Zinc Finger Proteins: A Bridge BetweenTransition Metals and Gene RegulationAndrew Green, 1,2 Maura Parker, 1,2 Damiano Conte, 1,2 andBibudhendra Sarkar 1,2*

1Department of Biochemistry Research, The Hospital for Sick Children, Toronto,Ontario, Canada2Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

The zinc finger motif occurs in many proteins that regulate eukaryotic gene expression. Thisfamily of proteins is expanding rapidly, and it is now clear that they are involved inbiological processes that go beyond the DNA-binding functions that they are best knownfor. Here we attempt to outline, in terms of both structure and function, some of thecharacteristics of the most thoroughly studied zinc finger proteins, including the classicalzinc finger family and the steroid hormone receptors. We will then move on to discuss someof the newer members of the zinc finger family, including GATA-1, the LIM domain,GAL4, the RING finger, and p53. Throughout this review we will keep in mind the rolemetals play in the biological activities of these proteins, and outline the means by whichtransition metals can affect the immensely important yet delicate biological tasks theseproteins carry out. J. Trace Elem. Exp. Med. 11:103–118, 1998.© 1998 Wiley-Liss, Inc.

Key words: zinc finger motif; zinc; DNA binding; transition metals

INTRODUCTION

Zinc is an essential element necessary for the growth and replication of all cells. Itis naturally incorporated into a number of important proteins, either functioning in theactive site of enzymes or providing structural support for intracellular proteins in-volved in molecular interactions. While it is similar to other transition metal atoms invarious respects, it is unlike many of them in that it is both abundant and nontoxic.Furthermore, zinc exists as a divalent cation, Zn2+, which is a completely filled d shellwith 10 d electrons. This electron configuration has four important ramifications.First, because of the filled d shell, Zn2+ has no ligand field stabilization energy whencoordinated by ligands in any geometry [1]. Thus, zinc is a flexible metal atom thatis able to adopt four, five, and six coordinate geometries when bound to proteins. Formetal ions with partially filled d shells, certain arrangements of ligands are favoredover others because of this electronic energy term. Second, Zn2+ is regarded as aborderline acid (in terms of hard–soft acid–base theory) [2]. Therefore, zinc caninteract strongly with a variety of ligand types including sulfur from cysteine, nitrogen

Contract grant sponsor: Medical Research Council of Canada; Contract grant sponsor: National CancerInstitute of Canada.

*Correspondence to: Bibudhendra Sarkar, Department of Biochemistry Research, The Hospital for SickChildren, 555 University Ave., Toronto, Ontario M5G 1X8, Canada.

Accepted 10 December 1997

The Journal of Trace Elements in Experimental Medicine 11:103–118 (1998)

© 1998 Wiley-Liss, Inc.

PROD #329

from histidine, and oxygen from aspartate, glutamate, and water. Third, zinc as adivalent ion is not redox active. Thus, unlike copper and iron, zinc does not participatein oxidation/reduction reactions. Last, Zn2+ is relatively labile in terms of kinetics,undergoing ligand exchange reactions relatively rapidly [3]. This appears to be at leastpartially responsible for the ease of uptake and release of this metal ion. Zinc bindingby proteins is not a recent discovery; it is abundant within cells, and for many yearstens of enzymes have been known to bind this metal. In the majority of these instancesthe zinc acts as an electrophilic catalyst. In some of these enzymes, however, zinc ionsare also seen to have a purely structural role. By tethering the protein at four or moredifferent points, zinc effectively acts as a structural cross-support.

In the last decade there has been an explosion of work in the zinc finger field, bothin terms of structure determination and functional analysis. The zinc finger is one ofthe major structural motifs involved in eukaryotic protein–nucleic acid interactions,and while only a few of these proteins that contain such fingers have been studied indetail, it appears that many of these zinc finger domains are involved in DNA binding.Proteins with zinc finger motifs are involved in many aspects of eukaryotic generegulation. For example, such fingers occur in proto-oncogenes, in proteins inducedby differentiation and growth signals, in general transcription factors, and in someregulatory genes of lower eukaryotic organisms.

To date, an ever-growing portion of the human genome encodes zinc-finger–containing proteins, including classical zinc fingers, the steroid hormone receptors,GATA-1 proteins, LIM domain proteins, RING fingers, and p53 (Fig. 1). It has beenestimated that those proteins which make up the classical zinc finger family alonemay constitute up to 1% of all human gene products [4]. Here we will attempt toprovide an overview of the major zinc finger families and their members by com-bining the major aspects of our knowledge in terms of their structure and function.Readers are directed to other outstanding reviews in this area, including those bySchwabe and Klug [5], Coleman [6], Pabo and Sauer [7], and Berg and Shi [8].

CLASSICAL ZINC FINGER FAMILY

A: TFIIIA

The original or ‘‘classical’’ zinc finger motif was identified from analyses of theamino acid sequence of theXenopus laevistranscription factor TFIIIA, which re-vealed a series of nine tandem repeats of the consensus sequence

(Y,F)-X-Cys-X2–4-Cys-X3-F-X5-C-L-X2-His-X3–5-His-X2–6

[9]. Of the 30 residues that make up the repeat, only seven of them and the spacingbetween them are conserved [10]. The four conserved cysteine and histidine residuesserve as zinc ligands, bringing together the three conserved hydrophobic residues toform a stable structural domain. Substitution of any of these residues results indestabilization of the structure [10]. Since the original proposal, a large number ofsequences that share the seven conserved positions of the TFIIIA motif have beenidentified. The classes of zinc fingers are now generally specified by the ligatingresidues that characterize them; thus the TFIIIA-like fingers are more correctly called

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(Cys)2(His)2 zinc fingers. Since the discovery of TFIIIA, numerous putative zincfinger proteins which contain this conserved motif have been discovered, the bestdocumented of which are Sp1, the Wilms’ tumor gene (WT1), the NGFI-A/Zif268/Egrl,2/Krox24 family, and the yeast regulatory gene ADR1. In the first part of this

Fig. 1. A growing number of important proteins ligate zinc in order to maintain their tertiary structureand thus function (adapted from Schwabe and Klug [5]).

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review, we will focus on those zinc fingers that are structurally homologous to theTFIIIA-like or ‘‘classical’’ zinc fingers.

As mentioned above, the first zinc finger of any family to be discovered wasXenopus laevisTFIIIA (Transcription Factor IIIA) in 1985 [9,11]. This factor is oneof at least three factors required to activate the gene that gives rise to 5S RNA. 2DNMR studies confirmed that the TFIIIA like ‘‘classical’’ zinc fingers contain anantiparallelb-sheet region and ana-helix [10]. These putative DNA binding struc-tures were initially identified as nine tandemly repeated sequences of approximately30 amino acid residues [12]. TFIIIA has been analyzed by metal replacement studies,atomic absorption spectroscopy, and EXAFS (extended X-ray absorption fine struc-ture), and based on these experiments it was postulated that the two cysteine and twohistidine residues in each finger bind a zinc ion and that the resulting metalloproteinstructures are involved in the sequence-specific binding of the protein to DNA. Milleret al. [9] observed that TFIIIA, which has nine fingers, interacts with a 50-bp regionin the 5S RNA gene, and they subsequently identified a 51⁄2-bp periodicity of guanineresidues in the TFIIIA-binding site. Based on these findings, it was postulated thateach finger motif binds to half a turn of DNA and that this 51⁄2-bp periodicity in thebinding site correlates with the interaction of contiguous finger structures to DNA[12]. The two cysteines, which are near the turn in theb-sheet region (essentiallyforming two b-sheet regions), and the two histidines, which are near thea-helix,coordinate the central zinc ion and hold these secondary structures together to forma compact globular domain [7]. This arrangement brings the three conserved hydro-phobic residues into contact with one another and places hydrophilic side chains(potential DNA-binding residues) on the exterior of the domain [10].

B: Sp1

Transcription factor Sp1 is a protein of 105 kDa (778 amino acids) which activatesa large subset of mammalian genes containing ‘‘GC box’’ (guanine/cytosine-rich)upstream promoter elements. The C-terminal 168 amino acid residues of Sp1 containthree contiguous (Cys)2(His)2 zinc fingers and have been shown to constitute theDNA-binding domain, while the N-terinal 610 amino acid residues form the trans-activating domain. Deletion mutations and domain swapping experiments on Sp1show that the two domains function independently [13]. Sp1 binds to some, but notall, DNA sequences that contain the asymmetric GGGCGG ‘‘GC box’’ [14]. Sp1 wasoriginally identified as a protein from HeLa cells that binds to multiple GGGCGGsequences in the 21-bp repeat elements of SV40 and activates in vitro transcriptionfrom the SV40 promoter [12]. Subsequently, a variety of cellular and viral promoterswere shown to be activated by Sp1 in vitro. These studies revealed that Sp1-responsive promoters usually contain multiple GC box recognition sites, although asingle binding site appears to be sufficient for a promoter to be stimulated by Sp1. Inaddition, Sp1 recognition sequences are often found near binding sites for othertranscription factors, such as CTF/NF-1 and AP-1 [12], which suggests that thesefactors may act in conjunction with each other to modulate transcription.

C: WT1

Perhaps one of the most striking examples of the ability of a zinc finger protein toplay a role in a disease state is that of the Wilms’ tumor protein. Nephroblastoma, or

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Wilms’ tumor, is a cancer thought to originate in primitive cells of the developingkidney and is associated with ridia, mental retardation, and urogenital functions [15].The gene believed to be responsible for the development of these tumors has beenisolated by a number of groups [16,17] and is expressed in high amounts in embryonickidney and adult spleen [15]. Known as WT1, the gene encompasses approximately50 kb of genomic DNA and encodes a 3-kb mRNA. The predicted protein sequenceshows that the product of the WT1 gene is a polypeptide containing four (Cys)2(His)2zinc finger domains clustered near the C terminus, with a proline- and glutanine-richdomain nearer to the N-terminal region [18]. An analysis of the DNA-binding sites forthis protein indicates that they are similar to the sequence recognized by the earlygrowth response (Egr-1) gene product [15]. This leads to the possibility that WT1 mayindeed act as a tumor suppressor gene by competing with other transcription factorsfor occupancy of their DNA site. Interestingly, a mutation identified in the WT1 geneof a Wilms’ tumor patient caused the deletion of 25 bp that included an exon–intronsplice junction and resulted in the deletion of the third zinc finger [15]. When a WT1polypeptide containing this mutation was constructed, expressed, and assayed forDNA binding with an Egr-1 binding site oligonucleotide, a severely reduced DNA-binding ability was observed [15], leading to speculation that perhaps loss of DNAbinding eventually leads to cell-growth deregulation and eventually malignancy.

D: NGFI-A/EGR/Krox 24/Zif 268 Family

NGFI-A (nerve growth factor), also known as Egr 1, Krox24, and Zif268, wasisolated from mouse and is the prototypic member of a family of immediate-earlytranscription factors (genes whose activation is generally very rapid, transient, andindependent of new protein synthesis in response to a multitude of stimuli). TheWilms’ tumor gene product (WT1) and the general transcription factor Sp1 possessDNA-binding domains that share a degree of homology with the DNA-binding do-mains of this family [19]. NGFI-A cDNA was isolated by differential cDNA screen-ing aimed at identifying genes upregulated by serum stimulation of quiescent fibro-blasts [20]. Transcription of this gene is induced in many cell types, including fibro-blasts, lymphocytes, and epithelial cells [21]. The transcript encodes an 80-kD nuclearphosphoprotein [22] with three tandem zinc fingers of the (Cys)2(His)2 subclass.NGFI-A is a trophic hormone that promotes the outgrowth of nerve fibers fromsympathetic and sensory ganglia [23]. The effects of NGFI-A on the differentiation ofsympathetic neurons are manifested by an accelerated outgrowth of nerve fibers andthe induction of several enzymes involved in neurotransmitter biosynthesis [24]. EGR2 and 3 have been identified as human homologs of the mouse NGF-IA/Egrl/Zif268/Krox24 family, all with remarkable amino acid sequence conservation in their zincfinger domains [25]. As mentioned above, this entire family of genes has beenobserved to be capable of coupling early biochemical second-messenger signals tolong-term changes in gene expression. They belong to a much larger family ofproteins (including c-fos and c-jun) that act as nuclear signal transducers by initiatinga cascade of gene–protein interactions.

The most detailed structural data of the (Cys)2(His)2 motif zinc finger family todate is that of the co-crystal structure of Zif268 with its consensus DNA site [26]. Themurine Zif268 contains three tandem zinc fingers and is known to participate in theearly development of mice. The concaternerized zinc fingers forming the DNA-

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binding domain wrap partly around the DNA, following the major groove of the DNAdouble helix. The amino-terminal end of thea-helix from each finger is tilted into themajor groove, which allows some of the amino acid side chains to form contacts withthe nucleotide bases, thus defining the specificity of the binding interaction. In the Zifcomplex, two antiparallelb-sheets are on the back of thea-helix (away from the basepairs) and are shifted toward one side of the major groove. The firstb-strand does notmake any contacts with the DNA, whereas the secondb-sheet contacts the sugarphosphate backbone. According to the crystal structure, 11 contacts are made with sixguanines on the primary strand of the DNA, with the most 58-DNA contact made bythe carboxyterminal (third) zinc finger and the most 38-DNA contact made by theamino-terminal (first) zinc finger. Additional non–sequence-specific contacts aremade by the protein with the backbone of the DNA. In each finger, an arginine in thesecondb-strand makes a contact to a phosphodiester oxygen, and the first zinc-binding histidine in thea-helix contacts another phosphodiester oxygen.

E: ADR1

ADR1 is a nuclear protein which is necessary for transcription of the glucose-repressible alcohol dehydrogenase II gene (ADH2) as well as glycerol kinase (GUT1)and catalase A (CTA1) in the yeastSaccharomyces cerevisiae[27]. ADR1 containstwo zinc finger domains of the (Cys)2(His)2 type. Point mutations in either fingerregion of ADR1 can give rise to null mutants which are incapable of activating ADH2expression [28]. ADR1 has been shown to bind with high affinity to an upstreamactivator sequence UASI, both in vitro and in vivo [29]. The minimal DNA-bindingdomain of ADR1, as identified by deletion mutagenesis, consists of two zinc fingermotifs and a short (approximately 17-bp) region just N-terminal to the fingers. Eventhough single molecules of ADR1 bind independently to each half of UAS1, thepresence of the two bound molecules is necessary to activate ADH2 expression [30].NMR and X-ray crystallographic structural studies of the two ADR1 zinc fingers havebeen conducted and show that this polypeptide consists of a two-strandedb-hairpinand a C-terminala-helix [31], which is not unexpectedly similar to the secondarystructure observed in the Zif268/DNA crystal structure. The side chains of aminoacids implicated by mutagenesis to be involved in DNA binding are on the finger tips,and the faces of alpha helices are exposed to solvent, as are equivalent residues in theZif268/DNA complex [32].

It is now commonly accepted that zinc ions in zinc fingers function to providestructural integrity to the protein such that it maintains the proper topology in orderto bind DNA. Some experiments have been conducted to determine if other divalentcations are capable of replacing zinc in these proteins and altering DNA-bindingactivity in some capacity. This has been postulated to give rise to possible mecha-nisms of metal-induced toxicity and/or carcinogenesis [33]. One group found throughequilibrium dialysis studies that TFIIIA was capable of being replaced by both Cd2+

and Ni2+ [34]. Similarily, another group found that Sp1 zinc could be replaced by Ni2+

with a subsequent alteration in DNA-binding specificity [35].

STEROID HORMONE RECEPTORS

Steroid hormone receptors are ligand-inducible transcription enhancers which bindspecifically to short DNA sequences or hormone response elements and regulate a

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number of genes. Each member of the superfamily contains two highly conserved(Cys)4-type zinc fingers [36,37]. Unlike the TFIIIA classical zinc fingers, the twohormone receptor zinc fingers fold together, forming an independent structural mod-ule called the hormone receptor DNA-binding domain. In general, the DNA-bindingdomain mediates specific DNA binding, contains a nuclear localization signal, andincludes a weak dimerization property [38]. The DNA-binding domain comprisesapproximately 80 amino acids and therein contains nine conserved cysteine residues[39] (Fig. 2).

The crystal structure of both the estrogen (ER) and glucocorticoid receptor (GR)revealed that eight of nine conserved cysteines are involved in coordinating two zincatoms tetrahedrally, forming two zinc fingers [40 and 41, respectively]. The two zincfingers differ from each other in both conformation and function. The first C-terminalfinger has a larger loop with ana-helix andb-sheet regions, while the second fingerhas twoa-helical segments. Three residues located within the first helix, positionedN-terminal to the first finger and designated the P-box, determine DNA binding

Fig. 2. Domain organization of nuclear hormone receptors and DNA-binding domain (domain C)sequences of selected steroid hormone receptors. A/B, cell- and promoter-specific activation region; C,DNA-binding and dimerization region; D, hinge region; E, ligand-binding, ligand-induced transactivation,and dimerization region; THRA, thyroid receptora; VERBA, v-erbA; THRB, thyroid receptorb; RARA,retinoic acid receptora; RARB, retinoic acid receptorb; RARG, retinoic acid receptorg; RXR, retinoidX receptor; H2RIIBP, H2RII gene-binding protein; ER, estrogen receptor; GR, glucocorticoid receptor;PR, progesterone receptor; MR, mineralocorticoid receptor; AR, androgen receptor; VDR, vitamin Dreceptor; ECR, ecdysone receptor (sequences from Laudet et al., [67]).

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specificity. The ER recognizes the DNA consensus half-site AGGTCA and differsfrom the GR half-site, AGAACA, by only two of the six base pairs. Mutagenesisexperiments demonstrated that replacing the three residues, glutamate, glycine, andalanine, within the ER DNA-binding domain P-box to glycine, serine, and valine,respectively, converts the DNA specificity of the ER to that of the GR. The specificDNA binding occurs within the DNA major groove, while sugar-phosphate contactsare made on either side of the major groove by the loops of both zinc fingers.

It has been found that most members of the steroid superfamily (including thethyroid, retinoic acid, and vitamin D3 receptors) bind to either the ER or GR half-sites,but each has a unique orientation and spacing, forming either homodimers or het-erodimers. Isolated ER or GR DNA-binding domains are monomeric in solution, yetform ‘‘head-to-head’’ homodimers on palindromic consensus DNA sequences sepa-rated by three base pairs [42]. The residues between the first two cysteines of thesecond zinc finger, designated the D-box, mediate the dimer contacts between the ERor GR DNA-binding domains. There is a tenfold higher propensity for the ER or GRDNA-binding domains to form head-to-head homodimers over just the monomercomplex with DNA. Since naturally occurring estrogen and glucocorticoid responseelements (ERE and GRE, respectively) commonly contain one perfect hexamer andone which is degenerate, this high propensity to cooperatively form homodimers mayhave a biological funcion. Experiments using degenerate and non-cognate EREs andGREs [43,44] clearly demonstrated the ability of the ER and GR DNA-bindingdomains to form non-cognate recognition complexes. In contrast to the ER and GR,the thyroid (TR), retinoic acid (RAR), and vitamin D3 (VDR) steroid receptors rec-ognize direct repeat half-sites, with the consensus motif of PuGGTCA, as het-erodimers with the retinoid X receptor (RXR) [45]. The RAR-RXR heterodimer bindsto the tandem direct repeat half-sites with either a 2- (DR2) or 5- (DR5) base pairspacer separating the half-sites. This base pair spacer is crucial for high-affinitybinding to occur. The RAR-RXR heterodimer is incapable of binding with highaffinity to the related tandem direct repeat half-sites of the TR or the BDR [45]. Aswell, it has been demonstrated that there is an order to the binding of the RAR andthe RXR to the DR2 and DR5, and this involves determinants adjacent to the RARand RXR DNA-binding domains [44]. The RXR occupies the 58-upstream half-site,and RAR the 38-downstream half-site of the direct repeat in both the DR2 and DR5.

As in the classical zinc fingers, the zinc atom does not directly interact with DNAbut instead acts as a type of scaffolding that confers the required conformation to theoverall structure, enabling the DNA-binding domain to bind DNA. This suggests thatany metal with similar coordination properties and ligand affinities to zinc may beable to replace it in the zinc finger. Experiments have demonstrated the ability ofcobalt, cadmium, and iron to structurally reconstitute the zinc finger motif of theDNA-binding domain [44,46,47]. On the other hand, nickel and copper were shownto bind to the ER DNA-binding domain, yet neither restored DNA-binding activity.In the case of ER DNA-binding domain iron-replaced zinc finger, it has been dem-onstrated that the iron finger complex binds the ERE with similar affinity as the nativefinger and under the appropriate conditions generates free radicals which damage theproximate ERE DNA [47]. Due to the close proximity of the zinc finger to DNA, itis postulated that iron substituted zinc fingers may generate free radicals while bound

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to genetic regulatory response elements, leading to adverse consequences such as ironinduced toxicity and/or carcinogenesis [47,48].

ERYTHROID TRANSCRIPTION FACTOR GATA-1

GATA-1 is a transcription factor that is expressed primarily in erythroid cellsregulating a diverse group of genes [49]. GATA-1 binds specifically as a monomer tothe asymmetric consensus target sequence (T/A)GATA (A/G). Members of theGATA-1 family of transcription factors contain two related but non-identical zincfingers. As found with the steroid receptors, both GATA-1 zinc fingers bind zinc witha four-cysteine motif [50]:

Cys-X2-Cys-X4-T-X-L-W-R-R-X3-G-X3-Cys-N-A-Cys

Unlike the steroid receptors, only one zinc finger, located in what is known as DomainII, and an adjacent C-terminal basic region, Domain I, are critical for DNA binding[51]. The minimal area required for specific binding, which encompasses Domains Iand II, is 59 residues in length. The other finger does not bind DNA, but is believedto stabilize the protein–DNA interaction. The nuclear magnetic resonance (NMR)structure of GATA-1 Domain I and II [52] illustrates a structural similarity betweenDomain I of GATA1 and the N-terminal zinc finger of the steroid receptors. Both ofthese zinc fingers have ana-helix attached to two anti-parallelb-sheets. However,unlike the steroid receptors, the twob-sheets and thea-helix of GATA-1 Domain Iinteract with the major groove of the DNA. Domain II, which is also necessary forspecific DNA recognition, wraps around the minor groove. The amino acids at po-sitions 54 to 59 (RNRKVS), within Domain I, were found to be necessary for specificDNA binding. An exceedingly basic region (KGKKRR), adjacent and just C-terminalto Domain I, has been suggested to be involved in nuclear localization [53].

As with the DNA-binding domain of the steroid receptors, the zinc finger motif inDomain I of GATA-1 has had its zinc replaced with iron, cobalt, and cadmium [54].GATA-1 functions in an iron-rich environment, suggesting that GATA-1 may haveevolved from early iron-binding factors.

THE LIM DOMAIN

The LIM domain is found within a wide variety of proteins of diverse function[55]. LIM consists of two zinc atoms bound by Cys-Cys-His-Cys and (Cys)4 motifs[56]. The LIM domain received its name from the first three transcription factors inwhich it was observed:Caenorhabditis elegansL in-ll, rat Is l-1, andC. elegansMec3 [55]. A majority of proteins containing the LIM motif are transcription factors, mostoften containing the DNA-binding homeodomain. LIM is also found in proteins withno additional DNA-binding motifs. However, there is no clear evidence that the LIMdomain has DNA-binding properties [57]. Most evidence suggests that LIM may beinvolved in protein–protein interactions.

The NMR structure of the LIM domain [58] demonstrates a novel overall fold. Yetthe C-terminal (Cys)4 motif of LIM folds in a similar manner to the GATA-1 DomainI and the N-terminal zinc finger of the steroid receptor DNA-binding domain. The

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LIM (Cys)4 zinc finger contains two anti-parallelb-sheets and ana-helical region,like that of the GATA-1 and steroid receptor DNA-binding domain zinc fingers. Aswell, several residues responsible for direct DNA contacts within GATA-1 Domain Iare also present in the LIM (Cys)4 zinc finger. This has led to the speculation that LIMmay have DNA-binding properties.

GAL4

GAL4 is a member of the group of fungal transcription factors, which, like manyother transcription factors, bind DNA by means of a conserved cysteine-rich motif.This motif contains six cysteine residues with the following consensus sequence:

Cys-X-X-Cys-X6-Cys-X6-Cys-X-X-Cys-X6-Cys

[59]. Two zinc atoms are tetrahedrally coordinated to the six cysteine residues as abinuclear zinc thiolate cluster. The structural elements of this DNA-binding regioninclude two shorta-helices, one of which is nucleated by the first cysteine residue andthe other by the fourth. Both zinc atoms interact with each of these two cysteineresidues, bringing the helices together in a rigid conformation. The C terminus of thefirst helix is oriented toward the DNA-binding site, allowing two lysine residues tomake base-specific contacts. This type of DNA interaction is in contrast to that of thesteroid hormone receptors, in which the N-terminal portion of thea-helix interactswith the DNA.

Recently, the co-crystal structure of the DNA-binding domain of another memberof this family, PPR1, bound to its cognate DNA, has been solved [60]. The structureis essentially identical with that of the GAL4 DNA-binding domain, with the majordifference being with the cognate DNA sequences. Both cysteine-rich domains rec-ognize a palindrome of the CGG triplet; however, PPR1 requires a 6–base pair spacer,whereas the GAL4 response element contains an 11–base pair spacer. This type ofselectivity mimics the steroid hormone receptor family in that the half-site sequencefor this particular family is the same, but the spacing of these half-sites determineswhich member of the family will bind to the DNA.

RING-FINGER

Recent additions to the growing family of zinc finger proteins include a group ofReally InterestingNew Gene (RING) products, rich in cysteine residues. TheseRING-finger proteins contain a zinc-binding motif with the consensus sequence:

Cys-X-(I/V)- Cys-X9–27-Cys-X1–3-His-X-(P/I/L)-Cys-(I/L/M)-X- Cys-X4–48-Cys-P-X-Cys

[61]. Two zinc atoms bind tetrahedrally to the motif by the conserved histidine andcysteine residues, effecting the formation of a single folded domain. This is similar tothe steroid hormone receptors and the LIM domain. The zinc is assumed to providethe protein with a stable secondary and tertiary structure, allowing the motif to bindDNA or participate in protein–protein or protein–membrane interactions. Other con-

112 Green et al.

served residues in the motif include a number of hydrophobic amino acids in positionsadjacent to coordinating cysteine and histidine residues. These conserved amino acidslikely function in two ways: to aid in the formation of the proper secondary structure,and to position the coordinating cysteine and histidine residues for the binding of zinc.This theory is supported by NMR and circular dichroism (CD) studies of two poly-peptides: the RING-finger domain of RING1 and the PML RING-finger [62,63].These studies have demonstrated that the RING-finger motif maintains a significantdegree of structure in the absence of zinc, but also that addition of zinc results in smallbut reproducible changes in structure. The RING-finger structure has been shown toconsist of a three-strandedb-sheet packed against ana-helix, a topology also seen inthe zinc-binding region of the regulatory domain of protein kinase C (PKC-RD).According to these results, there appears to be an increase inb-sheet content, as wellas a small increase in the amount ofa-helix present in the polypeptide subsequent tothe addition of zinc. Moreover, results from the NMR studies indicate that the mostsignificant changes induced by zinc involve the linking of structural elements togetherto form a functional motif configuration.

Currently, there are three RING-finger proteins motifs: the RING-finger motif(described above), the RING-H2 motif and the B box motif. The original RING-fingermotif is generally found within proteins involved in processes such as gene regulation,DNA recombination, and DNA repair. It is not known whether all of these proteinsbind to DNA directly, or instead, interact with DNA by associating with proteinsalready bound to the DNA. For example, the immediate-early herpes proteins, IEBHVand IE110, have both been shown to activate a variety of promoters in transientexpression assays, yet there is no direct evidence to show that these proteins bind toDNA. The difference between the second RING-finger protein motif, the RING-H2motif, and the original RING-finger, is the replacement of the fourth conservedcysteine by a second histidine residue. Proteins with this motif generally functionthrough protein–protein or protein–membrane interactions. For example, the postsyn-aptic membrane protein (PSMP), involved in the clustering and aggregation of ace-tylcholine receptors, is associated with the plasma membrane. Other RING-H2 pro-teins, PEP3/Vps 18p and PEP5/Vps1/END1, have been found to be involved invacuolar biogenesis. Since these proteins are localized to vacuoles, it can be assumedthat there is no DNA interaction, and that function involves an association withanother protein or with a membrane.

The third protein motif, the B-box motif, defines a group of proteins with one ortwo cysteine-rich motifs (the B-box) adjacent to the RING-finger. The B-box con-sensus sequence and spacing is similar to that of GAL4:

Cys-X-X-His-X7-Cys-X7-Cys-X-X-Cys-X5-His-X-X-His

[61]. B1 and B2 are two B-box associated motifs with a more variable consensussequence:

Cys-X2–4-Cys/His-X7–9-Cys-X-X-Cys-X4-Cys-X-X-Cys-X3–6-His-X2–8-Cys/His

[61]. This B-box motif is also characterized by a coiled-coil dimerization domainlocated on the C-terminal side of the cysteine-rich motifs, again reminiscent of the

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GAL4 protein motif. Many of the proteins with this motif have cellular transformationpotential when found as a fusion protein. For example, a translocation between humanchromosomes 15 and 17 results in the fusion of PML, a RING-finger protein of theB-box family, with RARa, an isoform of the retinoic acid receptor [64]. Fusion of theRING-finger with RAR disrupts the formation of PML nuclear bodies and is asso-ciated with the onset of acute promyelocytic leukemia.

p53

Zinc-binding proteins appear to play various roles in the development and sup-pression of cancer and leukemia. Another recently identified zinc-binding protein thathas been shown to operate as one of the central supressors of cancer development isp53 [65]. This important protein has been christened ‘‘guardian of the genome.’’ Ithas been found that approximately half of human cancers involve a p53 dysfunction,either by mutation or deletion, or by the inactivation of p53 through binding of anoncoprotein. Normally, p53 acts to induce cell cycle arrest upon identification ofgenomic DNA damage. Mutations in p53 alter this response and therefore result in anincreased frequency of genomic rearrangements. The most common mutations occurin the central region of the protein, containing the sequence-specific DNA-bindingactivity. This results in an inactivation of the protein’s transcriptional activity. Thus,expression of genes possibly regulated by p53 may be altered, resulting in an over- orunderexpression of important proteins involved in the cell cycle.

This core portion of p53 folds into a compact structural domain with the assistanceof a zinc atom. This domain consists of a sandwich of two anti-parallelb-sheets,containing four and fiveb-strands, and a loop-sheet-helix motif which packs againstone end of the so-calledb-sandwich. At this end of the sandwich, there are two largeloops held together in part by the tetrahedral coordination of a zinc atom to threecysteines and a histidine residue. Therefore, the zinc atom, in concert with theb-sandwich, provides the necessary scaffolding, allowing the loop-sheet-helix motif andone of the two large loops to bind to DNA.

Although p53 appears to contain a novel and distinct DNA-binding motif, it alsoincludes characteristics that resemble other zinc fingers. p53 binds to the majorgroove of DNA by means of ana-helix, a feature reminiscent of the steroid hormonereceptors, and the RING-finger motif [66]. Additional contacts with the major grooveof the DNA involve loop packing at the N terminus of thea-helix, a property sharedwith the GATA-1 zinc finger. In contrast with the other DNA-binding zinc fingersdiscussed, p53 forms a tetramer and binds to a DNA element consisting of four copiesof a consensus pentamer.

CONCLUSION

Zinc in zinc fingers serves a critical role in maintaining the structure and hencefunction of these proteins. Various zinc-ligating motifs are known to exist in severalproteins that regulate gene expression, including (Cys)2(His)2, (Cys)4 and(Cys)3(His). Thus, a question arises: Are these different motifs interchangeable in agiven protein, or is it that the positioning of the metal-ligating amino acids in thetertiary structure of the protein determines the ultimate nature of the ligating motif?

114 Green et al.

Studies along these lines will not only provide worthwhile insight into the flexibilityand interchangeability of zinc-ligating motifs in zinc finger proteins, but will help tobetter define the surrounding primary structural constraints allowed by the variousmetal-ligating groups. Furthermore, zinc finger motifs provide an excellent frame-work for designing newer hybrid proteins which will have DNA-binding propertiescoupled with other functionalities. Such studies are well underway. While most zincfinger proteins are presumed to bind zinc in vivo, data are lacking in delineating ifother transition metals in these fingers could also serve to maintian DNA-bindingactivity. Recent results of metal replacement, especially by iron, raise importantquestions of DNA damage by free radical generation, and its relevance in metal-induced toxicity and/or carcinogenesis. As a transcription factor, zinc fingers play aprofound role in the control and regulation of gene expression. The structure andfunction of these proteins in relation to zinc as a scaffolding and itx exchangeabilitywith other metals opens up an unknown territory for future research which may haveimportant implications in diverse biological systems.

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