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

Structural Analyses of Finger Domains for Specific Interactions with DNA Ki Seong Eom1, Jin Sung Cheong2,3, and Seung Jae Lee3*

1Department of Neurosurgery, School of Medicine and Hospital, Wonkwang University, Iksan 54538, Republic of Korea 2Department of Neurology, School of Medicine and Hospital, Wonkwang University, Iksan 54538, Republic of Korea 3Department of Chemistry and Research Institute of Physic and Chemistry, Chonbuk National University, Jeonju 54896, Republic of Korea

Received: September 19, 2016 Revised: September 26, 2016 are among the most extensively applied in the field of Accepted: September 28, 2016 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 . Two

First published online and two residues (Cys2His2) coordinate to the zinc ion for the structural October 6, 2016 functions to generate a ββα fold, and this secondary structure supports specific interactions

*Corresponding author with their binding partners, including DNA, RNA, , proteins, and small molecules. In Phone: +82-63-270-3412; this account, the structural similarity and differences of well-known Cys2His2-type zinc fingers Fax: +82-63-260-3407; such as zinc interaction factor 268 (ZIF268), factor IIIA (TFIIIA), GAGA, and Ros E-mail: [email protected] 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 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 pISSN 1017-7825, eISSN 1738-8872 complex structures and binding affinities aid in the design area.

Copyright© 2016 by The Korean Society for Microbiology Keywords: Zinc finger proteins, metalloproteins, classical zinc finger, transcriptional regulator and Biotechnology

Introduction 57, 68]. Early studies of zinc fingers have been limited to DNA binding ability and its transcriptional regulation, but One of the most well-known groups of transcriptional recent applications of zinc fingers in various fields are of factors in eukaryotes is the zinc finger proteins, which major interest owing to their specific interactions for coordinate zinc ions to achieve a folded structure that is cognate DNA, RNA, proteins, lipids, and small molecules crucial for interactions with its binding partners in through hydrogen bonds and hydrophobic interactions [32, physiological systems [20, 34, 36, 38-40, 43, 48, 51, 68]. 43, 56, 68]. The binding affinities of zinc fingers to their Biochemical and biophysical studies of these metalloproteins binding partners are significantly high, and preliminary have focused on eukaryotes owing to their critical functions reports proved that these proteins bind to their specific in signaling cascades. The growing evidence and genomic nucleic acids with sub-nanomolar ranges of dissociation advances proved that zinc fingers perform crucial roles in constants (Kd). The elaborate control of transcriptional maintaining physiological processes from prokaryotes to regulation is triggered by this tight binding, and gene eukaryotes, including the plant kingdom [2, 28, 31, 42, 52, expression is up-regulated through this complex generation

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[16, 29, 54]. Primary Structures of the Cys2His2-Type Zinc Structural domains of zinc fingers have been applied to Finger Motif gene-editing technologies to improve low specificity in vitro and in vivo for the use of clinical trials in the last decade Classical Zinc Finger Domains and Metal Coordination [20, 48, 74]. This technology has received considerable The zinc finger domain coordinates to cysteine (Cys) and attention because zinc fingers can improve specific binding histidine (His) side chains, and the secondary structure is abilities to target nucleic acids as a part of nucleases. Most generated only in the presence of the zinc ion (Zn2+). This zinc finger proteins have more than one domain, and many metal ion is structurally important in generating the biophysical reports proved that one zinc finger domain is secondary structure of both classical (Cys2His2) and not enough to obtain specific bindings [60, 76], although nonclassical (other zinc coordinating combinations except one GAGA from Drosophila has a Cys2His2) zinc fingers [17, 34, 43, 49, 61, 66, 68]. Two β- significantly high binding affinity to its cognate sequence strands and one α-helix is a general zinc finger domain of DNA [53]. To understand these tight bindings at the from classical zinc fingers (Fig. 1). Zinc interacting factor atomic level, interaction details of zinc fingers have been 268 (ZIF268), a member of the classical zinc finger group widely studied using nuclear magnetic resonance (NMR) and a transcriptional regulator, has three zinc finger and X-ray crystallography techniques. Unfortunately, the domains, and their interaction details were studied by gel structures of zinc fingers still need to be investigated mobile shift assay and X-ray crystallography [3, 4, 25, 62]. because of the limited solubility of full sequences of zinc The three zinc fingers generate tight binding compared finger proteins and difficulties of crystallization. Many zinc with that of one zinc finger domain, as is present in other fingers have limited solubility due to zinc finger domains zinc finger proteins. Many approaches were applied to and other parts of the proteins, and this limited solubility modify in the last decade, and these classical zinc retards structural and functional studies. fingers are extensively applied to biotechnology in the field In this account, the authors would like to discuss of gene editing. For these reasons, ZIF268 was applied to interaction details of Cys2His2-type zinc finger domains control site-specific DNA interactions as binding 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 Classical zinc finger domain (Cys His -type) coordinates compare the structural aspects of various zinc finger domains, Fig. 1. 2 2 with the zinc ion (cyan ball), and the secondary structures are from metal coordination to nucleic acid interactions. The generated rapidly. structural similarity and sequence variations are reviewed Four residues, Cys107, Cys112, His125, and His 129, coordinate to the to give valuable information for the selection and zinc ion with tetrahedral geometry. The first zinc finger domain from application of suitable candidates among zinc fingers for synthesized ZIF268 coordinates with the zinc ion to form a ββα structure bioengineering. to interact with dsDNA (PDB accession: 4X9J) through an α-helix.

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including zinc finger nucleases (ZFN) and transcription The structural and functional studies of nonclassical zinc -like effector nucleases (TALENs) [5, 27, 48]. finger domains showed many interesting aspects based on

Although cleavage domains have powerful activity, they the combinations of Cys and His residues such as Cys4, should recognize specific sequences to perform their Cys3His1, Cys1His3, and Cys2His1(His1)Cys1 types of zinc cleavages to their target . These applications of finger domains [43, 54, 68]. binding domains have powerful advantages in terms of safety and toxicity when they are applied to in vivo systems. Structures of Classical Zinc Fingers

The zinc ion generates tetrahedral geometry (Td) rapidly Classical zinc fingers are the largest family among zinc when it is applied to the apo-zinc finger domain in vitro finger proteins, and a single domain usually has 28-30 system to recognize target DNA [19, 23, 24, 46, 51]. The side amino acids involved in their structural role to perform chains of Cys107 and Cys112 coordinate with zinc ion in specific DNA interactions, although some of them generate the first and second β-strands, respectively (Fig. 1). Two tight binding with RNA, proteins, and lipids [43-45, 73]. histidine residues, His125 and His129 on the α-helix, Each zinc finger is modular and can fold independently in coordinate to zinc ion and these finally aid in generation of the presence of a zinc ion, although some cases do not proper folding of the zinc finger domain [78]. Reports have achieve suitable binding affinities. The structures of ZIF268 discovered that zinc finger domains show significantly and its cognate DNA complex have been intensively tight binding affinity with their physiological ions, Zn2+ studied by X-ray crystallography, and the specific roles of (femto- to picomolar dissociation constant), compared with each finger have been discovered [25, 62, 78]. ZIF268 other metal ions such as cobalt ion (Co2+, micromolar regulates the memory storage and recall through specific dissociation constant) [10, 11, 41, 49, 50, 63, 68, 69]. Several mechanisms of neural plasticity in the human brain. studies, however, have reported that toxic heavy metals, Studies at the molecular and cellular levels demonstrated such as Cd2+, can bind tighter than the zinc ion [37, 55, 72]. 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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behavior were generated through ZIF268, an activity- participates in this coordination, and the second His dependent transcriptional factor, and this zinc finger is completes a stable ββα fold in the presence of a zinc ion [26, considered as a central regulator of neural plasticity [3, 4]. 42, 58, 77]. This zinc finger is also called early growth reponse-1 (EGR-1), In most organisms, TFIIIA has been widely studied and and these proteins show rapid and transient responses detected owing to the structural and functional importance against extracellular stresses [21, 35, 67, 75]. ZIF268 consists of the cellular system, as it requires complex generation of a threonine (Thr)- and serine (Ser)-rich N-terminal with 5S ribosomal RNA (5S rRNA) for transcriptional domain and a Thr-, Ser-, and proline (Pro)-rich C-terminal activity. Among different organisms, interestingly, all domain. ZIF268 shows high sequence similarities among TFIIIAs have nine consecutive zinc fingers except that of these three domains, although their recognitions to their Schizosaccharomyces pombe, which has a tenth domain in the binding partners are quite specific, since each zinc finger C-terminus with a significant space [42]. The general shows different DNA binding specificity and different sequence of TFIIIA includes phenylalanine (Phe), isoleucine binding affinities [53]. The primary structures of these (Ile), and/or leucine (Leu), and these residues have specific fingers show that Cys-X4-Cys-X12-His-X3-His (X represents interactions with specific sites in an internal control region any amino acids) generates the appropriate secondary (ICR) of 5S rRNA genes. The mutational studies proved structures (Fig. 2). There are linker regions between each that all zinc finger domains of TFIIIA contribute to DNA zinc finger that usually show repeated patterns in eukaryotes. interactions, although they have different binding affinities These linker regions have a TGE(Q)KP sequence that to cognate DNAs [22]. TFIIIA has the same general generates loops between each domain. Three domains in sequence as ZIF268 (C-X4-C-X12-H-X3-H), but the last zinc ZIF268 generate crucial binding with 5’-GCGG(T)GGGCG finger has one more between the first and to control transcription at promoter regions in the nucleus second zinc-coordinating histidine residues. The overall α- for the regulation of the nervous system [53]. The structural helix structure is not affected owing to one more amino information from X-ray crystallography indicates that the acid, but the last turn toward the C-terminus shows a

Cys2His2 motif coordinates to the zinc ion with a stable ββα wider turn compared with those of the first and second fold, as shown in Fig. 2B, which interact tightly with the zinc fingers. major groove of DNA [25]. The preliminary studies showed that ZIF268, as a transcriptional regulator, binds to the Specific Interaction with Single Zinc Finger major groove of DNA and wraps around the double helix. Most zinc finger proteins require two or more zinc finger Each finger interacts with three or four bases in DNA. domains for specific interactions, but Pedone et al. [59, 64,

The sequence Cys-X4-Cys-X12-His-X3-His is a general 65] proposed that the from GAGA (Fig. 2B), pattern of zinc fingers in the eukaryotic kingdom. a single Cys2His2-type zinc finger isolated from Drosophila, IIIA (TFIIIA) is the first identified zinc has sufficient affinity to bind DNA in the GA(CT)-rich region finger domain, which was discovered from oocytes of of the promoter. The GAGA finger has 82 residues at the African frogs, and this zinc finger has a sequence pattern end of the N-terminal region that generate 5’-GAGAGAG like that of ZIF268, as shown in Fig. 2A [7, 8, 18, 30, 33, 34, near transcription start sites. The minimal DNA binding

56, 70]. The overall secondary structures of both ZIF268 domain shows a very low dissociation constant (Kd ~ 5 nM) and TFIIIA are a ββα fold, but the sequences of these two to (GA)n sequences. In the N-terminus of the GAGA zinc zinc fingers show low sequence homology. TFIIIA has nine finger domain, there are two basic regions called basic zinc fingers, and initial studies proved that these modular region 1 (BR1) and basic region 2 (BR2). Although single fingers have approximately 30 amino acids and a zinc ion zinc finger domains show tight binding to DNA, these in each finger. This protein, TFIIIA, consists of a zinc finger basic residues are critical for complex formation. BR1 and with an N-terminal region containing 280 amino acids for BR2 have highly conserved arginine (Arg) and lysine (Lys) DNA or RNA binding regions and 65 amino acids that residues, and BR2 residues generate an α-helix. This N- interact with other transcriptional factors in the C-terminus. terminal extension is unique in its binding compared with The folding mechanism of classical zinc fingers proposed other zinc fingers. The structural studies show that the that coordination is initiated by two in the β- GAGA DNA binding domain generates hydrogen bonds strands with Zn2+ through the support of the hydrophobic through the major and minor grooves [59, 64]. As shown in core (Fig. 2B). Computational studies proposed a metal- Fig. 2A, GAGA has a general X3-C-X2-C-X12-H-X4-H-X4 coordinating mechanism. The first His in the α-helix sequence. Compared with other eukaryotic zinc finger

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domains, GAGA has one less amino acid between two zinc- helical structures from ZIF268, TFIIIA, and GAGA have coordinating Cys residues (C-X2-C) and one more amino more than three turns, but the same regions of Ros have acid between two His residues (C-X4-C). These additional two turns due to a lower number of amino acids (Fig.2). interactions with basic residues are an interesting primary The α-helix looks more tilted owing to the second His structure and a main reason that GAGA binds specifically residue. For the generation of tetrahedral (Td) geometry to GAGA-rich sequences with one zinc finger domain. around the zinc ion with ε-nitrogen, the His residue The basic residues, including Lys13, Arg14, and Lys23, requires more space, which ultimately causes the different generate contacts with the minor groove. There is an angles of the α-helix to the β-sheet (Fig. 2B). Although extended α-helix (Arg27~Ser32) and secondary structure of different numbers of amino acids are placed between these amino acids that participate in interactions with eukaryotic and prokaryotic zinc fingers, the overall pattern major grooves. Arg27, Ser28, and Ser30 interact with the of His residues is almost similar [47]. The front view of the bases, phosphate, and deoxyribose sugar ring in double- zinc fingers shows that the side chains of the first and stranded DNA [59]. The well-elaborated sequences of second His residues are positioned almost perpendicular to primary structures for the functional activity as a each other for the generation of Td geometry. transcriptional regulator with an extended N-terminus are crucial for the complex generation of protein-DNA. The Structural Similarity among Cys2His2-Type Zinc GAGA zinc finger domain has one structural motif, and it Fingers can be limited to the ββα secondary structures, but a single domain of GAGA cannot generate satisfactory interactions Zinc Finger Domain as a Transcriptional Regulator without the help of extended regions of the N-terminus. A single domain in zinc finger proteins usually has 20- Hydrogen bonds from basic residues and structural features 30 amino acids that compose the structural motif critical for from the α-helix aid in specific and tight binding with one its biological functions [9, 11, 12]. These domains, in many single zinc finger domain to its binding partner. cases, show sequence homology and structural similarity in zinc finger proteins. X-Ray crystallography provides Classical Zinc Fingers in Prokaryotes structural details of ZIF268 when it is bound to its The first identified zinc finger domain in prokaryotes is interaction partner, although most structural information Ros from Agrobacterium tumefaciens and this 15.5 kDa protein, about the zinc finger motif was obtained by NMR studies with a coordinating zinc ion, serves as a transcriptional owing to instabilities when this structural motif did not regulator. Ros mutational studies proved that the zinc bind to its interaction partners [25, 62, 67, 76]. There are finger domain negatively controls virC and virD operons three domains that bind to the major groove of dsDNA, that regulate the oncogene-bearing region in the T-DNA of and these show sequence and structural similarities in the T1 plasmid [1, 14]. This signaling cascade finally ZIF268 (Figs. 2A and 3A). Their interactions through suppresses ipt in the plant. Ros affects a large number of hydrogen bonds and hydrophobic stacking induce diverse plants, and it was proposed that horizontal gene transfer interactions in the finger domains, although their sequence occurred from bacteria to plants, although there is a second homologies in each domain are highly conserved. All three opinion for this proposal. The primary sequence of this domains from the first (gray) to the third (magenta) zinc zinc finger domain shows that only nine amino acids are fingers show high structural similarity, more than those of placed between the second Cys and the first His residue their sequence homologs (Fig. 3A). Two β-strands and a (Fig. 2A). single α-helix show high structural similarity because the This classical and prokaryotic zinc finger shows significant root mean square deviation (r.m.s.d.) values are less than differences in structural and functional aspects compared 0.6 when they are superimposed upon each other. The with those of eukaryotic zinc fingers. The apo-domain, calculated r.m.s.d. values when the first and second, first which is the nonmetal-coordinating state, of Ros cannot and third, and second and third zinc fingers were generate typical secondary structures like the other zinc superimposed were 0.529, 0.364, and 0.404, respectively fingers, including the classical and the nonclassical fingers. (Fig. 3A). The linker region between the two β-strands has NMR studies revealed that two β-strand regions show high six amino acids in the first zinc finger in ZIF268, because structural similarity compared with those of eukaryotes, the first β-strand (Tyr5, Ala6, and Cys6) and the second β- although the α-helix structures of zinc fingers from strand (Arg14, Arg 15, and Phe 16) have two more amino eukaryotes demonstrate different patterns [47]. The α- acids compared with those of the second zinc finger in

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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 those of ZIF268, although they have the same sequence third β-strands show high homologies. The first β-strand patterns (C-X4-C-X12-H-X4-H). The slightly high values of starts with aromatic amino acid residues (Tyr5, Phe35, and r.m.s.d. were generated from the β-sheet and α-helix in this Phe63), and the first residues of the second β-strand of each domain. The two β-strands of ZIF268 were well matched in zinc finger starts with Arg residues (Arg14, Arg41, Arg70). space, although first zinc finger has one more amino acid Aromatic and hydrophobic residues are crucial for the compared with those of the second and third zinc fingers folding of zinc fingers, and polar and hydrophilic residues (Figs. 2A and 3B). In addition, structures of the helical are crucial for the folding for the generation of specific turns from the three zinc fingers of ZIF268 are well interactions through the α-helix [25, 35]. superimposed, and these will accelerate the interactions to TFIIIA, the first identified zinc finger, is one of the most the major groove of dsDNA [15, 25, 62]. The β-strands of well-known and investigated structural motifs from different the second zinc finger of TFIIIA are not positioned in the organisms and species. This zinc finger protein includes same space compared with those of first and third zinc several interesting aspects owing to its extraordinarily fingers, and this is observed by slightly higher r.m.s.d. strong binding affinities to DNAs and . In addition, values compared with those of ZIF268 (Fig. 3B). In addition, this zinc finger protein has a large number of domains and the α-helix of the second zinc finger is positioned in a they show very poor sequence homologies through species different space. These factors made the differences of and organisms. The first three zinc fingers are critical for r.m.s.d. of these three zinc fingers. Subtle differences from the interactions, and these residues are overlapped and structural motifs generate specific interactions with strong show high structural similarity, as shown in Fig. 3B. binding affinities with their binding partners, and it Preliminary studies support that this N-terminus has three proved that slight differences in structure distinguishes zinc fingers that are necessary for the specificity of binding. their cognate binding partners. The preliminary studies proved that the first and third fingers interact with 5’-TGGATGGGAGACC in Box C at Structural Similarity in a Single Zinc Finger ICR. Nine zinc fingers were considered to bind to BOX A, The structural similarity is important for generating intermediate element (IE), and Box C at ICR [58, 71]. The specific interactions in this case. They recognize different superimposed structures of the N-terminus three zinc sequences from different nucleic acids, but structural fingers show high structural homology, as demonstrated similarities are monitored in zinc fingers. The first three by the alpha carbon (Cα) aligned r.m.s.d. value. The fingers from TFIIIA show high structural similarity as superimposed structures with Cα aligned structures of the described, although the r.m.s.d. values of these are slightly first-second, first-third, and secnd-third finger domains lower than those of ZIF268 [42, 58, 76, 77]. The significant of TFIIIA have r.m.s.d. values of 1.082, 0.634, and 0.780, changes are not monitored, but the position of the second respectively. These values are slightly high compared with β-strand of the second zinc finger is slightly different

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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 Fig. 4. Two Arg residues are conserved in ZIF268 for the these zinc fingers were studied to understand the structural induction of hydrogen bonds. similarity, although their sequences show differences (A) The first Arg residues (114, 142, and 170) generate hydrogen (Fig. 3C). The numbers of amino acids between the Cys and bonds with the phosphate backbone of DNA. (B) The other conserved His residues that coordinate the zinc ion show the same Arg residues (118, 146, and 174) show well-superimposed side chains in space. These residues induce hydrogen bonds with the nitrogen patterns between these two zinc fingers. The Cα-aligned structure has an r.m.s.d. value of 0.584, and the overall and/or oxygen atoms in bases (PDB accession: 1AAY). The first, structures are well superimposed between these two zinc second, and third finger domains are represented as pink, green, and blue, respectively. The nitrogen atoms are presented as dark blue. fingers, although low sequences are monitored except for the linker regions. These results demonstrated that the structures are more conserved compared with that of the three domains that generate complexes with its cognate sequences, but the specificity of the sequences may be a sequence of DNA. The first conserved Arg residues are critical factor for the recognition of its binding partners. located in the second β-strand, and they point in the same Zinc fingers as transcriptional and translational regulators directions in space; these residues are Arg114, Arg142, and require specific binding affinity when signals are triggered, Arg170 (Fig. 4A). The second set of interesting Arg residues and these events activate enzyme activation immediately in include Arg118, Arg146, and Arg174, positioned at the our biological systems [43]. starting N-terminus of the α-helix, and these residues play pivotal roles in generating specific interactions (Fig. 4B). Protein and DNA Interactions These residues face the same directions when they are superimposed, demonstrating that these Arg residues in Classical zinc fingers naturally acquire zinc ions to each finger domain perform similar interactions. recognize DNAs through folding processes in physiological The first conserved set of Arg residues (Arg114, Arg142, systems [6, 9, 12, 13, 33]. Among these various complexes, and Arg117) positioned at the second β-strand of the ZIF268 is one of the most intensively investigated to domains, and these amino acids generate hydrogen bonds, understand the interactions between protein and nucleic not with bases of DNA but through the phosphate backbone acids because the high-resolution crystal structures have or ribose atoms within a 2.5-3.5 Å distance (Fig. 4A). This been reported for more than decades. Pabo and co-workers second β-strand faces the major groove DNA and supports have made significant progress to achieve elaborate DNA- strong interactions with the α-helix. The linker placed ZIF268 structures [25, 60, 62, 76]. These improvements between finger 1-2 and finger 2-3 twist for the fitting, and enhance the understanding of the biological implications of its flexibility is necessary to obtain the high association transcriptional factors. The initial crystal structure proposed constant of this protein-DNA complex. The superimposed that the finger domains of ZIF268 wrap up specific structures of side chains of the first conserved set of Arg sequences of DNA, which fit nicely into the major groove residues appear slightly different on space, but this is [62]. Each finger holds tightly to three base pairs, and high- required for the suitable hydrogen bonds. resolution X-ray crystallography proved the details of the The second conserved set of Arg residues (Arg118, Arg146, interactions [25]. Water molecules between ZIF268 and and Arg174) located at the starting point of the α-helix are DNA generate hydrogen bonds, and this successfully critical for overall complex generation with hydrogen explained how these fine bindings are generated between bonds in ZIF268 (Fig. 4B). All three of these residues certain sequences of DNA and other proteins. There are generate hydrogen bonds with purine or pyrimidine atoms. two conserved Arg residues in each ZIF268 domain among Arg residues generate more than one hydrogen bond

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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 superimpose studies in this account (vide supra) summarized proved that repeated zinc fingers from ZIF268 interact with that the binding affinities of classical zinc fingers are quite 3bp DNA, and the functional consequences are quite different although their structures are almost the same. repeated. The major contacts between ZIF268 and DNA Similar structures of zinc finger domains with different depend on the bases and a few hydrogen bonds with the amino acids can recognize their binding partners for specific DNA backbone and are not critical for interactions. The interactions. These binding events are well explained complex structure demonstrated that the bases of DNA through the complex of GAGA and DNA [59, 64, 65]. One regulate the orientation of each zinc finger. These binding zinc finger usually does not have strong enough binding events control the partial structures in zinc finger proteins, for DNA, and two or more zinc fingers are usually included and the secondary structures are tightly controlled in an in zinc finger proteins. For this reason, the extended interaction-based manner. N-terminal regions increase binding affinity through interactions from side chains and the base of DNA to Conclusions and Perspectives achieve specific interactions. For applications in the diverse fields of bioengineering The binding affinity of the three zinc fingers of ZIF268 to technology, the zinc finger proteins, especially classical -10 its DNA show quite strong interactions (Kd = 1.7 × 10 M) zinc fingers, have been widely studied to understand their with its target sequence of DNA [25]. Three zinc fingers biological implications and biochemical details [11, 43, 68]. show repeated fits into the major groove of DNA, and these Classical zinc finger proteins, the second largest family of interactions are through mostly one strand (Fig. 5A). The all proteins, participate in a wide spectrum of cellular GAGA zinc finger protein has one zinc finger domain, and activities, including differentiation, development, and it undergoes specific interactions with its binding partner suppression of tumors. The metal binding aspect and their -9 (Kd = 5.3 ± 0.7 × 10 M), although it has extended regions, specific folding process show totally different structural including BR1 and BR2 as described in Fig. 5B [64]. The patterns compared with other metalloproteins, since the

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