Structural Analyses of Zinc Finger Domains for Specific ... · Zinc finger proteins are among the most extensively applied metalloproteins in the field of biotechnology owing to their

December 2016⎪Vol. 26⎪No. 12

J. Microbiol. Biotechnol. (2016), 26(12), 2019–2029http://dx.doi.org/10.4014/jmb.1609.09021 Research Article jmbReview

Structural Analyses of Zinc Finger Domains for Specific Interactionswith DNAKi Seong Eom1, Jin Sung Cheong2,3, and Seung Jae Lee3*

1Department of Neurosurgery, School of Medicine and Hospital, Wonkwang University, Iksan 54538, Republic of Korea2Department of Neurology, School of Medicine and Hospital, Wonkwang University, Iksan 54538, Republic of Korea3Department of Chemistry and Research Institute of Physic and Chemistry, Chonbuk National University, Jeonju 54896, Republic of Korea

Introduction

One of the most well-known groups of transcriptionalfactors in eukaryotes is the zinc finger proteins, whichcoordinate zinc ions to achieve a folded structure that iscrucial for interactions with its binding partners inphysiological systems [20, 34, 36, 38-40, 43, 48, 51, 68].Biochemical and biophysical studies of these metalloproteinshave focused on eukaryotes owing to their critical functionsin signaling cascades. The growing evidence and genomicadvances proved that zinc fingers perform crucial roles inmaintaining physiological processes from prokaryotes toeukaryotes, including the plant kingdom [2, 28, 31, 42, 52,

57, 68]. Early studies of zinc fingers have been limited toDNA binding ability and its transcriptional regulation, butrecent applications of zinc fingers in various fields are ofmajor interest owing to their specific interactions forcognate DNA, RNA, proteins, lipids, and small moleculesthrough hydrogen bonds and hydrophobic interactions [32,43, 56, 68]. The binding affinities of zinc fingers to theirbinding partners are significantly high, and preliminaryreports proved that these proteins bind to their specificnucleic acids with sub-nanomolar ranges of dissociationconstants (Kd). The elaborate control of transcriptionalregulation is triggered by this tight binding, and geneexpression is up-regulated through this complex generation

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 Cys2His2-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

2020 Eom et al.

J. Microbiol. Biotechnol.

[16, 29, 54]. Structural domains of zinc fingers have been applied to

gene-editing technologies to improve low specificity in vitroand in vivo for the use of clinical trials in the last decade[20, 48, 74]. This technology has received considerableattention because zinc fingers can improve specific bindingabilities to target nucleic acids as a part of nucleases. Mostzinc finger proteins have more than one domain, and manybiophysical reports proved that one zinc finger domain isnot enough to obtain specific bindings [60, 76], althoughone GAGA binding domain from Drosophila has asignificantly high binding affinity to its cognate sequenceof DNA [53]. To understand these tight bindings at theatomic level, interaction details of zinc fingers have beenwidely studied using nuclear magnetic resonance (NMR)and X-ray crystallography techniques. Unfortunately, thestructures of zinc fingers still need to be investigatedbecause of the limited solubility of full sequences of zincfinger proteins and difficulties of crystallization. Many zincfingers have limited solubility due to zinc finger domainsand other parts of the proteins, and this limited solubilityretards structural and functional studies.

In this account, the authors would like to discussinteraction details of Cys2His2-type zinc finger domainsbased on structural features that are widely studied andapplied to recent advances in biotechnologies. There aregrowing reports of zinc finger domains in most kingdoms,including archaic, prokaryotic, and eukaryotic species,and their physiological and biochemical features need tobe discovered for successful applications in terms ofbiotechnologies, including gene editing, therapeutictargets, alteration of enzymatic pathways, and improvingthe yields of specific metabolites. The zinc ion is a structuralfactor in zinc finger proteins that regulates transcriptionaland translational pathways [38, 43]. These structuraldomains are essential in biological systems, includingnormal processes to maintain routine pathways and torespond to specific signals that can be artificially controlledbased on the primary to quaternary structures of thesedomains. Cys2His2-type domains have been extensively andintensely investigated over the last three decades in orderto understand their binding events and affinity againsttheir cognate binding partners. This article will furthercompare the structural aspects of various zinc finger domains,from metal coordination to nucleic acid interactions. Thestructural similarity and sequence variations are reviewedto give valuable information for the selection andapplication of suitable candidates among zinc fingers forbioengineering.

Primary Structures of the Cys2His2-Type Zinc

Finger Motif

Classical Zinc Finger Domains and Metal Coordination

The zinc finger domain coordinates to cysteine (Cys) andhistidine (His) side chains, and the secondary structure isgenerated only in the presence of the zinc ion (Zn2+). Thismetal ion is structurally important in generating thesecondary structure of both classical (Cys2His2) andnonclassical (other zinc coordinating combinations exceptCys2His2) zinc fingers [17, 34, 43, 49, 61, 66, 68]. Two β-strands and one α-helix is a general zinc finger domainfrom classical zinc fingers (Fig. 1). Zinc interacting factor268 (ZIF268), a member of the classical zinc finger groupand a transcriptional regulator, has three zinc fingerdomains, and their interaction details were studied by gelmobile shift assay and X-ray crystallography [3, 4, 25, 62].The three zinc fingers generate tight binding comparedwith that of one zinc finger domain, as is present in otherzinc finger proteins. Many approaches were applied tomodify genes in the last decade, and these classical zincfingers are extensively applied to biotechnology in the fieldof gene editing. For these reasons, ZIF268 was applied tocontrol 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 2021


including zinc finger nucleases (ZFN) and transcriptionactivator-like effector nucleases (TALENs) [5, 27, 48].Although cleavage domains have powerful activity, theyshould recognize specific sequences to perform theircleavages to their target DNAs. These applications ofbinding domains have powerful advantages in terms ofsafety and toxicity when they are applied to in vivo systems.

The zinc ion generates tetrahedral geometry (Td) rapidlywhen it is applied to the apo-zinc finger domain in vitrosystem to recognize target DNA [19, 23, 24, 46, 51]. The sidechains of Cys107 and Cys112 coordinate with zinc ion inthe first and second β-strands, respectively (Fig. 1). Twohistidine residues, His125 and His129 on the α-helix,coordinate to zinc ion and these finally aid in generation ofproper folding of the zinc finger domain [78]. Reports havediscovered that zinc finger domains show significantlytight binding affinity with their physiological ions, Zn2+

(femto- to picomolar dissociation constant), compared withother metal ions such as cobalt ion (Co2+, micromolardissociation constant) [10, 11, 41, 49, 50, 63, 68, 69]. Severalstudies, however, have reported that toxic heavy metals,such as Cd2+, can bind tighter than the zinc ion [37, 55, 72].

The structural and functional studies of nonclassical zincfinger domains showed many interesting aspects based onthe combinations of Cys and His residues such as Cys4,Cys3His1, Cys1His3, and Cys2His1(His1)Cys1 types of zincfinger domains [43, 54, 68].

Structures of Classical Zinc Fingers

Classical zinc fingers are the largest family among zincfinger proteins, and a single domain usually has 28-30amino acids involved in their structural role to performspecific DNA interactions, although some of them generatetight binding with RNA, proteins, and lipids [43-45, 73].Each zinc finger is modular and can fold independently inthe presence of a zinc ion, although some cases do notachieve suitable binding affinities. The structures of ZIF268and its cognate DNA complex have been intensivelystudied by X-ray crystallography, and the specific roles ofeach finger have been discovered [25, 62, 78]. ZIF268regulates the memory storage and recall through specificmechanisms of neural plasticity in the human brain.Studies at the molecular and cellular levels demonstratedthat 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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behavior were generated through ZIF268, an activity-dependent transcriptional factor, and this zinc finger isconsidered 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 responsesagainst extracellular stresses [21, 35, 67, 75]. ZIF268 consistsof a threonine (Thr)- and serine (Ser)-rich N-terminaldomain and a Thr-, Ser-, and proline (Pro)-rich C-terminaldomain. ZIF268 shows high sequence similarities amongthese three domains, although their recognitions to theirbinding partners are quite specific, since each zinc fingershows different DNA binding specificity and differentbinding affinities [53]. The primary structures of thesefingers show that Cys-X4-Cys-X12-His-X3-His (X representsany amino acids) generates the appropriate secondarystructures (Fig. 2). There are linker regions between eachzinc finger that usually show repeated patterns in eukaryotes.These linker regions have a TGE(Q)KP sequence thatgenerates loops between each domain. Three domains inZIF268 generate crucial binding with 5’-GCGG(T)GGGCGto control transcription at promoter regions in the nucleusfor the regulation of the nervous system [53]. The structuralinformation from X-ray crystallography indicates that theCys2His2 motif coordinates to the zinc ion with a stable ββαfold, as shown in Fig. 2B, which interact tightly with themajor groove of DNA [25]. The preliminary studies showedthat ZIF268, as a transcriptional regulator, binds to themajor 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 generalpattern of zinc fingers in the eukaryotic kingdom.Transcription factor IIIA (TFIIIA) is the first identified zincfinger domain, which was discovered from oocytes ofAfrican frogs, and this zinc finger has a sequence patternlike that of ZIF268, as shown in Fig. 2A [7, 8, 18, 30, 33, 34,56, 70]. The overall secondary structures of both ZIF268and TFIIIA are a ββα fold, but the sequences of these twozinc fingers show low sequence homology. TFIIIA has ninezinc fingers, and initial studies proved that these modularfingers have approximately 30 amino acids and a zinc ionin each finger. This protein, TFIIIA, consists of a zinc fingerwith an N-terminal region containing 280 amino acids forDNA or RNA binding regions and 65 amino acids thatinteract with other transcriptional factors in the C-terminus.The folding mechanism of classical zinc fingers proposedthat coordination is initiated by two cysteines in the β-strands with Zn2+ through the support of the hydrophobiccore (Fig. 2B). Computational studies proposed a metal-coordinating mechanism. The first His in the α-helix

participates in this coordination, and the second Hiscompletes a stable ββα fold in the presence of a zinc ion [26,42, 58, 77].

In most organisms, TFIIIA has been widely studied anddetected owing to the structural and functional importanceof the cellular system, as it requires complex generationwith 5S ribosomal RNA (5S rRNA) for transcriptionalactivity. Among different organisms, interestingly, allTFIIIAs have nine consecutive zinc fingers except that ofSchizosaccharomyces pombe, which has a tenth domain in theC-terminus with a significant space [42]. The generalsequence of TFIIIA includes phenylalanine (Phe), isoleucine(Ile), and/or leucine (Leu), and these residues have specificinteractions with specific sites in an internal control region(ICR) of 5S rRNA genes. The mutational studies provedthat all zinc finger domains of TFIIIA contribute to DNAinteractions, although they have different binding affinitiesto cognate DNAs [22]. TFIIIA has the same generalsequence as ZIF268 (C-X4-C-X12-H-X3-H), but the last zincfinger has one more amino acid between the first andsecond zinc-coordinating histidine residues. The overall α-helix structure is not affected owing to one more aminoacid, but the last turn toward the C-terminus shows awider turn compared with those of the first and secondzinc fingers.

Specific Interaction with Single Zinc Finger

Most zinc finger proteins require two or more zinc fingerdomains 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 regionof the promoter. The GAGA finger has 82 residues at theend of the N-terminal region that generate 5’-GAGAGAGnear transcription start sites. The minimal DNA bindingdomain shows a very low dissociation constant (Kd ~ 5 nM)to (GA)n sequences. In the N-terminus of the GAGA zincfinger domain, there are two basic regions called basicregion 1 (BR1) and basic region 2 (BR2). Although singlezinc finger domains show tight binding to DNA, thesebasic residues are critical for complex formation. BR1 andBR2 have highly conserved arginine (Arg) and lysine (Lys)residues, and BR2 residues generate an α-helix. This N-terminal extension is unique in its binding compared withother zinc fingers. The structural studies show that theGAGA DNA binding domain generates hydrogen bondsthrough the major and minor grooves [59, 64]. As shown inFig. 2A, GAGA has a general X3-C-X2-C-X12-H-X4-H-X4

sequence. Compared with other eukaryotic zinc finger



domains, GAGA has one less amino acid between two zinc-coordinating Cys residues (C-X2-C) and one more aminoacid between two His residues (C-X4-C). These additionalinteractions with basic residues are an interesting primarystructure and a main reason that GAGA binds specificallyto GAGA-rich sequences with one zinc finger domain.

The basic residues, including Lys13, Arg14, and Lys23,generate contacts with the minor groove. There is anextended α-helix (Arg27~Ser32) and secondary structure ofthese amino acids that participate in interactions withmajor grooves. Arg27, Ser28, and Ser30 interact with thebases, phosphate, and deoxyribose sugar ring in double-stranded DNA [59]. The well-elaborated sequences ofprimary structures for the functional activity as atranscriptional regulator with an extended N-terminus arecrucial for the complex generation of protein-DNA. TheGAGA zinc finger domain has one structural motif, and itcan be limited to the ββα secondary structures, but a singledomain of GAGA cannot generate satisfactory interactionswithout the help of extended regions of the N-terminus.Hydrogen bonds from basic residues and structural featuresfrom the α-helix aid in specific and tight binding with onesingle zinc finger domain to its binding partner.

Classical Zinc Fingers in Prokaryotes

The first identified zinc finger domain in prokaryotes isRos from Agrobacterium tumefaciens and this 15.5 kDa protein,with a coordinating zinc ion, serves as a transcriptionalregulator. Ros mutational studies proved that the zincfinger domain negatively controls virC and virD operonsthat regulate the oncogene-bearing region in the T-DNA ofthe T1 plasmid [1, 14]. This signaling cascade finallysuppresses ipt in the plant. Ros affects a large number ofplants, and it was proposed that horizontal gene transferoccurred from bacteria to plants, although there is a secondopinion for this proposal. The primary sequence of thiszinc finger domain shows that only nine amino acids areplaced between the second Cys and the first His residue(Fig. 2A).

This classical and prokaryotic zinc finger shows significantdifferences in structural and functional aspects comparedwith those of eukaryotic zinc fingers. The apo-domain,which is the nonmetal-coordinating state, of Ros cannotgenerate typical secondary structures like the other zincfingers, including the classical and the nonclassical fingers.NMR studies revealed that two β-strand regions show highstructural similarity compared with those of eukaryotes,although the α-helix structures of zinc fingers fromeukaryotes demonstrate different patterns [47]. The α-

helical structures from ZIF268, TFIIIA, and GAGA havemore than three turns, but the same regions of Ros havetwo turns due to a lower number of amino acids (Fig. 2).The α-helix looks more tilted owing to the second Hisresidue. For the generation of tetrahedral (Td) geometryaround the zinc ion with ε-nitrogen, the His residuerequires more space, which ultimately causes the differentangles of the α-helix to the β-sheet (Fig. 2B). Althoughdifferent numbers of amino acids are placed betweeneukaryotic and prokaryotic zinc fingers, the overall patternof His residues is almost similar [47]. The front view of thezinc fingers shows that the side chains of the first andsecond His residues are positioned almost perpendicular toeach 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 20-30 amino acids that compose the structural motif critical forits biological functions [9, 11, 12]. These domains, in manycases, show sequence homology and structural similarity inzinc finger proteins. X-Ray crystallography providesstructural details of ZIF268 when it is bound to itsinteraction partner, although most structural informationabout the zinc finger motif was obtained by NMR studiesowing to instabilities when this structural motif did notbind to its interaction partners [25, 62, 67, 76]. There arethree domains that bind to the major groove of dsDNA,and these show sequence and structural similarities inZIF268 (Figs. 2A and 3A). Their interactions throughhydrogen bonds and hydrophobic stacking induce diverseinteractions in the finger domains, although their sequencehomologies in each domain are highly conserved. All threedomains from the first (gray) to the third (magenta) zincfingers show high structural similarity, more than those oftheir sequence homologs (Fig. 3A). Two β-strands and asingle α-helix show high structural similarity because theroot mean square deviation (r.m.s.d.) values are less than0.6 when they are superimposed upon each other. Thecalculated r.m.s.d. values when the first and second, firstand third, and second and third zinc fingers weresuperimposed were 0.529, 0.364, and 0.404, respectively(Fig. 3A). The linker region between the two β-strands hassix amino acids in the first zinc finger in ZIF268, becausethe first β-strand (Tyr5, Ala6, and Cys6) and the second β-strand (Arg14, Arg 15, and Phe 16) have two more aminoacids compared with those of the second zinc finger in

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ZIF268 (Fig. 2A). The sequences of the first, second, andthird β-strands show high homologies. The first β-strandstarts with aromatic amino acid residues (Tyr5, Phe35, andPhe63), and the first residues of the second β-strand of eachzinc finger starts with Arg residues (Arg14, Arg41, Arg70).Aromatic and hydrophobic residues are crucial for thefolding of zinc fingers, and polar and hydrophilic residuesare crucial for the folding for the generation of specificinteractions through the α-helix [25, 35].

TFIIIA, the first identified zinc finger, is one of the mostwell-known and investigated structural motifs from differentorganisms and species. This zinc finger protein includesseveral interesting aspects owing to its extraordinarilystrong binding affinities to DNAs and RNAs. In addition,this zinc finger protein has a large number of domains andthey show very poor sequence homologies through speciesand organisms. The first three zinc fingers are critical forthe interactions, and these residues are overlapped andshow high structural similarity, as shown in Fig. 3B.Preliminary studies support that this N-terminus has threezinc fingers that are necessary for the specificity of binding.The preliminary studies proved that the first and thirdfingers interact with 5’-TGGATGGGAGACC in Box C atICR. Nine zinc fingers were considered to bind to BOX A,intermediate element (IE), and Box C at ICR [58, 71]. Thesuperimposed structures of the N-terminus three zincfingers show high structural homology, as demonstratedby the alpha carbon (C

α) aligned r.m.s.d. value. The

superimposed structures with Cα aligned structures of the

first-second, first-third, and secnd-third finger domainsof TFIIIA have r.m.s.d. values of 1.082, 0.634, and 0.780,respectively. These values are slightly high compared with

those of ZIF268, although they have the same sequencepatterns (C-X4-C-X12-H-X4-H). The slightly high values ofr.m.s.d. were generated from the β-sheet and α-helix in thisdomain. The two β-strands of ZIF268 were well matched inspace, although first zinc finger has one more amino acidcompared with those of the second and third zinc fingers(Figs. 2A and 3B). In addition, structures of the helicalturns from the three zinc fingers of ZIF268 are wellsuperimposed, and these will accelerate the interactions tothe major groove of dsDNA [15, 25, 62]. The β-strands ofthe second zinc finger of TFIIIA are not positioned in thesame space compared with those of first and third zincfingers, 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 adifferent space. These factors made the differences ofr.m.s.d. of these three zinc fingers. Subtle differences fromstructural motifs generate specific interactions with strongbinding affinities with their binding partners, and itproved that slight differences in structure distinguishestheir cognate binding partners.

Structural Similarity in a Single Zinc Finger

The structural similarity is important for generatingspecific interactions in this case. They recognize differentsequences from different nucleic acids, but structuralsimilarities are monitored in zinc fingers. The first threefingers from TFIIIA show high structural similarity asdescribed, although the r.m.s.d. values of these are slightlylower than those of ZIF268 [42, 58, 76, 77]. The significantchanges are not monitored, but the position of the secondβ-strand of the second zinc finger is slightly different

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.



compared with those of the first and the third. In addition,the turns of the α-helix show different lengths of pitchesamong zinc fingers. These subtle changes in structures cangenerate specific interactions among different fingers in thesame proteins.

The structures of zinc fingers are usually characterizedthrough NMR studies, but the structures of ZIF268-DNAand TFIIIA-DNA complexes were discovered by X-raycrystallography owing to the stability from the zinc fingerdomains and the DNA complexes. The first domains ofthese zinc fingers were studied to understand the structuralsimilarity, although their sequences show differences(Fig. 3C). The numbers of amino acids between the Cys andHis residues that coordinate the zinc ion show the samepatterns between these two zinc fingers. The C

α-aligned

structure has an r.m.s.d. value of 0.584, and the overallstructures are well superimposed between these two zincfingers, although low sequences are monitored except forthe linker regions. These results demonstrated that thestructures are more conserved compared with that ofsequences, but the specificity of the sequences may be acritical factor for the recognition of its binding partners.Zinc fingers as transcriptional and translational regulatorsrequire specific binding affinity when signals are triggered,and these events activate enzyme activation immediately inour biological systems [43].

Protein and DNA Interactions

Classical zinc fingers naturally acquire zinc ions torecognize DNAs through folding processes in physiologicalsystems [6, 9, 12, 13, 33]. Among these various complexes,ZIF268 is one of the most intensively investigated tounderstand the interactions between protein and nucleicacids because the high-resolution crystal structures havebeen reported for more than decades. Pabo and co-workershave made significant progress to achieve elaborate DNA-ZIF268 structures [25, 60, 62, 76]. These improvementsenhance the understanding of the biological implications oftranscriptional factors. The initial crystal structure proposedthat the finger domains of ZIF268 wrap up specificsequences of DNA, which fit nicely into the major groove[62]. Each finger holds tightly to three base pairs, and high-resolution X-ray crystallography proved the details of theinteractions [25]. Water molecules between ZIF268 andDNA generate hydrogen bonds, and this successfullyexplained how these fine bindings are generated betweencertain sequences of DNA and other proteins. There aretwo conserved Arg residues in each ZIF268 domain among

the three domains that generate complexes with its cognatesequence of DNA. The first conserved Arg residues arelocated in the second β-strand, and they point in the samedirections in space; these residues are Arg114, Arg142, andArg170 (Fig. 4A). The second set of interesting Arg residuesinclude Arg118, Arg146, and Arg174, positioned at thestarting N-terminus of the α-helix, and these residues playpivotal roles in generating specific interactions (Fig. 4B).These residues face the same directions when they aresuperimposed, demonstrating that these Arg residues ineach finger domain perform similar interactions.

The first conserved set of Arg residues (Arg114, Arg142,and Arg117) positioned at the second β-strand of thedomains, and these amino acids generate hydrogen bonds,not with bases of DNA but through the phosphate backboneor ribose atoms within a 2.5-3.5 Å distance (Fig. 4A). Thissecond β-strand faces the major groove DNA and supportsstrong interactions with the α-helix. The linker placedbetween finger 1-2 and finger 2-3 twist for the fitting, andits flexibility is necessary to obtain the high associationconstant of this protein-DNA complex. The superimposedstructures of side chains of the first conserved set of Argresidues appear slightly different on space, but this isrequired 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 arecritical for overall complex generation with hydrogenbonds in ZIF268 (Fig. 4B). All three of these residuesgenerate hydrogen bonds with purine or pyrimidine atoms.Arg residues generate more than one hydrogen bond

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.

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through nitrogen atoms in its side chains. These aspectsproved that repeated zinc fingers from ZIF268 interact with3 bp DNA, and the functional consequences are quiterepeated. The major contacts between ZIF268 and DNAdepend on the bases and a few hydrogen bonds with theDNA backbone and are not critical for interactions. Thecomplex structure demonstrated that the bases of DNAregulate the orientation of each zinc finger. These bindingevents control the partial structures in zinc finger proteins,and the secondary structures are tightly controlled in aninteraction-based manner.

Conclusions and Perspectives

The binding affinity of the three zinc fingers of ZIF268 toits DNA show quite strong interactions (Kd = 1.7 × 10-10 M)with its target sequence of DNA [25]. Three zinc fingersshow repeated fits into the major groove of DNA, and theseinteractions are through mostly one strand (Fig. 5A). TheGAGA zinc finger protein has one zinc finger domain, andit 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

superimpose studies in this account (vide supra) summarizedthat the binding affinities of classical zinc fingers are quitedifferent although their structures are almost the same.Similar structures of zinc finger domains with differentamino acids can recognize their binding partners for specificinteractions. These binding events are well explainedthrough the complex of GAGA and DNA [59, 64, 65]. Onezinc finger usually does not have strong enough bindingfor DNA, and two or more zinc fingers are usually includedin zinc finger proteins. For this reason, the extendedN-terminal regions increase binding affinity throughinteractions from side chains and the base of DNA toachieve specific interactions.

For applications in the diverse fields of bioengineeringtechnology, the zinc finger proteins, especially classicalzinc fingers, have been widely studied to understand theirbiological implications and biochemical details [11, 43, 68].Classical zinc finger proteins, the second largest family ofall proteins, participate in a wide spectrum of cellularactivities, including differentiation, development, andsuppression of tumors. The metal binding aspect and theirspecific folding process show totally different structuralpatterns compared with other metalloproteins, since the

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).



zinc finger domains can fold only in the presence of metalions. In addition, extraordinarily strong binding affinitiesto their cognate partners are promising for applications inthe fields of engineered zinc fingers. There are someapplications for the inhibition and activation of specific genes,including HIV-1, herpes simplex virus, VEGF-A, and theregulation of small molecules [68]. Scientific achievementin the realm of zinc fingers is still needed, specifically todiscover the huge number of zinc finger proteins and tounderstand the transcriptional progress against diverseresponses. The small region of domains from these largeproteins is a powerful candidate to provide a key for generegulation 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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Structural Analyses of Zinc Finger Domains for Specific ... · Zinc finger proteins are among the most extensively applied metalloproteins in the field of biotechnology owing to their