The structural role of the zinc ion can be dispensablein prokaryotic zinc-finger domainsIlaria Baglivoa,1, Luigi Russoa,1, Sabrina Espositoa, Gaetano Malgieria, Mario Rendaa, Antonio Salluzzob,Benedetto Di Blasioa,2, Carla Iserniaa, Roberto Fattorussoa,3, and Paolo V. Pedonea,3

aDipartimento di Scienze Ambientali, Seconda Universita degli Studi di Napoli, Via Vivaldi 43, 81100 Caserta, Italy; and bDepartment of Environment, GlobalChange, and Sustainable Development, Portici Research Center, Italian National Agency for New Technologies, Energy, and the Environment, Via VecchioMacello, 80055 Portici, Italy

Edited by Gary Felsenfeld, National Institutes of Health, Bethesda, MD, and approved March 4, 2009 (received for review October 8, 2008)

The recent characterization of the prokaryotic Cys2His2 zinc-fingerdomain, identified in Ros protein from Agrobacterium tumefa-ciens, has demonstrated that, although possessing a similar zinccoordination sphere, this domain is structurally very different fromits eukaryotic counterpart. A search in the databases has identified�300 homologues with a high sequence identity to the Rosprotein, including the amino acids that form the extensive hydro-phobic core in Ros. Surprisingly, the Cys2His2 zinc coordinationsphere is generally poorly conserved in the Ros homologues,raising the question of whether the zinc ion is always preserved inthese proteins. Here, we present a functional and structural studyof a point mutant of Ros protein, Ros56–142C82D, in which thesecond coordinating cysteine is replaced by an aspartate, 5 previ-ously-uncharacterized representative Ros homologues from Me-sorhizobium loti, and 2 mutants of the homologues. Our resultsindicate that the prokaryotic zinc-finger domain, which in Rosprotein tetrahedrally coordinates Zn(II) through the typicalCys2His2 coordination, in Ros homologues can either exploit aCysAspHis2 coordination sphere, previously never described inDNA binding zinc finger domains to our knowledge, or lose themetal, while still preserving the DNA-binding activity. We demon-strate that this class of prokaryotic zinc-finger domains is struc-turally very adaptable, and surprisingly single mutations can trans-form a zinc-binding domain into a nonzinc-binding domain andvice versa, without affecting the DNA-binding ability. In light ofour findings an evolutionary link between the prokaryotic andeukaryotic zinc-finger domains, based on bacteria-to-eukaryotahorizontal gene transfer, is discussed.

Cys2His2 zinc finger � DNA binding proteins � metal binding proteins �Ros protein

In eukaryotic organisms the abundant Cys2His2 zinc-fingerdomain, involved in relevant protein–nucleic acid and protein–

protein interactions, consists of �30 aa and a zinc ion, tetrahe-drally coordinated by 2 histidine nitrogens and 2 cysteine sulfursand essential for stabilizing the ��� fold (1–5). In prokaryoticorganisms, the first Cys2His2 zinc-finger domain has been iden-tified only recently in the transcriptional regulator Ros fromAgrobacterium tumefaciens (6). The Ros zinc-binding domaincontains 2 cysteines occupying the first 2 coordinating positionsand 3 histidines (His-92, His-96, and His-97; see Fig. 1). We (7)demonstrated that in the Ros protein the histidines involved inzinc coordination are His-92, acting as the third coordinatingresidue, and His-97 as the fourth; when His-97 is mutated in Ala,the protein is still able to bind zinc and DNA with His-96 actingas the fourth coordinating residue. The NMR structure of RosDNA-binding domain (Ros56–142) has shown that the prokary-otic Cys2His2 zinc-finger domain, although having a similar zinccoordination sphere, possesses a novel protein fold, which is verydifferent from that of the eukaryotic counterpart (8). In partic-ular, Ros56–142 globular domain consists of 58 aa, arranged in a����� topology and stabilized by an extensive 15-residue hy-drophobic core. Furthermore, basic residues flanking the zinc

binding region on either side have been demonstrated to beessential for Ros DNA binding (7). In recent years, �300 Roshomologues have been identified in a large number of bacteria,mostly belonging to the � subdivision of proteobacteria (9, 10).The identity of the homologous sequences relative to Ros ismainly between 35% and 80% and generally increases when thesole zinc-finger domain sequence is considered. Interestingly,our extended database analysis of Ros homologous sequencesconfirms the first observation reported by Moreira and Rodri-guez-Valera (10) that the 4 zinc-coordinating residues in Ros areall together poorly preserved with only the first coordinatinghistidine conserved in almost all of the Ros homologues. Inparticular, the first cysteine is replaced by a serine in �40% ofthe homologues and the second cysteine is replaced by anaspartate in �60%; the 2 possible positions for the fourthcoordinating residue, which in Ros are occupied by the 2adjacent histidines (7, 8), in 50% of the homologues are bothreplaced by different pairs of amino acids, such as arginine–glycine, glutamine–tyrosine, lysine–tyrosine, leucine–glycine,and others. Such a high variability in the putative coordinationsphere raises the question of whether the zinc ion is present inall of the Ros homologues.

To investigate functional and structural properties of the Roshomologues we characterized the DNA-binding activity and theability to bind Zn(II) of, respectively, a point mutant of Rosprotein, Ros56–142C82D, in which the second coordinating cys-teine is replaced by an aspartate, 5 representative Ros homo-logues from Mesorhizobium loti, and 2 of their mutants. Ourresults indicate that the prokaryotic zinc-finger domain, which inRos protein tetrahedrally coordinates Zn(II) through the typicalCys2His2 coordination, in Ros homologues can either exploit aCysAspHis2 coordination sphere, previously never described inDNA binding zinc finger domains to our knowledge, or lose themetal, while still retaining the DNA-binding activity. Moreover,we obtained, through a Chemical Shift Index analysis based onthe backbone and C� NMR resonance assignments, the second-ary structure prediction of the DNA-binding domain of azinc-lacking homologue (named Ml4), which appears to beidentical to that of Ros. An evolutionary link between theprokaryotic and eukaryotic zinc finger domains, based on abacteria-to-eukaryota horizontal gene transfer (HGT), is finallyconsidered in light of our findings.

Author contributions: B.D.B., C.I., R.F., and P.V.P. designed research; I.B., L.R., S.E., M.R., andA.S. performed research; I.B., L.R., S.E., G.M., M.R., A.S., B.D.B., C.I., R.F., and P.V.P. analyzeddata; and I.B., L.R., S.E., G.M., R.F., and P.V.P. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1I.B. and L.R. contributed equally to this work.

2Deceased September 2008.

3To whom correspondence may be addressed. E-mail: roberto.fattorusso@unina2.it orpaolov.pedone@unina2.it.

This article contains supporting information online at www.pnas.org/cgi/content/full/0810003106/DCSupplemental.

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ResultsCysAspHis2 Zinc Coordination Sphere in Prokaryotic Zinc Finger Do-mains. Among the substitutions of the 4 Ros zinc coordinatingresidues that are present in Ros homologues, the mutation of thesecond cysteine with an aspartate is the most frequently ob-served. To define whether an aspartic acid can substitute thesecond coordinating cysteine in a prokaryotic zinc-finger motifwithout affecting its correct functional fold and its capability tobind Zn(II) we have produced a point mutant of the Ros protein(Ros56–142C82D) in which the cysteine in position 82 (see Fig. 1)

is substituted by an aspartic acid. Ros56–142C82D has been testedby EMSA using the VirC oligonucleotide recognised by the Rosprotein (7) or an oligonucleotide with an unrelated sequence(NS), as probes (Fig. 2A, lanes 1 and 2). The results showed thatthe protein is able to bind specifically the VirC DNA sequence.As reported for the WT Ros protein (7), addition of EDTA tothe binding reaction abolishes the DNA-binding activity ofRos56–142C82D (Fig. 2 A, lane 3), indicating that even for themutant protein DNA binding is zinc-dependent. The protein zinccontent has been analyzed by inductively coupled plasma massspectrometry (ICP-MS), demonstrating a ratio between the

Fig. 1. Sequence alignment of the 10 Ros homologues identified in the M. loti genome. The accession numbers of the proteins that have been analyzed arein italics, and the names used in this study are in parentheses. The arrows and the boxes indicate the positions corresponding to the coordination residues in Ros.The positions corresponding to Ros His-96 and His-97 residues, able to function alternatively as the fourth coordinating position in Ros (7), are both indicated.In bold are indicated the amino acids constituting the Ros hydrophobic core (8). The basic residues shown to be important for Ros DNA binding (7) are underlined.The asterisk indicates a conserved residues; the column indicates a conservative substitution; a period indicates a semiconservative substitution.

Fig. 2. EMSA of protein DNA binding. The proteins used are indicated; residues corresponding to the coordinating positions in the Ros protein are inparentheses, and the residues occupying the 2 possible fourth coordinating positions are in italics. DNA-binding specificity has been demonstrated by incubatingthe proteins with the labeled VirC oligonucleotide (VirC, lanes 1) or a labeled oligonucleotide with an unrelated sequence (NS, lanes 2). EDTA (50 mM) has beenadded to Ros56–142C82D and Ml153–149 (lanes 3) to investigate the zinc requirement for DNA binding.

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Zn(II) and the protein equals to 1:1 (see Table 1). A 1H-15NHSQC J-18 experiment of Ros56–142C82D, allowing the coher-ence transfer from H�1 and H�2 protons to N�2 and N�1 nitrogensthrough the 2JHN coupling constants (11), has been acquired toderive the tautomeric and zinc-binding states of the histidine sidechains (ref. 12 and Fig. 3). The inspection of the spectrum showsthat in Ros56–142C82D the pattern of histidine side-chain chem-ical shifts is very similar to that observed in Ros56–142 WT. In

particular, 2 of the 4 histidines present in the entire protein areN�1-H tautomers (i.e., with the N�2 unprotonated), commonlyfound to bind Zn(II) in zinc-finger domain. Moreover, both N�2and N�1 chemical shifts of these residues are typical of zinc-binding histidine side chains. These results indicate that the zincion in Ros56–142C82D is still bound by His-92 and His-97 andtherefore very likely it is tetrahedrally coordinated with the firstand second coordination positions occupied by Cys-79 andAsp-82. This hypothesis is supported by the assignment of Asp-82�-protons, whose chemical shift resonates at 2.9 and 3.0 ppm.These chemical shifts are downfield-shifted with respect to therandom coil values (2.7 and 2.8 ppm; ref. 13), as expected forZn(II) electron-drawing effect, and are very similar to thoseobserved for an aspartic acid tetrahedrally coordinating the zincion (14). Interestingly, aspartic acid has never been found to bindthe zinc ion in zinc-finger DNA-binding domains (15–18).

DNA Binding and Zinc Coordination in M. loti Ros Homologues. Toaddress the role of the zinc ion in the Ros homologues and testtheir DNA binding activity we focused our attention on thoseidentified from M. loti genome (Fig. 1). The identity with Ros ofeach of these previously uncharacterized proteins is �40% andis rather uniform along the whole sequence, including the basicregions that in Ros are necessary for DNA recognition (Fig. 1).Oppositely, 3 of the 4 zinc-coordinating residues, as generallyobserved for all of the Ros homologues, are variable. The firstcysteine is replaced by a serine in 3 of the 10 proteins, while thesecond cysteine is always replaced by an aspartate. Finally, only5 of the 10 Ros homologues in M. loti show the conservation ofat least 1 of the 2 histidines occupying the 2 possible positions ofthe fourth coordinating residue. We tested 5 of the Ros homo-logues from M. loti (named Ml1, Ml2, Ml3, Ml4, and Ml5; see Fig.1) for their ability to bind DNA and coordinate Zn(II). Amongthe M. loti Ros homologues, these proteins are representative ofall of the different substitutions observed in the putative zinc-coordinating residues. We produced all of the M. loti proteins asN-terminal deletion mutants corresponding to the Ros DNA-binding domain (Ml153–149, Ml258–141, Ml355–150, Ml456–151, andMl556–154), and performed EMSA experiments to test theirDNA-binding activities (Fig. 2 B–F). All of the proteins wereable to bind specifically the VirC oligonucleotide, suggesting asimilar fold of the DNA-binding domains. To exclude thepossibility that the GST moiety could influence the DNA-binding capability of the fusion proteins, DNA binding was alsoconfirmed with all of the proteins expressed without the tag.Interestingly, ICP-MS experiments demonstrated that Ml153–149,Ml258–141, and Ml355–150, presenting a cysteine in the positioncorresponding to the first zinc-coordinating residue in Ros, areable to bind Zn(II) in a �1:1 metal/protein ratio (Table 1).Analogously to what was previously reported for Ros protein (7)and Ros56–142C82D (Fig. 2 A), addition of EDTA to the bindingreaction abolishes the DNA-binding activity of the Ml153–149protein (Fig. 2B, lane 3), confirming that even in the naturalproteins bearing an aspartate as the second coordinating residuethe metal ion is required for DNA binding. Moreover, the 1H-15NHSQC J-18 experiments indicated that Ml153–149 and Ml258–141contain 2 zinc-coordinating histidine side chains, whereas Ml355–150contains only 1 coordinating histidine side chain (see Fig. S1). Theseresults suggest that in Ml1 and Ml2, analogously to Ros56–142C82D,the zinc coordination involves the side chains of 1 cysteine,1 aspartate, and 2 histidines. In Ml3, which has 1 histidine presentin the protein sequence occupying the third coordinating position(Fig. 1), the fourth coordinating residue remains to be defined.

Differently, ICP-MS experiments conducted on Ml456–151 andMl556–154, which have a serine corresponding to the first cysteinecoordinating residue of Ros, showed that these proteins do notbind either zinc ion (Table 1) or other metals such as cadmium,cobalt, copper, manganese, molibdenum, nickel, and magne-

Fig. 3. Definition of the role of the His residues in zinc coordination byNMR analysis in Ros56 –142C82D. A portion of the 1H-15N HSQC J18 spectrumof Ros56–142C82D acquired at pH 6.8 is shown. The gray rectangles indicate thenitrogen chemical-shift ranges typical for His involved in zinc ion coordination.

Table 1. ICP-MS analysis of protein zinc content

Protein Zn2�:protein

Ros56–142 C82D (CDHHH) 1:1Ml153–149 (CDHHH) 1:1Ml258–141 (CDHHY) 0.8:1Ml355–150 (CDHDY) 1:1Ml456–151 (SDHLG) 0.0:1Ml556–154 (SDHHY) 0.0:1Ml556–154 S77C (CDHHY) 0.8:1Ml258–141 C79S* (SDHHY) 0.0:1

Residues corresponding to the coordinating positions in theRos protein are indicated in parentheses; the residues occupyingthe 2 possible fourth coordinating positions are indicated initalics.*The Ml258–141C79S protein has been expressed as GST fusion.

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sium. Accordingly, the 1H-15N HSQC J-18 experiments didconfirm that neither the single histidine in Ml456–151 nor the 2histidines in Ml556–154 have side chains involved in zinc coordi-nation (Fig. 4 A and B). To verify whether the zinc coordinationin the prokaryotic zinc-finger domain was related to the presenceof a cysteine in the first coordinating position, we first substitutedin Ml556–154, which does not bind Zn(II), the serine 77 with acysteine (Ml556–154S77C) to reproduce the zinc coordinationsphere, and tested this mutant for its ability to bind DNA andZn(II). Interestingly, the Ml556–154S77C mutant was still able tobind specifically DNA and did bind zinc (Fig. 2G and Table 1).Moreover, the 1H-15N HSQC J-18 experiment of Ml556–154S77Cnicely revealed that after the serine-to-cysteine substitution the2 histidine side chains acquire chemical shifts and a tautomericstate typical for zinc coordination (Fig. 4 compare B and C). Wethen produced a Ml258–141 point mutant in which the cysteine ofthe zinc-binding domain was mutated to serine (Ml258–141C79S)and tested this protein for its ability to bind DNA and Zn(II).Ml258–141C79S is still able to bind specifically DNA but does notbind zinc (see Fig. 2H and Table 1). The ICP-MS experiment onthe Ml258–141C79S mutant was conducted on the protein ex-pressed as a GST fusion, and the 1H-15N HSQC J-18 experimentof Ml258–141C79S was not performed because this mutant couldnot be expressed at a sufficient concentration.

Interestingly, the basic residues outside of the zinc-binding

region, essential for Ros DNA binding, are highly conserved inall of the M. loti proteins (Fig. 1). Deletions of the basic regionsat the N terminus (Ml569–154) and the C terminus (Ml556–133) ofMl556–154 DNA-binding domain abolish DNA binding (Fig. S2),just as observed in Ros, further indicating that these proteinsshare with Ros a similar DNA-binding domain, independently oftheir zinc-binding capability.

Structural Roles of the Zinc Ion and the Hydrophobic Core in Ros andIts Homologues. To get more structural insights into the zinc-lacking members of this group of Ros homologues, we undertooka NMR solution characterization of Ml456–151, the most distanthomologue with respect to the zinc coordination residues. Inparticular, we obtained the complete assignment of HN, H�, C�,and C� chemical shifts of Ml456–151. On the basis of the ChemicalShift Index (19) we derived a secondary structure prediction,shown in Fig. 5, which is essentially identical to that obtained forRos and consistent with its high-resolution structure (8). Thisfinding, together with the observation that the amino acidsinvolved in the extensive Ros hydrophobic core are highlyconserved (Fig. 1), strongly suggests that the DNA-bindingdomain can adopt a similar functional tertiary fold in Ros and itshomologues lacking the zinc ion. The role of the hydrophobiccore versus that of the zinc ion in stabilizing the global fold ofRos and its zinc-binding homologues was evaluated by removing

Fig. 4. Definition of the role of the His residues in zinc coordination by NMR analysis in Ml456–151, Ml556–154, and Ml556–154 S77C. Portions of the 1H-15N HSQCJ18 spectra acquired at pH 6.8 are shown. The proteins analyzed are indicated. The gray rectangles indicate the nitrogen chemical-shift ranges typical for Hisinvolved in zinc ion coordination. (A) The single His present in Ml456–151 is a N�2-H tautomer and does not coordinate Zn(II). (B and C) Upon mutation of Ser-77to Cys in Ml556–154, the 2 His residues change their WT N�2-H tautomeric forms (B) to N�1-H tautomeric forms and their nitrogen chemical shifts fall in the typicalregions of chemical shifts for His involved in zinc ion coordination (C).

Fig. 5. Secondary structure elements (�-strands and helices) in Ml456–151 sequence as derived by the Chemical Shift Index, based on H�, C� and C� resonanceassignments.

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the zinc ion from Ros56–142 through an acidic reversible unfold-ing. The structural changes of Ros56–142 have been followed via1H-15N HSQC spectra and the protonation state of the coordi-nating histidines was followed via 1H-15N HSQC J-18 experi-ments (Fig. S3). At pH 5.1 the histidines were completelyprotonated and the zinc ion was released, while the cysteineswere kept reduced, as demonstrated by the reversibility of theunfolding. In these mildly acidic conditions, the 1H-15N HSQCspectrum of Ros56–142 shows that the folding is in a major partlost, even though a meaningful residual structure is maintainedand involves residues of either the �-hairpin and the 2 helicescontributing to the hydrophobic core. Lowering the pH to 3.7,the 1H-15N HSQC spectrum does not change significantly,indicating that the first major structural change is very likelycaused by the loss of the metal ion. These results are inagreement with the data describing the loss of Ros DNA-bindingactivity in the presence of an excess of EDTA (7).

DiscussionRos homologues contain important structural peculiarities asour study of M. loti proteins reveals. Although their DNA-binding domains share a high sequence identity, they can besubdivided into 2 groups based on their ability to chelate zincions. In particular, the typical Cys2His2 coordination spherefound in the Ros zinc-finger domain is not conserved in the M.loti Ros homologues that bind zinc, where the second cysteine isalways replaced by an aspartate. This finding, together with ourresults on the Ros56–142C82D mutant, demonstrates that pro-karyotic zinc fingers can also tetrahedrally coordinate zincthrough a unique CysAspHis2 sphere (15, 18). Interestingly, inWT Ros homologues where the first coordinating cysteine isreplaced by a serine, the zinc ion is lost but the protein still foldsinto a functional protein. Moreover, we were able, with singlemutations (Cys to Ser and Ser to Cys) in this coordinatingposition, to transform a zinc-binding domain into a nonzinc-binding domain and vice versa without affecting the DNA-binding activity. These surprising results reveal that the presenceof a cysteine in the first coordinating position is strictly relatedto the presence of the zinc ion. Indeed, very few cases are knownof proteins with �35% sequence identity to one another thatdiffer in whether they bind metal with structural role (20–25). Inparticular, zinc ion loss occurs rarely and when it does it isgenerally a result of deletion of the surrounding secondarystructure elements and/or loops whose structure the metalstabilizes (20). In the cases of the prokaryotic zinc-finger do-main, the present study demonstrates that the loss of metalbinding occurs when the 2 coordinating cysteines are bothreplaced with serine and aspartate and does not cause a signif-icant change in the secondary structures and DNA-bindingactivity. Differently, removal of the zinc ion in Ros produces aconsiderable loss of the protein fold that is partially retained byamino acids constituting the hydrophobic core. These resultsindicate that in Ros and its zinc-binding homologues the zinc ionand the hydrophobic core are both essential for stabilizing thestructure of the DNA binding domain, whereas in the homo-logues lacking zinc binding, the role played by the zinc ion musthave been replaced by new interactions. Overall, this class ofprokaryotic zinc-finger domains appears to be structurally veryflexible, because it is not restricted to the Cys2His2 zinc coor-dination sphere and can overcome the metal requirement forproper folding and DNA-binding activity.

The observation that all of the Ros homologues are able tointeract with the same DNA sequence strongly suggests thatthese proteins display similar functions in vivo whether or notthey bind zinc. Why does only a subset of proteins require zinc?The correct folding of proteins in which zinc ions are keystructural components strictly depends on zinc homeostasis andcellular redox potential (26). Prokaryotic organisms are single-

cell entities that have immediate contact with their proximalenvironment and are separated from it by a set of cellularenvelopes. Moreover, many bacteria are likely to face acutemetal stress caused by changes in extracellular environment thataffect metal availability or alter intracellular physiology (26).Possibly, zinc-plus and zinc-minus DNA binding proteins mightbe involved in the regulation of gene expression under differentphysiological conditions, notably the redox potential of cellsand/or fluctuating zinc availability in the environment. The factthe zinc-dependent and zinc-independent Ros homologues shareextensive sequence identity and, particularly important, thatshifting from one to the other condition is obtained by substi-tution of a single residue, makes it extremely difficult to establishwhich one is more primitive. Considering that zinc-independentprotein folding might have been an advantageous character in thosebacterial cells lacking efficient mechanisms of zinc homeostasis, itis possible that zinc-dependent Ros homologues are a derivedcharacter that appeared after the evolution of more efficientmechanisms to optimize the intracellular availability of the metalion. However, this hypothesis needs further substantiation.

Along with the functional and structural characterization ofthe prokaryotic zinc finger domain (6–8) this study allows us toformulate a comparison between the main features of zinc-fingerdomains in prokaryota and eukaryota. Definitely, the 2 domainshave a very similar zinc coordination sphere (8) arranged in a��� motif, but their global folds are significantly different. Theeukaryotic Cys2His2 zinc finger is a cylindrical ��� domain of�30 residues that is completely unstructured in the absence ofthe metal ion (1, 2). The prokaryotic counterpart is a globular����� domain of �60 residues stabilized by an extendedhydrophobic core; it retains some of its structure in the absenceof zinc and can functionally fold in the absence of the metal ionwhen appropriate amino acids are substituted for the coordi-nating cysteines. The eukaryotic Cys2His2 zinc fingers recognizespecific DNA targets through variable amino acids of the �-helixand their number in a single protein varies from 1 (27–30) to�30, suggesting that duplication of this autonomously-foldedprotein domain has been a simple mechanism by which eukary-otic proteins were able to acquire new functions during evolu-tion. Variation in the �-helix sequence of Cys2His2 zinc fingersallows them to be targeted to different locations in the genome,whereas variation in the number of fingers allows cooperativebinding to different 6- to 12-bp DNA-target sequences (1, 2).This provides great versatility for recognizing different DNAsequences, obtained at the cost of a total dependence on bindingzinc ions. In contrast, the prokaryotic zinc-finger domain appearsnot to be widespread in prokaryotes. It is generally present as asingle copy in bacterial proteins and most likely uses a larger proteinsurface, including the N-terminal region, the first �-helix, and theC-terminal flexible tail, to recognize the DNA target (8).

Despite the significant differences between the prokaryoticand the eukaryotic zinc-finger domains, an evolutionary linkbetween them is likely to exist. The observation that Cys2His2zinc finger transcriptional regulators are present mostly inspecies living in intimate ecological relationship with eukaryotes,led Kado and coworkers (6, 31) to propose that these bacteriaacquired Cys2His2 zinc-finger proteins by HGT from eukaryotes.More recently, Moreira and Rodriguea-Valera (10) extended theanalysis of the Ros homologues present in the genome databanks and noticed that the bacterial species carrying these genesbelong to the �-subdivision of the proteobacteria from whichmitochondria originated. On the basis of this observation, theyproposed an alternative evolutionary hypothesis, bacteria-to-eukaryota HGT, implying that Cys2His2 zinc-finger proteinsappeared as an innovation in proteobacteria and were inheritedvia mitochondrial endosymbiosis.

The ��� prokaryotic zinc-binding motif, located in the N-terminal region of a larger DNA-binding domain stabilized by an

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extended hydrophobic core, bears a significant resemblance tothe eukaryotic Cys2His2 zinc finger domain and differs from itin the spacer that separates the second coordinating residuefrom the third: 9 aa versus 12. Two sets of properly-spacedmetal-chelating ligands appear then to be required for theautonomous folding of the eukaryotic Cys2His2 zinc-finger do-main. Thus, a bacterial proto-zinc finger motif might haveacquired the ability to fold in a zinc-dependent fashion auton-omously from the rest of the protein simply by acquiring the rightspacer between the metal-chelating ligands. Interestingly, therecently-reported sequence of a gene from Candidatus Liberib-acter asiaticus (ZP�03287372), which encodes a putative tran-scription regulator protein with a 52% sequence identity to theRos protein, seems to support this hypothesis (Fig. S4). Besidesthe residues Cys-81, Asp-84 and His-94, and His-99, which alignwith the coordinating residues in Ros, Ml1, Ml2, and Ml3, thisprotein contains an extra cysteine, Cys-78, located exactly 3residues ahead of the putative zinc-coordinating motif (Fig. S4).This results in a protein sequence that can be aligned with theeukaryotic Cys2His2 zinc-finger motif, with a precise 12-aaspacer separating the second Cys from the first His. Thisobservation suggests an evolutionary mechanism by which asingle point mutation introducing an extra cysteine in a nonau-tonomously-folding bacterial zinc-finger motif with the sequenceCysX2AspX9HisX3– 4His could have transformed it into a CysX2CysX12HisX3–4Hiszinc-finger motif. This event might have been the first steptoward a more compact zinc-finger domain able to fold auton-omously from the rest of the protein. In this regard, it is worthnoticing that an aspartic acid has a lower affinity for zinc comparedwith cysteine at physiological pH and its presence in the secondcoordinating position could have favored this transition.

If one considers that the genes found in bacteria are too highlydiverged from their eukaryotic homologues, phylogenetic anal-ysis is unlikely to resolve a mithocondrial origin as opposed to aneukaryotic origin for the Cys2His2 zinc-finger proteins (31).Independent of phyletic relationships, the structural and func-

tional analysis reported here supports the hypothesis that a ���motif, whose fold does not strictly depend on zinc coordination,originated in �-proteobacteria as a part of a larger DNA bindingdomain. A simple variant of this motif, whose fold was strictlyzinc-dependent but autonomous from the rest of the protein, hadthen an unprecedented evolutionary success in eukaryotes,which have a more sophisticated metal chaperone system foracquiring and storing zinc (26).

Materials and MethodsEMSAs. All of the proteins used for the DNA-binding experiments wereexpressed and purified from Escherichia coli BL21 host strain as GST fusions,after cloning the coding sequence in the pGEX-6P-1 expression vector (see SIText). Five hundred nanograms of the purified proteins was tested by EMSAexperiments as reported (7), with the exception of Ml153–149 for which 100 ngwas used.

ICP-MS Analysis. The amount of zinc contained in the proteins (for theirexpression see SI Text) was measured by ICP-MS. Samples of 20 mL containing30.6 nmol of the proteins and 6.5% HNO3 were hydrolized by the MilestoneMLS-1200 Mega microwave laboratory system. The hydrolyzed samples wereanalyzed by using Elan 6000 ICP-MS (PerkinElmer) equipped with a cross-flowNebulyzer. For all of the proteins, except Ml258–141C79S, a sample without theprotein was prepared in the same way and used as a blank, while a samplecontaining the GST was prepared and used as a blank for Ml258–141C79S.

NMR Spectroscopy. The NMR experiments were recorded with 0.8 mM samplesof uniformly 15N- and 15N-13C-labeled proteins (for their expression and theacidic reversible titration see SI Text), containing 20 mM phosphate buffer and0.2 M NaCl, at pH 6.8 and 25 °C, in a H2O/2H2O 90:10 mixture. 1H15N-HSQC J-18spectra were acquired as described (7). To obtain sequence-specific backboneand C� resonances assignment, a standard set of triple-resonance NMR exper-iments was collected on a 15N-13C-labeled sample of Ml456–151.

ACKNOWLEDGMENTS. We thank Dr. Emilio Cuoco, Dr. Vincenzo Piscopo, Mr.Maurizio Muselli, and Mr. Marco Mammucari for excellent technical assistanceand Prof. Roberto Ligrone for helpful discussions and careful reading of themanuscript. This work was partially funded by Ministero dell’Istruzione,dell’Universita e della Ricerca Grants PRIN 2007 (to C.I.), PRIN 2006 (to R.F. andP.V.P.), and FIRB 2003 (to P.V.P), and Regione Campania Grant L.R.5 2003 (toP.V.P). This article is dedicated to the memory of Prof. Benedetto Di Blasio forhis humanity and scientific competence.

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