Proc. Nati. Acad. Sci. USAVol. 91, pp. 10655-10659, October 1994Biochemistry

The LIM/double zinc-finger motif functions as a proteindimerization domainRON FEUERSTEIN*, XINKANG WANG*, DECHENG SONG*, NANCY E. COOKE*, AND STEPHEN A. LIEBHABER*t#*Departments of Genetics and Medicine and the tHoward Hughes Medical Institute, University of Pennsylvania School of Medicine,Philadelphia, PA 19104-6145

Communicated by Y. W. Kan, June 28, 1994

ABSTRACT Protein-protein interactions resulting indimerization and heterodimerization are of central importancein the control ofgene expression and cell function. Proteins thatshare the 52-residue LIM/double zinc-finger domain areinvolved in a wide range ofdevelopmental and cellular controls.Some of these functions have been hypothesized to involveprotein dimerization. In the present report we demonstrate,using both in vitro and cell-based studies, that a representativeLIM protein, human cysteine-rich protein (hCRP), can effi-ciently homodimerize. The dimerization ability of hCRP ismapped to the LIM domains, can be transferred to an unre-lated protein by fusion of a single minimal LIM/doublezinc-finger segment, occurs in the absence as well as thepresence of DNA, and appears to depend on coordination oftwo zinc atoms in the finger doublet. These observationssupport a specific role for protein dimerization in the functionof proteins containing the LIM/double zinc-finger domain andexpand the general spec of potential interactions mediatedby zinc-finger motifs.

Proteins capable of dimerization tend to belong to extendedfamilies that share particular protein-protein interaction mo-tifs. These motifs, which can mediate both homo- and het-erodimerization, including the leucine zipper (1, 2), helix-loop-helix (3, 4), ankyrin (5), and PAS (6) domains. The LIMprotein family, named for three of the originally identifiedmembers, lin-11 (7), isl-1 (8), and mec-3 (9), is defined by thepresence of one to three repeats of a 52-residue segmentcontaining two adjacent zinc fingers separated by a two-residue linker, (CX2CX17HX2C)-X2-(CX2CX17CX2C/H/D).Of the 12 LIM proteins defined thus far, 7 contain a DNA-binding homeodomain adjacent to the LIM domains, and theremaining 5 members lack a homeodomain. The LIM pro-teins are involved in a wide range of cell functions, includingtranscription activation [isl-1, lmx-1 (10)], somatic patterning[lin-11, mec-3, apterous (11)], focal cell adhesion [zyxin (12)],oncogenic transformation [rhombotin (13, 14)], and immedi-ate-early response to serum stimulation [cysteine-rich pro-tein (CRP) (15)]. There have been reports suggesting theinvolvement of LIM proteins in protein-protein interactions(10, 16, 17). In one case two LIM proteins lacking homeo-domains, CRP and zyxin, copurified as a complex from focaladhesion plaques (12). Although a "docking model" has beenputforward which hypothesizes that LIM proteins can dimer-ize (18), it has not been demonstrated that this occurs, nor hasit been shown that the LIM domain directly mediates suchinteractions. In the present study we demonstrate that arepresentative LIM protein, CRP (19), a highly conserved,widely distributed, immediate-early gene containing two re-peated LIM/double zinc-finger domains, can homodimerizewhen tested in vitro and in vivo and that the LIM domain isnecessary and sufficient for this activity.

MATERIALS AND METHODSGeneration of cDNA Subclones and Fusion Constructs.

cDNA fragments corresponding to each of the desired re-gions of human CRP (hCRP) were generated by the poly-merase chain reaction (PCR) using primers (sequences avail-able upon request) containing EcoRP restriction sites. Forsynthesis of glutathione S-transferase (GST) fusion proteins,the pGEX2T(128/129) vector (gft ofM. Blanar, University ofCalifornia, San Francisco) was used.

Site-directed mutagenesis was carried out by PCR usingoligonucleotide primers (sequences available upon request)containing the appropriate point substitutions at amino acids9 and 62. Each mutation was verified by DNA sequenceanalysis.Antibody Production. A synthetic peptide representing

residues 83-99 of hCRP was conjugated to keyhole limpethemocyanin by glutaraldehyde crosslinking and used to im-munize rabbits (Hazelton Research Products, Lenexa, KS).Clarified antiserum was used without further purification.

Expression and Analysis of Recombinant Proteins. GSTfusion proteins expressed and purified from clarified bacteriallysate, as described (20), were incubated with a 50% slurry ofglutathione agarose beads (Sigma) for 15 min at 40C andwashed three times with phosphate-buffered saline (PBS),and the fusion proteins were eluted by incubating beads with2 bead vol of 10mM glutathione/50mM Tris-HCl, pH 8.0, for5 min at room temperature. Approximately 5 pg of eachprotein was loaded on a 12% polyacrylamide/SDS gel andelectrophoresed for 3 hr at 200 V followed by electrotransferto a nitrocellulose membrane and immunoblotting usingstandard techniques. Antibody binding on Western blots wasdetected by using the ECL system (Amersham).

Generation of 32p Labed hCRP Fragments. For radiola-beling, bacterially expressed GST-hCRP fusion proteinswere incubated with glutathione-agarose beads, and theloaded beads were then washed and labeled for 60 min at 370Cin 2 bead vol of lx HMK buffer (21) containing cAMP-dependent protein kinase (Sigma) at 1 unit/pl, ['y.32P]ATP(5000 Ci/mmol, 10 mCi/ml; Amersham; 1 Ci = 37 GBq) at 10puCi/pl, and 1 mM dithiothreitol. After incubation the beadswere washed five times with PBS. For cleavage, the labelingmix was washed once in thrombin cleavage buffer (50 mMTris HCl, pH 8.0/150 mM NaCl/2.5 mM CaCl2/0.1% 2-mer-captoethanol) followed by incubation in 2 bead vol of lxthrombin cleavage buffer containing human thrombin (Sig-ma) at a 1:10 wt ratio of enzyme to substrate for 40 min atroom temperature. The beads were sedimented and thelabeled CRP fragments were collected from the supernatant.

Abbreviations: CRP, cysteine-rich protein; hCRP, human CRP;GST, glutathione S-transferase; CAT, chloramphenicol acetyltrans-ferase.tTo whom reprint requests should be addressed at: Department ofGenetics, University of Pennsylvania, Room 437A Clinical Re-search Building, Philadelphia, PA 19104.

10655

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. Filter overlay assay demonstrating specificity of CRP dimerization; localization of the interacting region to the LIM domain. (A)Diagram of CRP. CRP contains two LIM/double zinc-finger domains (15, 19, 24). A glycine-rich region of undetermined function is locatedimmediately carboxyl-terminal to each LIM/double finger domain. The position of peptide A, synthesized for epitope-specific antiserumproduction, is shown. The extent of each CRP segment that was fused to GST is indicated. (B) Expression and analysis of GST/CRP fusionproteins. (Upper) Coomassie blue-stained, 12% polyacrylamide/SDS gel ofpurifiedGSTand five differentGST-hCRPfusion products. Positionsof the molecular mass markers (in kDa) are shown to the left of the gel. (Lower) Western blot analysis of the fusion protein gel shown in Upperwith anti-hCRP polyclonal antisera specific to the peptide A epitope. (C) Generation of 32P-labeled hCRP proteins. Bacterially expressedGST-hCRP41-193) fusion protein was labeled by incubation with bovine heart muscle kinase (HMK) and [t32P]ATP, then cleaved with thrombinand analyzed on a 12% polyacrylamide/SDS gel. The diagram of the GST-hCRP fusion protein indicates the approximate positions of thethrombin cleavage site and the HMK phosphorylation site. The 32P-hCRP-(118-171) and 32P-hCRP-(62-118) probes were generated in identicalfashion. (D) Overlay binding assay for CRP-CRP interaction. GST-CRP proteins were electrotransferred from 12% polyacrylamide/SDS gelsto nitrocellulose membranes, incubated with 32P-labeled hCRP42-193) or the indicated subfiagments in the presence or absence of unlabeledcompetitors, washed, and autoradiographed. The identity ofeachfusion protein is indicated at the tops ofthe lanes; the positions of size markers(kDa) are noted. The identity of the 32P-labeled probe used in each gel is shown at its top. With the 32P-hCRP-(118-171) probe, the middle andbottom membranes were preincubated with 5 pAM unlabeled GST-hCRP-(118-171) or GST-hCRP-(80-118) (estimated 15,000-fold molar excessof competitor).

Filter Overlay Binding Assay. Electrotransfer from SDS/polyacrylamide gels to nitrocellulose membranes was carriedout at 20 V for 30 min, followed by incubation of themembranes for 12-15 hr at room temperature in blockingsolution (5% bovine serum albumin/50 mM Tris HCl, pH7.5/150 mM NaCi). When a competition experiment wasperformed, the filters were incubated with unlabeled com-petitors in overlay buffer (OB) (20mM Hepes, pH 7.5/0.25%gelatin/0.5% bovine serum albumin/0.1% 2-mercaptoetha-nol/1 mM EGTA/10 mM NaCi) for 2 hr at room temperaturefollowed by 4 hr in OB containing probe at 600,000 cpm/ml.After incubation, membranes were washed twice in universalbuffer (50mM Tris HCl, pH 7.5/150mM NaCl) for 5 min andonce in Sarkosyl buffer (1M NaC1/0.5% N-lauroylsarcosine/0.25% gelatin/50 mM Tris HCI, pH 7.5) for 30 min. Mem-branes were then dried and autoradiographed.

Two-Hybrid Interaction Assay. VP16 and GAL4 expressionvectors were gifts of C. Dang (Johns Hopkins University).Each ofthecDNA frMgments used in this assay was generatedby PCR (see above) and cloned in plasmid pNLVP, whichcontains the transcriptional activating domain for the Herpessimplex viral protein VP16. DNA encoding full-length hCRP-(1-193) was inserted into pGAL4 plasmid, which encodesamino acids 1-147 of the DNA-binding domain of the yeastprotein GAL4. The full-length hCRP-(1-193) construct in-cludes an additional 12 amino acids between GAML and hCRPencoded by the hCRP 5' nontranslated region. NIH 3T3 cellsat 90%o confluency were transfected with combinations ofsupercoiled plasmids (total of 30 .g) by Ca3(P4)2 coprecip-itation and assayed for chloramphenicol acetyltransferase(CAT) activity by the standard technique (23) after 2 days inculture. Plasmid pCH110, which contains the /-galacto-

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Proc. Nad. Acad Sci. USA 91 (1994)

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Proc. Natl. Acad. Sci. USA 91 (1994) 10657

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FIG. 2. Analysis of CRP-CRP interaction by the two-hybrid interaction assay. (A) Two-hybrid protein interaction assay. In the upper partof the diagram, the DNA-binding domain ofGAL4 and the transcriptional activation domain of VP16 do not directly interact and the minimalE1B promoter controlling CAT gene expression is not activated. In the lower part of the diagram, CAT gene transcription is activated ifGAL4and VP16 can be brought together by their fusion protein partners. The fusion protein extension on each is a representation of theLIM/double-finger domain. (B) Map of the hCRP cDNA fiagments fused to VP16 and GAL4 fragments. The first two subclones, (7-79) and(62-118), contain the LIM and inter-LIM regions, respectively, while the third, (2-188), encompasses both regions. (C) Two-hybrid interactionresults. Each bar on the histogram represents an average taken from at least three separate experiments run in duplicate. A representative CATanalysis is shown below the histogram. Each lane is labeled with the chimeric plasmids transfected alone or in combination into NIH 3T3 cells.Negative controls were included to be sure that hCRP does not bind directly to GALA tGAL4 + VP16-hCRP-(2-188)], to VP16 [GAL4-hCRP-(1-193) + VP16], or act as a transcriptional activator on its own [GAL4-hCRP-(1-193)]. The level of activity of the GAL4-hCRP-(1-193) withVP16-hCRP-(2-188) was 20%6 of that generated by fusing the same GAL4 and VP16 regions to the leucine zipper domains of fos and jun,respectively (data not shown).

sidase coding sequence under the control of the simian virus40 early promoter, was cotransfected along with each set oftest plasmids, and the amount of cell lysate used for individ-ual CAT assays was adjusted to the relative levels of (-ga-lactosidase activity in the extract. CAT assays were quanti-tated by PhosphorImager (Molecular Dynamics) analysis.Means and standard errors for data points were calculated (n2 3) by using the paired t test.

RESULTSThe LIM/Double Zinc-Flnger Doman Mediates Protein-

Protein Interaction in Vitro. Full-length hCRP and four hCRPLIM/double-finger and inter-LIM subsegments were synthe-sized as GST fusion proteins (Fig. 1A). Sizes of each fusionprotein expressed in Escherichia coli were in agreement withthe predicted structure: GST, 28 kDa; GST-hCRP-(2-193), 53kDa; GST-hCRP-(2-110), 42 kDa; GST-hCRP-(118-171), 36kDa; GST-hCRP-(62-118), 36 kDa; and GST-hCRP-(80-118),34 kDa (Fig. 1B Upper). As a second check on structure, eachof the fusion proteins was probed with a polyclonal antibodyraised against 83-99 of hCRP. As expected, four of the fivefusion proteins contained this inter-LIM epitope (peptide A;

Fig. 1). GST and each of the five GST fusion proteins wereelectrotransferred from a polyacrylamide gel to a nitrocellu-lose membrane, allowed to renature in situ, and then incu-bated with a series of 32P-labeled protein probes representingfull-length hCRP-(2-193), the isolated second LIM/double-finger domain hCRP-(118-171), and the inter-LIM segmenthCRP-(62-118) (Fig. 1C). The first two LIM/double-finger-containing probes [hCRP-(2-193) and hCRP-(118-171)]bound efficiently to each of the GST fusion constructscontaining one or both LIM domains but bound only atbackground (GST) levels to the two inter-LIM fiagments(Fig. 1D, Top Left and Center). In contrast, the inter-LIMprobe hCRP-(62-118) bound only weakly and equivalently toall fragments at levels comparable to GST binding, indicatinglack of specificity (Fig. 1D, Top Right).To document the specificity of the LIM-LIM interaction,

binding competitions were carried out (Fig. 1D, Middle andBottom). Unlabeled LIM/double-finger domain (118-171) orinter-LIM segment (80-118) fusion proteins were incubatedwith the filter prior to addition of the labeled LIM probe(118-171). The unlabeled LIM fragment blocked virtually allspecific binding of the LIM probe, while the inter-LIMsegment was ineffective.

Biochemistry: Feuerstein et al.

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FiG. 3. Fidelity of the zinc fingers is necessary for CRP-CRP interaction. (A) Schematic representation of hCRP, the LIM/double-fingerdomain subfiagment, and the mutants that were studied. The position(s) of the cysteine-to-serine substitution(s) is shown. Each fragment wasligated into the VP16 vector and then cotransfected along with the GAL4-hCRP-(1-193) plasmid to assess dimerizing potential by the level ofCAT activity. (B) The two-hybrid protein interaction assay was carried out and analyzed as described for Fig. 2. Each bar on the histogramrepresents an average taken from at least three separate experiments run in duplicate. A representative CAT analysis is shown below thehistogram. The signal intensity for (3AL4-hCRP-(1-193) cotransfected with VP16hCRP-(ZnCc) was set at 1.0 in each experiment, and all othervalues were taken as a ratio of this value. The intensity of CAT activity generated with VP16hCRP-(ZnCC) could not be clearly distinguishedfrom VP16-hCRP-(2-188) (P > 0.1) but differed significantly from that obtained with each of the cysteine-to-serine mutants [P < 0.001 (**)].

LIM/Double Zinc-Fine-Mediated Protein Interaction inIntact Cells. The studies summarized in Fig. 1 demonstratethat the LIM/double-finger domain can mediate protein-protein interaction in vitro. This interaction was furthertested by using a two-hybrid interaction assay (25-27) dia-grammed in Fig. 2A. In the initial experiment dimerization ofthe full-length hCRP was tested by fusing hCRP to both theGAL4 DNA-binding domain and the VP16 transcriptionalactivation domain (Fig. 2B)' and determining if these twofusion proteins were brought together by their respectivehCRP extensions. A set of negative controls demonstratedthat these fusion proteins did not individually activate thereporter (Fig. 2C, lanes 1-4). When the GAL4-hCRP chimerawas coexpressed with the VP-hCRP-(2-188) chimera, a sig-nificant increase inCAT reporter activity was generated (lane5). To localize the interacting segments of hCRP, the firstLIM/double-finger domain, (7-79), and the inter-LIM seg-ment, (62-118), were separately fused to VP16 and tested fortheir interactions with GAIA-hCRP (Fig. 2 B and C). Incontrast to the minimal interaction by the inter-LIM domain,the isolated LIM/double-finger domain (7-79) mediatesdimerization with hCRP-(1-193) at a level comparable to thatof the full-length hCRP (Fig. 2C, lanes 5-7). Although thelevel of (62-118) expression in the. transfected cells is notdirectly demonstrated, the parallel absence of significantinteraction of the. (62-118) segment with full-length hCRP' inthe in vitro binding assay confirms the conclusion'that theinter-LIM domain is not necessary to mediate CRP-CRPinteraction.

The Zinc-Finger Doublet Is Necessry and Sufcint forProtein-Protein Interaction. The dimerization- efficiency of aminimal LIM/double-fmnger domain extending from the firstto the last cysteine in the zinc-finger doublet (9-62) was nexttested by using the two-hybrid assay (Fig. 3A; ZnCC). Re-markably, this minimal domain was sufficient to mediateinteraction with the GAL4-hCRP-(1-193) fusion protein at70% of full-length hCRP (Fig. 3B, lanes 1 and 2). Theimportance of the zinc fingers was then directly tested byinterrupting the coordination of the zinc atoms. Cysteine-to-serine substitutions have been shown to result in a 95%reduction in zinc coordination to zinc fingers (28, 29) (Fig.3A). When the first, last, or both first and last cysteines oftheminimal LIM domain were replaced by serines, the protein-protein interaction was decreased 5-fold (Fig. 3B, lanes 3-5).The equivalent impact of all three cysteine-to-serine substi-tutions suggests that both zinc fingers are necessary forefficient protein-protein interaction.

DISCUSSIONThe in vitro and cell-based studies presented in this reportdemonstrate that the LIM/double zinc-finger domain ofhCRP is necessary and sufficient for protein dimer formation.In vitro dimerization of hCRP was demonstrated in a geloverlay binding assay using GST fusion constructs (Fig. 1).Protein-protein interaction was specific for the presence ofthe LIM/double-finger domain and could be reduced bycompetition with unlabeled LIM-containing protein seg-

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ments. Furthermore, the fact that the hCRP-(118-171) boundwith equal intensity to both the first [GST-hCRP-(2-110)] andsecond [GST-hCRP-(118-171)] LIM domains suggests that,at least under the conditions of the gel overlay assay, thereis no discrimination between the two LIM motifs of hCRP intheir protein-binding efficacy. The dihybrid interaction ap-proach was utilized to determine whether this in vitro assayreflects the situation in the intact cell and whether thisinteraction is mediated by the LIM domain. The results (Fig.2) demonstrate not only that CRP-CRP protein interaction isdependent on the LIM region but also that this region is fullysufficient for this interaction, as it is possible to transfer thisinteractive property to an unrelated protein by the transfer ofthe minimal LIM/double zinc-finger domain [GAL4-hCRP-(9-62)].The structure of the LIM domain has been recently shown

to form a zinc-finger domain, as was originally hypothesizedon the basis of primary structure comparisons (19). Theevidence supporting this structural conformation consists ofstudies demonstrating an appropriate stoichiometry ofzinc toLIM domain (2:1) (12, 30) and the solution of the structure ofthis domain by NMR (31). Whether the protein interactiveproperty of this region reflected intact zinc-finger structurewas studied by selectively destabilizing the zinc finger bysite-directed mutation of the coordinating cysteines. Theresults ofthese studies (Fig. 3) demonstrate that dimerizationis dependent on the intact nature and full coordination ofbothof the adjacent zinc fingers. The sum of these results allowsus to conclude that the double zinc-finger domain functionsin protein-protein interactions.The in vitro evidence and the in vivo evidence for the

protein interactive property of the LIM domain presented inthis report are mutually supportive. On the basis of thestructure of the proteins in the LIM family and precedents inother systems, it has been suggested that such interactionsare central to the functioning of the LIM proteins in theirnative cellular roles (18). For example, there are strikingparallels between the proteins in the helix-loop-helix (HLH)family and the LIM family. Both the LIM and HLH proteinscan be divided into two classes: those that contain and thosethat lack definable DNA-binding domains. These DNA-binding domains are homeodomains in the LIM family andbasic domains in the HLH family (22). MyoD, from the HLHfamily, contains a basic DNA-binding domain and activatestranscription when dimerized with a second HLH proteinalso containing a DNA-binding domain. This interaction isblocked when MyoD dimerizes with Id, an HLH proteinlacking a DNA-binding domain (4). LIM proteins mightinteract similarly: LIM/homeodomain proteins might trans-activate by forming dimers and might be silenced by com-bining with a LIM protein lacking a homeodomain (18). Suchcombinatorial interactions of proteins in the LIM familymight modulate signal transduction and gene expression. Thepresent data establish a possible mechanism by providingevidence that the LIM domain can mediate protein-proteinassociation. In this way these findings also serve to extend

the potential roles for zinc fingers beyond the establishedDNA/RNA-binding activities.

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