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

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J. Microbiol. Biotechnol. (2016), 26(12), 2019–2029 http://dx.doi.org/10.4014/jmb.1609.09021 Research Article Review jmb Structural Analyses of Zinc 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 Zinc finger proteins are among the most extensively applied metalloproteins 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 eukaryotes. Two First published online cysteine and two histidine 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, lipids, 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), transcription 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 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 pISSN 1017-7825, eISSN 1738-8872 complex structures and binding affinities aid in the protein 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 December 2016 ⎪ Vol. 26⎪ No. 12 2020 Eom et al. [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 binding domain 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 genes 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. J. Microbiol. Biotechnol. Structural Analyses of Zinc Finger Domains for Specific Interactions with DNA 2021 including zinc finger nucleases (ZFN) and transcription The structural and functional studies of nonclassical zinc activator-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 DNAs. 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
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