Sticky fingers: zinc-fingers asprotein-recognition motifsRoland Gamsjaeger*, Chu Kong Liew*, Fionna E. Loughlin, Merlin Crossley andJoel P. Mackay

School of Molecular and Microbial Biosciences, University of Sydney, NSW 2006, Australia

Review TRENDS in Biochemical Sciences Vol.32 No.2

Zinc-fingers (ZnFs) are extremely abundant in highereukaryotes. Once considered to function exclusively assequence-specific DNA-binding motifs, ZnFs are nowknown to have additional activities such as therecognition of RNA and other proteins. Here we discussrecent advances in our understanding of ZnFs as specificmodules for protein recognition. Structural studies ofZnF complexes reveal considerable diversity in terms ofprotein partners, binding modes and affinities, andhighlight the often underestimated versatility of ZnFstructure and function. An appreciation of the structuralfeatures of ZnF–protein interactions will contribute toour ability to engineer and to use ZnFs with tailoredprotein-binding properties.

IntroductionIn the 20 years since zinc-binding repeats were firstidentified in a protein (TFIIIA [1]), the DNA-bindingproperties of zinc-fingers (ZnFs) have been explored inexquisite detail [2], and it is now even possible to generateor indeed to purchase ‘designer’ ZnF proteins that willtarget specific genomic sites. These TFIIIA-type or ‘classi-cal’ ZnFs have proved to be common in complex organisms:in humans, >15 000 such domains are predicted to exist in�1000 different proteins [3]. More than 20 additionalclasses of structurally distinct modules, also termed ZnFs,have been identified [4], but it should be noted thatmany ofthese are related to classical ZnFs only by the existence ofthe structural zinc atom or atoms. Nevertheless, roles innucleic acid recognition are almost invariably postulatedwhen these domains are first described. In some cases,these predictions have been borne out: both GATA- andGal4-type ZnFs recognize DNA in a sequence-specificmanner [5,6], and several ZnF domains can bind to RNA(reviewed in Refs [7–10]).

It has also emerged, however, that ZnFs can mediateprotein–protein interactions [11]. In the past 3–5 years,detailed structural and functional data have been reportedfor several ZnF–protein interactions (Table 1), showingunequivocally that several classes of ZnFs (including clas-sical ZnFs) can function as protein recognitionmotifs. Herewe review the current understanding of ZnFs as proteinrecognition motifs with a strong emphasis on those ZnFclasses for which the structural basis has been determined.

Corresponding author: Mackay, J.P. (j.mackay@mmb.usyd.edu.au).*Authors contributed equally.Available online 8 January 2007.

www.sciencedirect.com 0968-0004/$ – see front matter � 2006 Elsevier Ltd. All rights reserve

One-zinc wonders: classical, GATA, RanBP and A20ZnFsIn the following sections, zinc-binding domains have beengrouped according to their zinc ligation topology. We firstconsider domains that coordinate a single zinc atom. Sev-eral of these classes of ZnFs are well known as DNArecognition motifs and, notably, the interface used by someof these domains to contact DNA often differs from thatinvolved in protein–protein interactions.

Classical ZnFs

Classical ZnFs comprise a short b hairpin and an a helixwith a zinc atom coordinated in either a Cys-Cys-His-His ora Cys-Cys-His-Cys manner (Figure 1a). The transcrip-tional regulator FOG-1 (‘Friend of GATA-1’) contains nineclassical ZnF domains (Figure 1a), at least five of which canmediate protein–protein interactions [12,13]. Remarkably,four of these ZnFs can independently bind the N-terminalGATA-type ZnF of GATA-1 (termed the ‘N-finger’;Figure 1a), and the recruitment of FOG-1 to gene promo-ters by GATA-1 gives rise to changes in gene expressionthat are essential for normal erythro- and megakaryopoi-esis in mammals [14]. The recent structure of the GATA-1N-finger in complex with a FOG-type finger (from theorthologous Drosophila protein U-shaped; Figure 2a)showed for the first time the molecular details of a proteininteraction mediated by classical and GATA-type ZnFs[15].

The GATA-1 N-finger also recognizes GATC sequencesin DNA [16] and the GATA–FOG-finger structure showsthat the predicted DNA recognition surface of the N-fingeris distinct from the FOG-binding surface (Figure 2a). Pull-down data confirm that the N-finger can interact with bothDNA and FOG-1 simultaneously [15] – a surprisinglydexterous feat for a single ZnF. By contrast, the surfaceused by FOG ZnFs to contact GATA-1 (Figure 2b) overlapsconsiderably with the highly conserved DNA recognitionface of classical ZnFs (Figure 2c). This observation, incombination with the fact that the GATA-binding ZnFsin FOG-1 are widely spaced in the protein (i.e. they are notpresent in the tandem arrays that are associated withDNA-binding activity), supports the possibility that theseFOG fingers are not typical DNA recognition modules butinstead have a primary role in protein recognition.

A tandem array of three classical ZnFs, termed ZnF2,ZnF3 and ZnF4, exists in FOG-1. Despite considerableefforts from more than one laboratory, however, no

d. doi:10.1016/j.tibs.2006.12.007

Table 1. Structures of ZnF–protein complexes with coordinates in the Protein Data Bank

ZnF Protein Partner protein Function Refs PDB code ID Kd (M)

GATA GATA-1 FOG Transcription regulation [15] 1Y0J 10�5

ZnF UBP IsoT Ubiquitin Deubiquitination [30] 2G43 10�6

RanBP Npl4 Ubiquitin Ubiquitination (proteolysis) [35] 1Q5W 10�4

A20 Rabex5 Ubiquitin Ubiquitination (proteolysis) [40] 2FID 10�5

LIM LMO2 (N terminus) Ldb1 Transcription regulation [78] 1J2O NA

LIM LMO4 (N terminus) Ldb1 Transcription regulation [78] 1M3V NA

LIM LMO4 Ldb1 Transcription regulation [46] 1RUT 10�9

LIM PINCH Nck2 (SH3 domain) Cell adhesion and migration [49] 1U5S 10�2

RING c-Cbl UbcH7 Ubiquitination (proteolysis) [60] 1FBV NA

RING Rbx1 Cul1 Ubiquitination (proteolysis) [61] 1LDJ NA

RING Cnot4 Ubch5B Ubiquitination (proteolysis) [79] 1UR6 NA

RING Bmi1 Ring1B Ubiquitination (proteolysis) [80,81] 2CKL, 2H0D NA

PHD ING2 H3K4me3 Chromatin regulation [65] 2G6Q 10�6

PHD BPTF H3K4me3 Chromatin regulation [66] 2FUU 10�6

TAZ1 CBP/p300 HIF-1a Cellular hypoxic response [73] 1L8C 10�7

TAZ1 CBP/p300 CITED2 Cellular hypoxic response [75,82] 1P4Q, 1R8U 10�8

Abbreviations: Kd, dissociation constant; NA, not applicable.

64 Review TRENDS in Biochemical Sciences Vol.32 No.2

DNA-binding activity has been demonstrated for this array(e.g. see Ref. [17]). Instead, ZnF3 is both necessary andsufficient to mediate an interaction with the coiled-coildomain of Transforming acidic coiled-coil protein 3(TACC3) [13]. The functional implications of this inter-action are not well defined, although cellular data suggestthat TACC3 might inhibit the nuclear localization of FOG-1 [18]. Structural and mutagenesis studies have localizedthe TACC3-bindingmotif to the a helix of FOGZnF3, againoverlapping with the traditional DNA-binding surface ofclassical ZnFs (Figure 2d).

Figure 1. Topology and structures of ZnF domains. The grouping of the ZnF domains ref

GATA (1GAT), ZnF UBP (2G43), RanBP (1Q5W), A20 (2FID). (b) LIM (1A7I) and MYND (

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Transcription factors of the Ikaros family, which haveessential roles in lymphoid development, contain twoclusters of classical ZnFs: an N-terminal cluster of 3–4ZnFs that binds DNA, and a C-terminal cluster of twoclassical ZnFs that mediates homo- and heterotypic inter-actions with other family members [19]. Self-associationis important for high-affinity DNA binding by Ikaros, andits C-terminal ZnFs behave as a dimer in gel-filtrationstudies [20]. By contrast, the C-terminal ZnFs of anotherfamily member, Eos, seem to form an oligomer containingapproximately nine monomers [21], although detailed

lects the order in which they are described in the text. (a) Classical (PDB code 1FU9),

1FV6). (c) RING (1CHC) and PHD (1XWH). (d) TAZ (1R8U).

Figure 2. Protein-binding surfaces of classical, GATA-1, RanBP and A20 ZnF domains. The zinc atoms and ligating side chains are shown in dark blue. (a) The GATA-1 N-

finger (light blue) in complex with the classical ZnF from Drosophila U-shaped (red; PDB code 1Y0J). FOG-binding residues of the GATA-1 N-finger are shown in green and

DNA-binding residues in yellow. (b) First ZnF of Drosophila U-shaped (1Y0J) with GATA-1-binding residues shown in green. (c) First ZnF of Zif268 (1AAY) with DNA-binding

residues shown in green. (d) Third ZnF of FOG-1 (1SRK) with TACC3-binding residues shown in green. (e) RanBP ZnF of Npl4 (light blue) in complex with ubiquitin (red; PDB

1Q5W). Ubiquitin-binding residues of Npl4 ZnF are shown in green. (f) A20 ZnF of Rabex-5 (light blue) in complex with ubiquitin (red; 2FID). Ubiquitin-binding residues of

the Rabex-5 ZnF are shown in green.

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structural information has been elusive for these proteinsbecause of solubility and aggregation issues. The structureof a single monomeric C-terminal ZnF from Eos is highlydynamic [22] andmight be stabilized on oligomerization. Amutagenic analysis [22] has shown that both ZnFs of Eosare required for binding and that, again, residues in the a

helix are important for the interaction.Like members of the Ikaros family, the transcriptional

regulator Roaz uses its N-terminal classical ZnFs to bindDNA and its C-terminal fingers to mediate homo- andheterodimerization [23]. Classical ZnFs in the Wilms’tumour protein WT1 seem to mediate interactions withbothDNAand theHMGbox of the transcription factor SRY[24]; however, no structural data are available for either ofthese interactions.

GATA-type ZnFs

The C-terminal ZnF or ‘C-finger’ of GATA-1 (which is theprimary DNA-binding domain of GATA-1) has also beenreported to mediate interactions with several transcrip-tional regulators, including CBP, Fli-1, Sp1, EKLF andPU.1 [25–28]. However, many of these interactions haveproved to be difficult to analyze in detail, and structuraldata are available only for the interaction between PU.1and GATA-1 [29]. PU.1 drives the development of myeloidcells, whereas GATA-1 regulates erythroid gene expres-sion. The two proteins each antagonize the activity of theother, and it is thought that their direct interactioncontributes to lineage determination [28]. The GATA-1C-finger interacts with the Ets domain of PU.1, itself a

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well-characterized DNA-binding domain [28]. Thisinteraction has an unexpectedly low affinity (with anassociation constant of �103 M�1), and a combination ofmutagenesis and NMR titration data has implicated boththe a helix and a C-terminal, disordered region of the C-finger in mediating the association [29]. This contrastswith the FOG-binding surface of the GATA-1 N-finger,which comprises residues from the b strands on the oppo-site side of the domain. Notably, some of the PU.1-bindingresidues of the GATA-1 C-finger are also used to contactDNA. Inspection of these residues in the complex betweenthe GATA-1 C-finger and DNA [6] shows that theyare relatively accessible, raising the possibility that theC-finger binds simultaneously to both PU.1 andDNA. Suchbinding, however, has not been demonstrated.

Interestingly, the deubiquitinating enzyme IsoTcontains a ubiquitin-binding domain, termed ZnF UBP,that is a remarkable elaboration of the GATA-type ZnFstructure, containing a 13-residue insertion between thethird and fourth zinc ligands, and considerable additionalstructure (Figure 1a). This domain, which is found inseveral other protein families, recognizes the C-terminaldiglycine of ubiquitin and contributes to the specificity ofIsoT activity in the recycling of polyubiquitin chains [30].

RanBP ZnFs

RanBP ZnFs are found in �17 human proteins, each ofwhich contains between one and eight RanBP ZnFdomains [31]. These domains are named for their presencein the nuclear export protein RanBP2, although little is

Figure 3. Diversity of protein-binding surfaces within the LIM domain family. The zinc atoms and ligating side chains are shown in dark blue. (a) Fusion protein containing

the two LIM domains of LMO4 (light blue) and ldb1-LID (red; PDB code 1RUT). Residues of LMO4 that participate in hydrophobic or electrostatic interactions are shown in

green. b Strands of LMO4 that pair up with a b strand from Ldb1-LID are shown in yellow. (b) LIM1 domain of PINCH (1G47) with ILK-binding residues shown in green. (c)

LIM4 domain of PINCH (light blue) in complex with the SH3 domain of Nck2 (red; 1U5S); the Nck2-binding residues are shown in green. For reference, domains in (b) and (c)

are shown in the same orientation as the LIM1 domain of LMO4 in (a).

66 Review TRENDS in Biochemical Sciences Vol.32 No.2

known about their function in this protein. RanBP ZnFsconsist of two short b hairpins that sandwich a single zincatom (Figure 1a) and are related to the zinc ribbon fold [32].Although it has been reported that a RanBP ZnF inZNF265 can recognize mRNA [33], a subset of these ZnFsbind ubiquitin [34].

Recently, a structure was reported for the complexformed between ubiquitin and the RanBP domain ofNpl4 [35] (Figure 2e), a protein with roles thatinclude endoplasmic-reticulum-associated degradationand nuclear-envelope reassembly [36]. A principaldeterminant of this interaction is a Thr-Phe dipeptidesituated between two of the zinc ligands in Npl4 (in aCys-Thr-Phe-Cys arrangement). Indeed, this dipeptideseems to define the ubiquitin-binding subset of RanBPZnFs, and introduction of the crucial contact residues intoa RanBP ZnF that does not bind ubiquitin is sufficient togenerate ubiquitin-binding activity [35].

A20 ZnFs

Ubiquitin-binding activity is also shown by thetreble-clef ZnFs from the A20 family (Figure 1a), whichwere originally identified in the protein A20 [37]. The A20ZnF from the guanine nucleotide exchange factor Rabex-5shows ubiquitin ligase activity [38], and two crystal struc-tures of this ZnF in complex with ubiquitin have beenrecently published [39,40] (Figure 2f). Most other ubiqui-tin-binding domains characterized so far (including theRanBP ZnF domain of Npl4 discussed earlier) contact ahydrophobic surface on ubiquitin that is centred on Ile44.Importantly, the A20 ZnF of Rabex-5 binds a completelydifferent surface on ubiquitin, centred on Asp58.

The existence of these two distinct interaction modes forubiquitin raises the possibility that a single molecule ofubiquitin might be recognized simultaneously by two

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different ubiquitin-binding receptors [39]. Interestingly,proteins containing A20 and RanBP ZnFs have opposingeffects on the NF-kB signalling pathway. The RanBP ZnFsof the adaptor proteins TAB2 and TAB3 interact withpolyubiquitin chains to activate signalling [41], whereasthe ZnF domains of A20 polyubiquitinate the mediatorprotein RIP, targeting it for degradation and therebydownregulating NF-kB signalling [42].

MYND your LIMsSeveral classes of ZnFs coordinate more than one zincatom. LIM domains (named after the three proteins,LIN-11, Isl1 and MEC-3, in which they were first discov-ered [43]) and MYND domains (named after the threeproteins myeloid translocation protein 8, Nervy andDEAF-1 [44]) both comprise two sequential zinc-bindingmotifs that pack against each other to make a singlecompact domain. Both of these domains mediateprotein–protein interactions.

LIM domains

LIM domains comprise two sequential zinc-bindingmodules that resemble the treble-clef GATA-type fold(Figure 1b). The consensus sequence is rather variable:cysteine, histidine, aspartate and glutamate residues areall known to be involved in zinc coordination, and the loopsbetween zinc ligands differ in length [45]. Fifty-eighthuman proteins contain LIM domains – these proteinseither consist completely of two or more LIM domains orcontain additional domains, including protein recognitionor catalytic modules. LIM proteins are involved in a rangeof biological functions, including gene expression, cytoske-leton organization and development [45]. Current dataindicate that LIM domains function predominantly asprotein recognition motifs, and it has been proposed that

Figure 4. RING and PHD domains. (a) Overall view of the ternary complex of c-Cbl

(E3), UbcH7 (E2) and phosphorylated ZAP-70 peptide (the substrate for

ubiquitination by E3; PDB code 1FBV). The c-Cbl domain, which includes the

RING domain, is shown in light blue, zinc atoms and ligating residues are in dark

blue, the UbcH7 domain in red and the ZAP-70 peptide in yellow. In the enlarged

image of the RING domain, E2-binding residues are shown in green. (b) PHD

domains of ING2 (2G6Q) and BPTF (2F6J) interacting with a six-residue H3K4me3

peptide. PHD domains are shown in light blue, the H3K4me3 peptide in red and the

methylated lysine in stick representation. Zinc atoms and ligating residues are dark

blue. The BPTF structure also has a bromodomain (C-terminal to the PHD domain)

that is not shown here.

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proteins that contain multiple LIM domains mightfunction as molecular bridges [45].

The LIM-only protein LMO4 and its binding partnerldb1 are both negative regulators of mammary epithelialdifferentiation. Their interaction is mediated by the twotandem LIM domains of LMO4 and the LIM interactiondomain (LID) from ldb1. The crystal structure of theLMO4–ldb1-LID complex [46] shows that ldb1-LID bindsin an extended form to LMO4, spanning thewhole length ofthe two LIM domains and forming a tight complex. A seriesof backbone hydrogen bonds is formed, whereby ldb1 effec-tively adds an additional b strand to four of the eight b

hairpins that make up the two LIM domains (Figure 3a).The nature of the side chain interactions between ldb1-LIDand LMO4 differ, however, for each of the two LIMdomains: the LIM1 interface is a mixture of ionic andhydrophobic interactions, whereas the LIM2 interface isprimarily hydrophobic.

The adaptor protein PINCH consists solely of five LIMdomains and has roles in cell adhesion, growth and differ-entiation [47]. PINCH interacts with the integrin-likekinase (ILK) and Nck2, and both interactions are essentialfor integrin signalling. Specifically, the LIM1 domain ofPINCH interacts with the ankyrin repeat domain of ILK,and this interaction is localized to the second zinc-bindingmodule of PINCH-LIM1. Chemical shift perturbation stu-dies have identified a hydrophobic surface patch, made upof residues from the C-terminal helix, as the likely bindingsite for ILK [48] (Figure 3b). PINCH-LIM4 mediates theinteraction with the third Src homology 3 (SH3) domain ofNck2 [49] and does so through a different binding mode. Inthis case, it is the first b hairpin of LIM4 that contacts theSH3 domain of Nck2 (Figure 3c), forming a weak complex(dissociation constant, Kd �3 � 10�3 M) in which two argi-nines from LIM4 are responsible for the formation of saltbridges and hydrophobic interactions with Nck2.

MYND domains

MYND domains also ligate two zinc atoms in a sequentialmanner (Figure 1b), but they are smaller than LIMdomains (�40 residues versus �55 residues for LIMdomains). The first zinc-binding module contains only ashort b hairpin, whereas the second zinc-binding moduleforms two short a helices [50]. Like LIM domains, MYNDdomains function primarily as protein-binding domainsand are present mainly in transcriptional regulators[51,52]. No structural information is available onMYND-mediated interactions, although a few MYNDdomains recognize a Pro-X-Leu-X-Pro (PXLXP) motif intheir partner proteins. For example, the MYND domain inthe transcriptional co-repressor BS69 interacts with theviral oncoproteins E1A and EBNA2 in addition to thetranscription factor MGA, all of which contain a PXLXPmotif [52]. Mutational data implicate a large, positivelycharged surface patch, which is conserved in PXLXP-bind-ing MYND domains [50].

Cross-brace yourself for PHD and RING actionLike LIM and MYND domains, RING (‘really interestingnew gene’ [53]) and PHD (‘plant homeodomain’; named forthe class of proteins in which it was first recognized [54])

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domains also coordinate two zinc atoms. For RING andPHD domains, however, the two sets of zinc ligands are notsequential, but instead are interdigitated (Figure 1c) or‘cross-braced’.

RING domains

Both RING and PHD domains function primarily asprotein–protein interaction motifs and are found in manyhuman proteins (�200 for RING and �70 for PHD) [31].Until recently, confusion has existed in the classification ofthese domains owing to their similar consensus sequencesand zinc-ligation topology, and several RING domainshave been mistakenly labelled as PHD domains [55].Now, with more sequence and structural information athand, the distinction is clearer. PHD domains contain acrucial tryptophan residue, located two residues N-term-inal to the seventh metal ligand, that is involved in thehydrophobic core; in RING domains, by contrast, the equiv-alent residue is solvent exposed [56]. Furthermore, RINGdomains seem to participate in a wider range of functions.For example, RING domains in KAP-1 and PML mediateinteractions with the transcription factor KOX-1 [57] andthe mRNA cap-binding protein eIF4E [58], respectively,thereby regulating gene repression and RNA transport.

Many RING domains are present in E3 ubiquitinligases, which catalyze the final step in the protein ubiqui-tination pathway. Ubiquitination targets proteins for pro-teosomal degradation and has been shown to function as asignalling module for the destruction of plasma membrane

Figure 5. Complexes of the TAZ1 domain of CBP. (a) TAZ1–HIF-1a complex (PDB

code 1L3E). (b) TAZ1–CITED2 complex (1P4Q). The two structures show that HIF-1a

and CITED2 compete for the same binding motif on the TAZ1 domain of CBP,

which is shown in green and consists of the sequence LP(E/Q)L. The TAZ1 domain

of CBP is shown in light blue, zinc atoms and ligating residues in dark blue, and

HIF-1a and CITED2 in red.

68 Review TRENDS in Biochemical Sciences Vol.32 No.2

proteins in vacuoles and lysosomes [59]. Several hundredproteins are predicted to function as E3 ligases, catalyzingthe transfer of ubiquitin units from E2 transferases to thetarget protein, and RING domains are present in a sub-stantial proportion of these proteins. The X-ray crystalstructure of the RING E3 ligase c-Cbl bound to its pairedE2 UbcH7 shows that the RING domain makes directcontact with UbcH7 through a shallow groove on the sur-face of the former [60] (Figure 4a). This groove seems to beconserved in other E3 ligase RING domains [61]. Interest-ingly, several RING domains from E3 ligases seem topossess intrinsic E3 ligase activity [62], although nomechanistic explanation for this activity is available atpresent.

PHD domains

Despite the discovery of PHD domains 14 years ago [54],detailed information on the function of these modulesbecame available only in 2006. Sequence and functionalanalyses of proteins that contain PHD motifs have longpointed to a role in the regulation of chromatin structure,and these suspicions have been finally confirmed with thepublication of four back-to-back papers in Nature on thePHD domains of ING2 and BPTF. Both of these proteinscooperate with p53 to induce cellular growth arrest andapoptosis, linking chromatin regulation with p53 functionand tumour suppression [63]; in addition, their PHDdomains have been shown to recognize specifically theN-terminal tail of histone H3 in which Lys4 is trimethy-lated (H3K4me3) [64–67]. Comparison of the structures ofthe ING2 and BPTF complexes reveals a similar bindingmode in which the trimethylated lysine side chain isrecognized by a ‘cage’ of, respectively, two and four aro-matic side chains (Figure 4b). A related mechanism is usedby the double chromodomains of CHD1 – a chromatinassembly factor that also binds methylated H3 tails [68].

The PHD domains of Pygopus, a Drosophila nuclearprotein, recruit the protein Legless [69] as part of the Wntsignalling pathway and seem, from mutagenic data, to dothis through residues in the loop between the fifth andsixth cysteines. Interestingly, this loop is located in aportion of the PHD domain that corresponds to the surfaceof E3 RING domains.

Fan-TAZ-tically differentTAZ (‘transcriptional adaptor zinc-binding’) domains seemto exist only in the transcriptional coactivator CBP, wheretwo ZnFs form a fold that is unrelated to all other ZnFs[70]: namely, three zinc atoms are present in a triangularstructure, where each zinc atom is present in a loop regionbetween two antiparallel a helices (Figure 1d).

CBP is an acetyltransferase that is recruited to DNAthrough interactions with a wide array of DNA-bindingtranscription factors [71]. The TAZ domains are respon-sible for mediating many of these interactions. Forexample, the more N-terminal TAZ1 domain binds toHypoxia-inducible factor 1a (HIF-1a); this factor recruitsCBP under conditions of oxygen deprivation to direct theexpression of genes necessary for the survival of thecell [72]. The structure of TAZ1 bound to a peptide fromHIF-1a [73,74] shows a surprising arrangement in which

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the HIF-1a peptide forms three helices that wrap almostcompletely around the TAZ1 domain (Figure 5a). Interest-ingly, CITED2 competes with HIF-1a for TAZ1 in a feed-back mechanism during the hypoxic response, binding in adifferent way but to an overlapping site on TAZ1 [75](Figure 5b). Subtle differences between the structures ofTAZ1 and TAZ2 (in the fourth a helix) prevent TAZ2 frombinding to HIF-1a [73,74], illustrating the specificity ofthese two domains for different transcription factor tar-gets.

Concluding remarksAlthough a model of ZnFs as DNA-binding domains wasestablished after their early characterization, numerousrecent studies have confirmed that a major function ofmany classes of ZnFs is to function as protein recognitionmodules. Indeed, even in the classes of ZnFs for whichDNA-binding activity is well established (e.g. GATA-typeand classical ZnFs), several protein recognition eventshave now been observed. Given that the functions of manythousands of classical ZnFs have not been experimentallyaddressed, and that a substantial portion of these domainsexist in isolation (rather than in tandem DNA-bindingarrays), it seems probable that many more classicalZnF-mediated protein interactions await discovery. Whena significant number of such interactions have been charac-terized, we might be in a position to predict with someaccuracy whether a given classical ZnF will bind to otherproteins or to DNA or to both.

Diversity is also apparent within a single class of ZnF inthat different members of that class adopt distinct protein-binding modes: both the classical ZnF and the LIMdomains are striking examples of this point. These obser-vations reinforce the idea that ZnF domains have evolvedas stable molecular scaffolds, onto which evolution hasgrafted different binding functions as required. One con-served feature among these complexes is that ZnFs tend toundergo little, if any, structural rearrangement on recog-nizing their protein partners. This lack of rearrangement ispresumably due in part to the stability afforded by zinc

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ligation. Another notable feature is that several of theseinteractions are modular in nature, similar to the mode ofinteraction observed for classical ZnFs binding to DNA. Forexample, the two LIM domains of LMO proteins are inde-pendently folded units that contact adjacent parts of theldb1-LID peptide. Similarly, it is possible that the tandemPHD domains of proteins such as Mi2b [76] will be shownto bind simultaneously to different methylated lysine resi-dues in the same (or different) histone tails. It will beinteresting to see whether other proteins with multipleZnFs adopt a related mechanism for recognizing extendedsubstrates.

As shown in Table 1, ZnF domains mediate interactionswitharangeofdifferentaffinities.These interactions tend tobe of moderate to weak affinity, which is perhaps not sur-prising considering the relatively small size of many ZnFdomains.Nevertheless, the biological importance ofmany ofthe weaker interactions (such as GATA-1–FOG-1, Npl4–ubiquitin and PINCH–Nck2) is indisputable, and despitetheir low affinity the interactions are also highly specific.These data emphasize the prevalence of weak interactionsin regulating cellular processes; it is likely that as structuralmethods become more sensitive we will see an increasingnumber of weak complexes characterized in moleculardetail. It is also possible that post-translational modifi-cations, such as lysine methylation, might increase theaffinity of some of these weak interactions in vivo.

Remarkable progress has been recently made in design-ing tandem arrays of ZnFs with tailored DNA-bindingspecificities, with the ultimate goal of using them as thera-peutics [77]. Although designing protein-binding ZnFswith tailored specificities also holds considerable thera-peutic potential, it is clearly a considerably more difficulttask. The greater variability and complexity of protein-binding interfaces, in addition to the small number ofstructural studies on protein-binding ZnFs, are currentlysignificant impediments. As we have highlighted here,however, progress is being made to address what was,until recently, a substantial deficiency in our understand-ing and, as more ZnF–protein interactions are discovered,our knowledge of such interactions and our potential tomanipulate them can only increase.

AcknowledgementsR.G. is supported by a fellowship from the Austrian Science Fund (J2474).C.K.L. is a C.J. Martin Fellow of the National Health and MedicalResearch Council (NHMRC) and F.E.L. holds an Australian PostgraduateAward. J.P.M. is an NHMRC Senior Research Fellow. This work wassupported by an NHMRC Program Grant to J.P.M. and M.C.

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