaction factor 268 (ZIF268), transcription factor IIIA (TFIIIA), GAGA, and Ros
will be explained. These proteins perform their specific roles in species from archaea to
eukaryotes and they show significant structural similarity; however, their aligned amino acids
present low sequence homology. These zinc finger proteins have different numbers of
domains for their structural roles to maintain biological progress through transcriptional
regulations from exogenous stresses. The superimposed structures of these finger domains
provide interesting details when these fingers are applied to specific gene binding and editing.
The structural information in this study will aid in the selection of unique types of zinc finger
applications in vivo and in vitro approaches, because biophysical backgrounds including
complex structures and binding affinities aid in the protein design area.
Keywords: Zinc finger proteins, metalloproteins, classical zinc finger, transcriptional regulator
Introduction
One of the most well-known groups of transcriptional
factors in eukaryotes is the zinc finger proteins, which
coordinate zinc ions to achieve a folded structure that is
crucial for interactions with its binding partners in
physiological systems [20, 34, 36, 38-40, 43, 48, 51, 68].
Biochemical and biophysical studies of these metalloproteins
have focused on eukaryotes owing to their critical functions
in signaling cascades. The growing evidence and genomic
advances proved that zinc fingers perform crucial roles in
maintaining physiological processes from prokaryotes to
eukaryotes, including the plant kingdom [2, 28, 31, 42, 52,
57, 68]. Early studies of zinc fingers have been limited to
DNA binding ability and its transcriptional regulation, but
recent applications of zinc fingers in various fields are of
major interest owing to their specific interactions for
cognate DNA, RNA, proteins, lipids, and small molecules
through hydrogen bonds and hydrophobic interactions [32,
43, 56, 68]. The binding affinities of zinc fingers to their
binding partners are significantly high, and preliminary
reports proved that these proteins bind to their specific
nucleic acids with sub-nanomolar ranges of dissociation
constants (Kd). The elaborate control of transcriptional
regulation is triggered by this tight binding, and gene
expression is up-regulated through this complex generation
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Eom et al.
[16, 29, 54].
Structural domains of zinc fingers have been applied to
gene-editing technologies to improve low specificity in vitro
and in vivo for the use of clinical trials in the last decade
[20, 48, 74]. This technology has received considerable
attention because zinc fingers can improve specific binding
abilities to target nucleic acids as a part of nucleases. Most
zinc finger proteins have more than one domain, and many
biophysical reports proved that one zinc finger domain is
not enough to obtain specific bindings [60, 76], although
one GAGA binding domain from Drosophila has a
significantly high binding affinity to its cognate sequence
of DNA [53]. To understand these tight bindings at the
atomic level, interaction details of zinc fingers have been
widely studied using nuclear magnetic resonance (NMR)
and X-ray crystallography techniques. Unfortunately, the
structures of zinc fingers still need to be investigated
because of the limited solubility of full sequences of zinc
finger proteins and difficulties of crystallization. Many zinc
fingers have limited solubility due to zinc finger domains
and other parts of the proteins, and this limited solubility
retards structural and functional studies.
In this account, the authors would like to discuss
interaction details of Cys2His2-type zinc finger domains
based on structural features that are widely studied and
applied to recent advances in biotechnologies. There are
growing reports of zinc finger domains in most kingdoms,
including archaic, prokaryotic, and eukaryotic species,
and their physiological and biochemical features need to
be discovered for successful applications in terms of
biotechnologies, including gene editing, therapeutic
targets, alteration of enzymatic pathways, and improving
the yields of specific metabolites. The zinc ion is a structural
factor in zinc finger proteins that regulates transcriptional
and translational pathways [38, 43]. These structural
domains are essential in biological systems, including
normal processes to maintain routine pathways and to
respond to specific signals that can be artificially controlled
based on the primary to quaternary structures of these
domains. Cys2His2-type domains have been extensively and
intensely investigated over the last three decades in order
to understand their binding events and affinity against
their cognate binding partners. This article will further
compare the structural aspects of various zinc finger domains,
from metal coordination to nucleic acid interactions. The
structural similarity and sequence variations are reviewed
to give valuable information for the selection and
application of suitable candidates among zinc fingers for
bioengineering.
J. Microbiol. Biotechnol.
Primary Structures of the Cys2His2-Type Zinc
Finger Motif
Classical Zinc Finger Domains and Metal Coordination
The zinc finger domain coordinates to cysteine (Cys) and
histidine (His) side chains, and the secondary structure is
generated only in the presence of the zinc ion (Zn2+). This
metal ion is structurally important in generating the
secondary structure of both classical (Cys2His2) and
nonclassical (other zinc coordinating combinations except
Cys2His2) zinc fingers [17, 34, 43, 49, 61, 66, 68]. Two βstrands and one α-helix is a general zinc finger domain
from classical zinc fingers (Fig. 1). Zinc interacting factor
268 (ZIF268), a member of the classical zinc finger group
and a transcriptional regulator, has three zinc finger
domains, and their interaction details were studied by gel
mobile shift assay and X-ray crystallography [3, 4, 25, 62].
The three zinc fingers generate tight binding compared
with that of one zinc finger domain, as is present in other
zinc finger proteins. Many approaches were applied to
modify genes in the last decade, and these classical zinc
fingers are extensively applied to biotechnology in the field
of gene editing. For these reasons, ZIF268 was applied to
control site-specific DNA interactions as binding domains
Fig. 1. Classical zinc finger domain (Cys2His2-type) coordinates
with the zinc ion (cyan ball), and the secondary structures are
generated rapidly.
Four residues, Cys107, Cys112, His125, and His 129, coordinate to the
zinc ion with tetrahedral geometry. The first zinc finger domain from
synthesized ZIF268 coordinates with the zinc ion to form a ββα structure
to interact with dsDNA (PDB accession: 4X9J) through an α-helix.
Structural Analyses of Zinc Finger Domains for Specific Interactions with DNA
including zinc finger nucleases (ZFN) and transcription
activator-like effector nucleases (TALENs) [5, 27, 48].
Although cleavage domains have powerful activity, they
should recognize specific sequences to perform their
cleavages to their target DNAs. These applications of
binding domains have powerful advantages in terms of
safety and toxicity when they are applied to in vivo systems.
The zinc ion generates tetrahedral geometry (Td) rapidly
when it is applied to the apo-zinc finger domain in vitro
system to recognize target DNA [19, 23, 24, 46, 51]. The side
chains of Cys107 and Cys112 coordinate with zinc ion in
the first and second β-strands, respectively (Fig. 1). Two
histidine residues, His125 and His129 on the α-helix,
coordinate to zinc ion and these finally aid in generation of
proper folding of the zinc finger domain [78]. Reports have
discovered that zinc finger domains show significantly
tight binding affinity with their physiological ions, Zn2+
(femto- to picomolar dissociation constant), compared with
other metal ions such as cobalt ion (Co2+, micromolar
dissociation constant) [10, 11, 41, 49, 50, 63, 68, 69]. Several
studies, however, have reported that toxic heavy metals,
such as Cd2+, can bind tighter than the zinc ion [37, 55, 72].
2021
The structural and functional studies of nonclassical zinc
finger domains showed many interesting aspects based on
the combinations of Cys and His residues such as Cys4,
Cys3His1, Cys1His3, and Cys2His1(His1)Cys1 types of zinc
finger domains [43, 54, 68].
Structures of Classical Zinc Fingers
Classical zinc fingers are the largest family among zinc
finger proteins, and a single domain usually has 28-30
amino acids involved in their structural role to perform
specific DNA interactions, although some of them generate
tight binding with RNA, proteins, and lipids [43-45, 73].
Each zinc finger is modular and can fold independently in
the presence of a zinc ion, although some cases do not
achieve suitable binding affinities. The structures of ZIF268
and its cognate DNA complex have been intensively
studied by X-ray crystallography, and the specific roles of
each finger have been discovered [25, 62, 78]. ZIF268
regulates the memory storage and recall through specific
mechanisms of neural plasticity in the human brain.
Studies at the molecular and cellular levels demonstrated
that activation and repression of diverse signals of brain
Fig. 2. Structures of classical zinc finger domains.
(A) The primary structures from eukaryotic (ZIF268, TFIIIA, and GAGA) and prokaryotic zinc fingers (Ros) show low sequence homology. The
eukaryotic zinc fingers have a Cys-X2-4-Cys-X12-His-X3-4-His pattern, whereas prokaryotic domain Ros has a Cys-X2-Cys-X9-His-X3-His pattern. (B)
The secondary structures of each domain represent zinc binding patterns and structural differences. These demonstrated that classical zinc fingers
generally show well-folded secondary structures. PDB accessions: ZIF268: 1AAY; TFIIIA: 1TF3; GAGA: 1YUI; and Ros: 2JSP.
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Eom et al.
behavior were generated through ZIF268, an activitydependent transcriptional factor, and this zinc finger is
considered as a central regulator of neural plasticity [3, 4].
This zinc finger is also called early growth reponse-1 (EGR-1),
and these proteins show rapid and transient responses
against extracellular stresses [21, 35, 67, 75]. ZIF268 consists
of a threonine (Thr)- and serine (Ser)-rich N-terminal
domain and a Thr-, Ser-, and proline (Pro)-rich C-terminal
domain. ZIF268 shows high sequence similarities among
these three domains, although their recognitions to their
binding partners are quite specific, since each zinc finger
shows different DNA binding specificity and different
binding affinities [53]. The primary structures of these
fingers show that Cys-X4-Cys-X12-His-X3-His (X represents
any amino acids) generates the appropriate secondary
structures (Fig. 2). There are linker regions between each
zinc finger that usually show repeated patterns in eukaryotes.
These linker regions have a TGE(Q)KP sequence that
generates loops between each domain. Three domains in
ZIF268 generate crucial binding with 5’-GCGG(T)GGGCG
to control transcription at promoter regions in the nucleus
for the regulation of the nervous system [53]. The structural
information from X-ray crystallography indicates that the
Cys2His2 motif coordinates to the zinc ion with a stable ββα
fold, as shown in Fig. 2B, which interact tightly with the
major groove of DNA [25]. The preliminary studies showed
that ZIF268, as a transcriptional regulator, binds to the
major groove of DNA and wraps around the double helix.
Each finger interacts with three or four bases in DNA.
The sequence Cys-X4-Cys-X12-His-X3-His is a general
pattern of zinc fingers in the eukaryotic kingdom.
Transcription factor IIIA (TFIIIA) is the first identified zinc
finger domain, which was discovered from oocytes of
African frogs, and this zinc finger has a sequence pattern
like that of ZIF268, as shown in Fig. 2A [7, 8, 18, 30, 33, 34,
56, 70]. The overall secondary structures of both ZIF268
and TFIIIA are a ββα fold, but the sequences of these two
zinc fingers show low sequence homology. TFIIIA has nine
zinc fingers, and initial studies proved that these modular
fingers have approximately 30 amino acids and a zinc ion
in each finger. This protein, TFIIIA, consists of a zinc finger
with an N-terminal region containing 280 amino acids for
DNA or RNA binding regions and 65 amino acids that
interact with other transcriptional factors in the C-terminus.
The folding mechanism of classical zinc fingers proposed
that coordination is initiated by two cysteines in the βstrands with Zn2+ through the support of the hydrophobic
core (Fig. 2B). Computational studies proposed a metalcoordinating mechanism. The first His in the α-helix
J. Microbiol. Biotechnol.
participates in this coordination, and the second His
completes a stable ββα fold in the presence of a zinc ion [26,
42, 58, 77].
In most organisms, TFIIIA has been widely studied and
detected owing to the structural and functional importance
of the cellular system, as it requires complex generation
with 5S ribosomal RNA (5S rRNA) for transcriptional
activity. Among different organisms, interestingly, all
TFIIIAs have nine consecutive zinc fingers except that of
Schizosaccharomyces pombe, which has a tenth domain in the
C-terminus with a significant space [42]. The general
sequence of TFIIIA includes phenylalanine (Phe), isoleucine
(Ile), and/or leucine (Leu), and these residues have specific
interactions with specific sites in an internal control region
(ICR) of 5S rRNA genes. The mutational studies proved
that all zinc finger domains of TFIIIA contribute to DNA
interactions, although they have different binding affinities
to cognate DNAs [22]. TFIIIA has the same general
sequence as ZIF268 (C-X4-C-X12-H-X3-H), but the last zinc
finger has one more amino acid between the first and
second zinc-coordinating histidine residues. The overall αhelix structure is not affected owing to one more amino
acid, but the last turn toward the C-terminus shows a
wider turn compared with those of the first and second
zinc fingers.
Specific Interaction with Single Zinc Finger
Most zinc finger proteins require two or more zinc finger
domains for specific interactions, but Pedone et al. [59, 64,
65] proposed that the structural motif from GAGA (Fig. 2B),
a single Cys2His2-type zinc finger isolated from Drosophila,
has sufficient affinity to bind DNA in the GA(CT)-rich region
of the promoter. The GAGA finger has 82 residues at the
end of the N-terminal region that generate 5’-GAGAGAG
near transcription start sites. The minimal DNA binding
domain shows a very low dissociation constant (Kd ~ 5 nM)
to (GA)n sequences. In the N-terminus of the GAGA zinc
finger domain, there are two basic regions called basic
region 1 (BR1) and basic region 2 (BR2). Although single
zinc finger domains show tight binding to DNA, these
basic residues are critical for complex formation. BR1 and
BR2 have highly conserved arginine (Arg) and lysine (Lys)
residues, and BR2 residues generate an α-helix. This Nterminal extension is unique in its binding compared with
other zinc fingers. The structural studies show that the
GAGA DNA binding domain generates hydrogen bonds
through the major and minor grooves [59, 64]. As shown in
Fig. 2A, GAGA has a general X3-C-X2-C-X12-H-X4-H-X4
sequence. Compared with other eukaryotic zinc finger
Structural Analyses of Zinc Finger Domains for Specific Interactions with DNA
domains, GAGA has one less amino acid between two zinccoordinating Cys residues (C-X2-C) and one more amino
acid between two His residues (C-X4-C). These additional
interactions with basic residues are an interesting primary
structure and a main reason that GAGA binds specifically
to GAGA-rich sequences with one zinc finger domain.
The basic residues, including Lys13, Arg14, and Lys23,
generate contacts with the minor groove. There is an
extended α-helix (Arg27~Ser32) and secondary structure of
these amino acids that participate in interactions with
major grooves. Arg27, Ser28, and Ser30 interact with the
bases, phosphate, and deoxyribose sugar ring in doublestranded DNA [59]. The well-elaborated sequences of
primary structures for the functional activity as a
transcriptional regulator with an extended N-terminus are
crucial for the complex generation of protein-DNA. The
GAGA zinc finger domain has one structural motif, and it
can be limited to the ββα secondary structures, but a single
domain of GAGA cannot generate satisfactory interactions
without the help of extended regions of the N-terminus.
Hydrogen bonds from basic residues and structural features
from the α-helix aid in specific and tight binding with one
single zinc finger domain to its binding partner.
Classical Zinc Fingers in Prokaryotes
The first identified zinc finger domain in prokaryotes is
Ros from Agrobacterium tumefaciens and this 15.5 kDa protein,
with a coordinating zinc ion, serves as a transcriptional
regulator. Ros mutational studies proved that the zinc
finger domain negatively controls virC and virD operons
that regulate the oncogene-bearing region in the T-DNA of
the T1 plasmid [1, 14]. This signaling cascade finally
suppresses ipt in the plant. Ros affects a large number of
plants, and it was proposed that horizontal gene transfer
occurred from bacteria to plants, although there is a second
opinion for this proposal. The primary sequence of this
zinc finger domain shows that only nine amino acids are
placed between the second Cys and the first His residue
(Fig. 2A).
This classical and prokaryotic zinc finger shows significant
differences in structural and functional aspects compared
with those of eukaryotic zinc fingers. The apo-domain,
which is the nonmetal-coordinating state, of Ros cannot
generate typical secondary structures like the other zinc
fingers, including the classical and the nonclassical fingers.
NMR studies revealed that two β-strand regions show high
structural similarity compared with those of eukaryotes,
although the α-helix structures of zinc fingers from
eukaryotes demonstrate different patterns [47]. The α-
2023
helical structures from ZIF268, TFIIIA, and GAGA have
more than three turns, but the same regions of Ros have
two turns due to a lower number of amino acids (Fig. 2).
The α-helix looks more tilted owing to the second His
residue. For the generation of tetrahedral (Td) geometry
around the zinc ion with ε-nitrogen, the His residue
requires more space, which ultimately causes the different
angles of the α-helix to the β-sheet (Fig. 2B). Although
different numbers of amino acids are placed between
eukaryotic and prokaryotic zinc fingers, the overall pattern
of His residues is almost similar [47]. The front view of the
zinc fingers shows that the side chains of the first and
second His residues are positioned almost perpendicular to
each other for the generation of Td geometry.
Structural Similarity among Cys2His2-Type Zinc
Fingers
Zinc Finger Domain as a Transcriptional Regulator
A single domain in zinc finger proteins usually has 2030 amino acids that compose the structural motif critical for
its biological functions [9, 11, 12]. These domains, in many
cases, show sequence homology and structural similarity in
zinc finger proteins. X-Ray crystallography provides
structural details of ZIF268 when it is bound to its
interaction partner, although most structural information
about the zinc finger motif was obtained by NMR studies
owing to instabilities when this structural motif did not
bind to its interaction partners [25, 62, 67, 76]. There are
three domains that bind to the major groove of dsDNA,
and these show sequence and structural similarities in
ZIF268 (Figs. 2A and 3A). Their interactions through
hydrogen bonds and hydrophobic stacking induce diverse
interactions in the finger domains, although their sequence
homologies in each domain are highly conserved. All three
domains from the first (gray) to the third (magenta) zinc
fingers show high structural similarity, more than those of
their sequence homologs (Fig. 3A). Two β-strands and a
single α-helix show high structural similarity because the
root mean square deviation (r.m.s.d.) values are less than
0.6 when they are superimposed upon each other. The
calculated r.m.s.d. values when the first and second, first
and third, and second and third zinc fingers were
superimposed were 0.529, 0.364, and 0.404, respectively
(Fig. 3A). The linker region between the two β-strands has
six amino acids in the first zinc finger in ZIF268, because
the first β-strand (Tyr5, Ala6, and Cys6) and the second βstrand (Arg14, Arg 15, and Phe 16) have two more amino
acids compared with those of the second zinc finger in
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Eom et al.
Fig. 3. Superimposed structures of classical zinc finger domains.
Each zinc finger domain of ZIF268 (A) and TFIIIA (B) shows high structural similarity. The first (gray), second (green), and third (magenta) zinc
fingers are aligned with the backbone (Cα). (C) The superimposed structure of the first zinc finger motif of ZIF268 (purple) and TFIIIA (pale
green). The two structures show structural homology.
ZIF268 (Fig. 2A). The sequences of the first, second, and
third β-strands show high homologies. The first β-strand
starts with aromatic amino acid residues (Tyr5, Phe35, and
Phe63), and the first residues of the second β-strand of each
zinc finger starts with Arg residues (Arg14, Arg41, Arg70).
Aromatic and hydrophobic residues are crucial for the
folding of zinc fingers, and polar and hydrophilic residues
are crucial for the folding for the generation of specific
interactions through the α-helix [25, 35].
TFIIIA, the first identified zinc finger, is one of the most
well-known and investigated structural motifs from different
organisms and species. This zinc finger protein includes
several interesting aspects owing to its extraordinarily
strong binding affinities to DNAs and RNAs. In addition,
this zinc finger protein has a large number of domains and
they show very poor sequence homologies through species
and organisms. The first three zinc fingers are critical for
the interactions, and these residues are overlapped and
show high structural similarity, as shown in Fig. 3B.
Preliminary studies support that this N-terminus has three
zinc fingers that are necessary for the specificity of binding.
The preliminary studies proved that the first and third
fingers interact with 5’-TGGATGGGAGACC in Box C at
ICR. Nine zinc fingers were considered to bind to BOX A,
intermediate element (IE), and Box C at ICR [58, 71]. The
superimposed structures of the N-terminus three zinc
fingers show high structural homology, as demonstrated
by the alpha carbon (Cα) aligned r.m.s.d. value. The
superimposed structures with Cα aligned structures of the
first-second, first-third, and secnd-third finger domains
of TFIIIA have r.m.s.d. values of 1.082, 0.634, and 0.780,
respectively. These values are slightly high compared with
J. Microbiol. Biotechnol.
those of ZIF268, although they have the same sequence
patterns (C-X4-C-X12-H-X4-H). The slightly high values of
r.m.s.d. were generated from the β-sheet and α-helix in this
domain. The two β-strands of ZIF268 were well matched in
space, although first zinc finger has one more amino acid
compared with those of the second and third zinc fingers
(Figs. 2A and 3B). In addition, structures of the helical
turns from the three zinc fingers of ZIF268 are well
superimposed, and these will accelerate the interactions to
the major groove of dsDNA [15, 25, 62]. The β-strands of
the second zinc finger of TFIIIA are not positioned in the
same space compared with those of first and third zinc
fingers, and this is observed by slightly higher r.m.s.d.
values compared with those of ZIF268 (Fig. 3B). In addition,
the α-helix of the second zinc finger is positioned in a
different space. These factors made the differences of
r.m.s.d. of these three zinc fingers. Subtle differences from
structural motifs generate specific interactions with strong
binding affinities with their binding partners, and it
proved that slight differences in structure distinguishes
their cognate binding partners.
Structural Similarity in a Single Zinc Finger
The structural similarity is important for generating
specific interactions in this case. They recognize different
sequences from different nucleic acids, but structural
similarities are monitored in zinc fingers. The first three
fingers from TFIIIA show high structural similarity as
described, although the r.m.s.d. values of these are slightly
lower than those of ZIF268 [42, 58, 76, 77]. The significant
changes are not monitored, but the position of the second
β-strand of the second zinc finger is slightly different
Structural Analyses of Zinc Finger Domains for Specific Interactions with DNA
compared with those of the first and the third. In addition,
the turns of the α-helix show different lengths of pitches
among zinc fingers. These subtle changes in structures can
generate specific interactions among different fingers in the
same proteins.
The structures of zinc fingers are usually characterized
through NMR studies, but the structures of ZIF268-DNA
and TFIIIA-DNA complexes were discovered by X-ray
crystallography owing to the stability from the zinc finger
domains and the DNA complexes. The first domains of
these zinc fingers were studied to understand the structural
similarity, although their sequences show differences
(Fig. 3C). The numbers of amino acids between the Cys and
His residues that coordinate the zinc ion show the same
patterns between these two zinc fingers. The Cα-aligned
structure has an r.m.s.d. value of 0.584, and the overall
structures are well superimposed between these two zinc
fingers, although low sequences are monitored except for
the linker regions. These results demonstrated that the
structures are more conserved compared with that of
sequences, but the specificity of the sequences may be a
critical factor for the recognition of its binding partners.
Zinc fingers as transcriptional and translational regulators
require specific binding affinity when signals are triggered,
and these events activate enzyme activation immediately in
our biological systems [43].
Protein and DNA Interactions
Classical zinc fingers naturally acquire zinc ions to
recognize DNAs through folding processes in physiological
systems [6, 9, 12, 13, 33]. Among these various complexes,
ZIF268 is one of the most intensively investigated to
understand the interactions between protein and nucleic
acids because the high-resolution crystal structures have
been reported for more than decades. Pabo and co-workers
have made significant progress to achieve elaborate DNAZIF268 structures [25, 60, 62, 76]. These improvements
enhance the understanding of the biological implications of
transcriptional factors. The initial crystal structure proposed
that the finger domains of ZIF268 wrap up specific
sequences of DNA, which fit nicely into the major groove
[62]. Each finger holds tightly to three base pairs, and highresolution X-ray crystallography proved the details of the
interactions [25]. Water molecules between ZIF268 and
DNA generate hydrogen bonds, and this successfully
explained how these fine bindings are generated between
certain sequences of DNA and other proteins. There are
two conserved Arg residues in each ZIF268 domain among
2025
Fig. 4. Two Arg residues are conserved in ZIF268 for the
induction of hydrogen bonds.
(A) The first Arg residues (114, 142, and 170) generate hydrogen
bonds with the phosphate backbone of DNA. (B) The other conserved
Arg residues (118, 146, and 174) show well-superimposed side chains
in space. These residues induce hydrogen bonds with the nitrogen
and/or oxygen atoms in bases (PDB accession: 1AAY). The first,
second, and third finger domains are represented as pink, green, and
blue, respectively. The nitrogen atoms are presented as dark blue.
the three domains that generate complexes with its cognate
sequence of DNA. The first conserved Arg residues are
located in the second β-strand, and they point in the same
directions in space; these residues are Arg114, Arg142, and
Arg170 (Fig. 4A). The second set of interesting Arg residues
include Arg118, Arg146, and Arg174, positioned at the
starting N-terminus of the α-helix, and these residues play
pivotal roles in generating specific interactions (Fig. 4B).
These residues face the same directions when they are
superimposed, demonstrating that these Arg residues in
each finger domain perform similar interactions.
The first conserved set of Arg residues (Arg114, Arg142,
and Arg117) positioned at the second β-strand of the
domains, and these amino acids generate hydrogen bonds,
not with bases of DNA but through the phosphate backbone
or ribose atoms within a 2.5-3.5 Å distance (Fig. 4A). This
second β-strand faces the major groove DNA and supports
strong interactions with the α-helix. The linker placed
between finger 1-2 and finger 2-3 twist for the fitting, and
its flexibility is necessary to obtain the high association
constant of this protein-DNA complex. The superimposed
structures of side chains of the first conserved set of Arg
residues appear slightly different on space, but this is
required for the suitable hydrogen bonds.
The second conserved set of Arg residues (Arg118, Arg146,
and Arg174) located at the starting point of the α-helix are
critical for overall complex generation with hydrogen
bonds in ZIF268 (Fig. 4B). All three of these residues
generate hydrogen bonds with purine or pyrimidine atoms.
Arg residues generate more than one hydrogen bond
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Eom et al.
Fig. 5. The complex structures of the zinc finger domains and DNA.
(A) ZIF268 binds with its target DNA through the major groove. The first (gray) to third (magenta) zinc fingers fold independently with the zinc
ion (yellow sphere). The α-helices face the nucleic acids for the generation of hydrogen bonds. (B) Single zinc finger domain of GAGA with an
extended region of the α-helix clamped efficiently through the major and minor grooves of DNA. This N-terminal extension includes highly basic
regions (BR1 and BR2).
through nitrogen atoms in its side chains. These aspects
proved that repeated zinc fingers from ZIF268 interact with
3 bp DNA, and the functional consequences are quite
repeated. The major contacts between ZIF268 and DNA
depend on the bases and a few hydrogen bonds with the
DNA backbone and are not critical for interactions. The
complex structure demonstrated that the bases of DNA
regulate the orientation of each zinc finger. These binding
events control the partial structures in zinc finger proteins,
and the secondary structures are tightly controlled in an
interaction-based manner.
Conclusions and Perspectives
The binding affinity of the three zinc fingers of ZIF268 to
its DNA show quite strong interactions (Kd = 1.7 × 10-10 M)
with its target sequence of DNA [25]. Three zinc fingers
show repeated fits into the major groove of DNA, and these
interactions are through mostly one strand (Fig. 5A). The
GAGA zinc finger protein has one zinc finger domain, and
it undergoes specific interactions with its binding partner
(Kd = 5.3 ± 0.7 × 10-9 M), although it has extended regions,
including BR1 and BR2 as described in Fig. 5B [64]. The
J. Microbiol. Biotechnol.
superimpose studies in this account (vide supra) summarized
that the binding affinities of classical zinc fingers are quite
different although their structures are almost the same.
Similar structures of zinc finger domains with different
amino acids can recognize their binding partners for specific
interactions. These binding events are well explained
through the complex of GAGA and DNA [59, 64, 65]. One
zinc finger usually does not have strong enough binding
for DNA, and two or more zinc fingers are usually included
in zinc finger proteins. For this reason, the extended
N-terminal regions increase binding affinity through
interactions from side chains and the base of DNA to
achieve specific interactions.
For applications in the diverse fields of bioengineering
technology, the zinc finger proteins, especially classical
zinc fingers, have been widely studied to understand their
biological implications and biochemical details [11, 43, 68].
Classical zinc finger proteins, the second largest family of
all proteins, participate in a wide spectrum of cellular
activities, including differentiation, development, and
suppression of tumors. The metal binding aspect and their
specific folding process show totally different structural
patterns compared with other metalloproteins, since the
Structural Analyses of Zinc Finger Domains for Specific Interactions with DNA
zinc finger domains can fold only in the presence of metal
ions. In addition, extraordinarily strong binding affinities
to their cognate partners are promising for applications in
the fields of engineered zinc fingers. There are some
applications for the inhibition and activation of specific genes,
including HIV-1, herpes simplex virus, VEGF-A, and the
regulation of small molecules [68]. Scientific achievement
in the realm of zinc fingers is still needed, specifically to
discover the huge number of zinc finger proteins and to
understand the transcriptional progress against diverse
responses. The small region of domains from these large
proteins is a powerful candidate to provide a key for gene
regulation in terms of biotechnology.
Acknowledgments
This research was supported by the Yuyu Pharma, Inc.
and Chonbuk National University research program in 2016.
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