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BIOLOGICAL SCIENCES / BIOCHEMISTRY
The prokaryotic Cys2His2 zinc-finger adopts a novel fold as revealed by the NMR structure of Agrobacterium tumefaciens Ros DNA-binding domain
*Dipartimento di Scienze Ambientali, Seconda Università degli Studi di Napoli, Via Vivaldi 43, 81100 Caserta, Italy; and Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche, Via Mezzocannone 16, 80134 Napoli, Italy
Edited by Gary Felsenfeld, National Institutes of Health, Bethesda, MD, and approved September 18, 2007 (received for review July 16, 2007)
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topology, and is stabilized by an extensive 15-residue hydrophobic core. A backbone dynamics study of Ros87, based on 15N R1, 15N R2, and heteronuclear 15N-{1H}-NOE measurements, has further confirmed that the globular domain is uniformly rigid and flanked by two flexible tails. Mapping of the amino acids necessary for the DNA binding onto Ros87 structure reveals the protein surface involved in the DNA recognition mechanism of this new zinc-binding protein domain.
DNA binding proteins | NMR spectroscopy | Ros protein
30 aa in which a zinc ion, crucial for its stability, is tetrahedrally coordinated by two cysteines and two histidines. Its amino acid consensus sequence is (F/Y)XCX2–5CX3(F/Y)X5
X2HX3–5H, where X represents any amino acid and is any hydrophobic amino acid; forms with the other two hydrophobic residues (F/Y) a small hydrophobic core that together with the zinc ion stabilizes a compact 3D structure, consisting in an antiparallel -sheet faced by an -helix ( fold) (2). The -helix is constituted of three turns including the two coordinating histidines on two successive turns at the C-terminal part of the finger, whereas the -sheet occurs at the N-terminal part and contains the two coordinating cysteines. Structural studies accomplished on classical zinc-finger protein–DNA complexes have revealed that sequence-specific recognition is achieved by contacts between the -helix of the zinc-finger and bases in the major groove of the DNA. A single zinc-finger domain in itself is not sufficient for high-affinity binding to a specific DNA target sequence. In fact, proteins containing multiple zinc-finger domains usually require a minimum of two zinc-fingers for high-affinity DNA binding (1, 6). Nevertheless, the single zinc-finger domain present in the Drosophila GAGA transcription factor (7, 8), as well as the QALGGH single zinc-finger domain of the Arabidopsis thaliana SUPERMAN protein (9, 10), are capable of sequence-specific DNA binding when flanked by basic regions.
Recently, the first putative prokaryotic Cys2His2 zinc-finger domain has been identified in a transcriptional regulator, the Ros protein, from Agrobacterium tumefaciens (11), indicating that the classical zinc-finger domain, originally thought to be confined to the eukaryotic kingdom, could be widespread throughout the living kingdom from eukaryotic, both animal and plant, to prokaryotic. A. tumefaciens is a Gram-negative bacterium able to infect a large number of plants. The infection leads to crown gall tumors caused by a horizontal transfer of genes, similar to bacterial conjugation (12), from the bacterium to the plant. The transferred genes, 25 kb called T-DNA and contained in the 200-kb Ti plasmid, encode products that catalyze the formation of plant growth hormones (indoloacetic acid and cytokinin) in the transformed plant cells (11).
The protein Ros negatively regulates the virC and virD operons (13), present on the Ti plasmid, whose products are involved in the processing of the T-DNA. It binds a 40-bp sequence, named Ros box, present in the promoter of virC and virD and in the promoter of ros gene itself (14, 15). Ros also regulates the expression of the ipt oncogene located on the T-DNA region (11). Mutation in the ros gene causes increased expression of virC and virD, cold temperature sensitivity, and derepression of the ipt oncogene (11).
Ros is a 15.5-kDa protein with an isoelectric point of 7.13. The N-terminal part of the protein is negatively charged and contains many hydrophobic amino acid residues whereas the C-terminal part is positively charged and hydrophilic. Analysis of Ros primary structure revealed the presence of the sequence IXCX2CX3FX2LX2HX3HH (Fig. 1), which significantly resembles the consensus sequence of an eukaryotic Cys2His2 zinc-finger domain. Interestingly, this zinc-finger-like domain contains three histidine residues, and the 9-aa region between the second cysteine and the first histidine is shorter than the canonical 12-aa spacer invariantly observed in eukaryotic zinc-finger. We have recently demonstrated (16) that the putative zinc-finger domain is essential for Ros DNA binding and is part of a larger DNA-binding domain (region 56–142, Ros87) that includes four basic regions located on either side of the finger, one at the N terminus and three at the C terminus. We have also shown that Cys-79 (Cys-24 in Ros87), Cys-82 (Cys-27), His-92 (His-37), and His-97 (His-42) are involved in the zinc coordination and that His-96 (His-41) can replace His-97 in the coordination sphere, when His-97 is mutated to alanine. In this article we report the NMR solution structure of the Ros DNA-binding domain, providing a structural characterization of a prokaryotic Cys2His2 zinc-finger domain. The obtained high-resolution structure shows that the putative zinc-finger sequence (Fig. 1) is part of a larger domain that assumes a fold very different from the classical fold reported for the eukaryotic classical zinc-finger. Ros DNA-binding domain, in fact, consists of a globular domain comprising 58 aa and stabilized by an extensive 15-residue hydrophobic core. A backbone dynamics study of Ros87, based on 15N R1, 15N R2, and heteronuclear 15N-{1H}-NOE measurements, has further confirmed that the globular domain is uniformly rigid, whereas the two tails are flexible. Mapping of the amino acids necessary for the DNA binding onto Ros87 structure reveals the protein surface involved in the DNA recognition mechanism of this new zinc-binding protein domain that by sequence alignment is shown to be highly conserved in a number of prokaryotic proteins identified so far.
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topology, which is flanked by a series of well defined -turns. The N-terminal region of the domain is constituted by a short loop (loop1, residues 9–13), followed by a distorted type I -turn (residues Val-13, Arg-14, Lys-15, and Ser-16) preceding the first -strand. 1 strand (1, formed by Val-17 and Gln-18), 2 strand (2, formed by His-21, Ile-22, and Val-23), and 3 strand (3, formed by Ser-30 and Phe-31) constitute an antiparallel -sheet that partially faces -helix 1 (1). The exposed surface of the -sheet is constituted by side chains of Gln-18, His-21, whose side chain is a N
1-H tautomer (16), stabilized by N1-H hydrogen bond with Asp-19 backbone carbonyl group (Fig. 3Right), Val-23, and Ser-30. 1 and 2 are connected by a type II -turn, which contains two acidic residues. The loop connecting 2 and 3 (loop 2) is in part constituted by a well defined type II -turn (formed by Cys-24, Leu-25, Glu-26, and Cys-27) and contains the two cysteines coordinating the zinc ion. A short two-residue loop (loop 3) links 3 and 1, which is constituted by slightly more than two turns, ranging from Leu-34 to His-42. The zinc ion resides on a tip of the globular fold and is tetrahedrally coordinated by Cys-24 and Cys-27 thiolates and by His-37 and His-42 side chain N
nitrogens (Fig. 2 Right). A three-residue loop (loop 4) connects 1 to -helix 2 (2), which includes residues from Pro-46 to Trp-53 and whose axis is nearly orthogonal with the 1 axis. 2 is followed by two tight turns, a type II -turn, formed by residues Leu-55, Pro-56, Val-57, and Asp-58, and a type I -turn, formed by residues Ala-63, Pro-64, Ala-65, and Tyr-66; those two turns are linked by a four-residue loop (loop 5), and this protein region is strongly anchored through a backbone hydrogen bond (Ala-63 HN 
Lys-32 CO) to loop 3. The hydrophobic core is well resolved in the solution structure and is constituted by 15 side chains of residues positioned quite uniformly along the entire domain backbone chain, particularly by Pro-9, Val-17, Ile-22, Leu-25, Phe-31, Leu-34, Leu-38, Met-44, Tyr-49, Trp-53, Leu-55, Pro-56, Tyr-59, Met-61, and Pro-64 (Fig. 3). The 67–72 region, although being predicted as an helix on the basis of the CSI (see SI Fig. 7), does not fold in any predominant secondary structure element and is, on the contrary, structurally disordered.
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Backbone Dynamics of Ros87.
The three relaxation parameters 15N R1, 15N R2, and heteronuclear 15N-{1H}-NOE of Ros87 have been measured. The graphs of the relaxation parameters vs. residue numbers are reported in SI Materials (see SI Fig. 8). Relaxation parameters are generally constant along the whole globular domain as expected for a rigid structure, whereas they are well below the mean values in the N- and C-terminal regions. Interestingly, R2 values higher than the mean have been found for backbone amides at the N-terminal region of the globular domain (residues 11–16). The measured relaxation data were used in the ModelFree software to determine the parameters characterizing the internal mobility. Five models were used to appropriately fit the dynamical parameters to the experimental relaxation data. The model selection strategy of Mandel et al. (20) was used to select the correct model for each residue (see SI Table 2 and SI Materials), and the axially symmetric diffusion tensor of the molecule has been chosen as the best fitting the collected relaxation data. The initial estimations of the overall molecular correlation time 
m (6.88 ± 0.1 ns) were calculated on the basis of R2/R1 ratio and later optimized with the ModelFree protocol; the calculated dynamics parameters, S2 and e, vs. the polypeptide sequence of the two proteins are reported in Fig. 4.
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topology (Fig. 2). To better appreciate the differences between prokaryotic and the eukaryotic Cys2His2 zinc-finger domains, we superimposed Ros87 globular domain with the first zinc-binding domain of Tramtrack (21), which possesses a triple -sheet similarly to Ros87, aligning their zinc-coordinating residues (Fig. 5). The two zinc coordination spheres are extremely similar; in particular, in Ros87 Cys-24 and Cys-27, located on the -hairpin, together with His-37 and His-42, positioned at the middle and at the C terminus of 1, tetrahedrally coordinate the zinc ion through their thiolate sulfurs and indole N nitrogens, respectively. His-41, able to coordinate the zinc ion when His-42 is mutated to Ala (16), is also included in 1 and is close to the coordination sphere, having the possible role to further protect the zinc ion from the water bulk (Fig. 2 Right). The relative orientation of Ros87 triple -sheet and 1 is also very similar to that observed in Tramtrack zinc-finger 1 (Fig. 5); on the contrary, 1 in the Ros structure is indeed one turn shorter than the -helix in Tramtrack and in all of the other eukaryotic classical zinc-finger domains. This missing turn is clearly due to the linker between the second cysteine and first histidine, which in Ros87 is three residues shorter but is still able to orient the four zinc-coordinating residues in the same relative orientation as in the eukaryotic zinc-finger domain. Moreover, in Ros87, 2 bends over the region with an axis nearly orthogonal to the 1 axis and contributes to form the enlarged compact hydrophobic core. In the eukaryotic Cys2His2 zinc-finger domain the zinc coordination and the small three-residue hydrophobic core contribute similarly to the fold stabilization, whereas Ros87 contains an extensive and highly conserved (Fig. 1) 15-residue hydrophobic core, which appears to play a major role in stabilizing the globular fold (Fig. 3 Right). Particularly, residues included in each of the secondary structure elements of the motif are involved in the hydrophobic core, and two hydrogen bonds anchor the 2 to the -hairpin, further stabilizing the globular domain (Fig. 3 Right). On the contrary, amino acids of the N-terminal (residues 1–8) and of the C-terminal (residues 67–87) tails do not make any relevant interaction with the globular domain and are almost completely disordered.
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m value of 6.6 ± 0.2 ns, corresponding, through the Debye equation, to a hydrodynamic radius (rh) value of 1.9 ± 0.1 nm, which is in a good agreement with the rh values derived from the DOSY translational diffusion coefficient (2.1 ± 0.1 nm) and from the Ros87 NMR structure analyzed with HYDRO software (2.1 ± 0.1 nm). The obtained S2 values (Fig. 4) in the 10–66 region (residue 9 is a proline) are uniformly rather high and significantly drop in the two terminal regions, confirming that Ros87 consists of a compact globular domain and two flexible tails. In particular, the global average S2 value is 0.86 ± 0.01 in the region 10–66 and 0.39 ± 0.05 and 0.50 ± 0.06 in the N and C termini, respectively. Moreover, exchange terms (Rex) are required for only two residues of the globular domain (0.351 ± 0.172 s–1 for Val-23 and 0.303 ± 0.169 s–1 for Gly-28), and effective internal correlation times (e) are needed for 4 and 11 residues of the N and C termini, respectively (Fig. 4). Interestingly, Arg-14 and Lys-15, which are necessary for DNA binding of Ros87 (Fig. 6, BR1), are included in a region that shows R2 values higher than the mean; therefore, they should be affected by chemical exchange processes occurring on slow microsecond-to-millisecond time scales, which have been already reported to characterize residues involved in nonspecific and specific protein–DNA interactions (22).
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1, and Arg-50 and Lys-52 in 2 (BR2) are included in the globular domain, whereas Arg-70 and Arg-72 (BR3) are just at the beginning of C-terminal tail and Arg-82, Arg-83, and Lys-84 (BR4) are at its end. BR2 side chains are involved in ionic interactions with Asp-58 and Glu-26 carboxylate oxygens, respectively, playing therefore a clear structural role in the stabilization of Ros87 globular domain. On the contrary, BR1, BR3, BR4, Lys-35, and Arg-36 side chains are solvent-exposed and form a basic face, as is shown in Fig. 6; as a result, their relevance in Ros87 DNA-binding activity could be well explained by a direct involvement in Ros87–DNA interaction. Interestingly, the BR3 region is included in the 67–72 fragment that has been shown by the CSI prediction (SI Fig. 7) to have some tendency to assume an helical conformation, which is not clearly present in Ros87 solution structure but could be further stabilized by the interaction with the DNA. We therefore propose that Ros87 interacts with its DNA specific target through a surface including the N-terminal region of the globular domain and the 1 and through its C-terminal tail that could wrap around the double helix. In this way, Ros87 could likely contact and recognize more than three DNA bases, not necessarily contiguous. Moreover, the sequence alignment of the Ros homologues (Fig. 1) indicates that they should preserve Ros87 globular domain, and they probably recognize very similar or even identical DNA target sequences, because the amino acids involved in the DNA recognition are highly conserved.
The eukaryotic classical zinc-finger domains recognize their specific target sequence mostly by contacts between the -helix and bases in the major groove of the DNA, with each finger being able to fold independent of the rest of the protein and contacting a triplet of the DNA target site; also in the Ros DNA-binding domain amino acids of 1 are important for high-affinity DNA binding, but the presence of amino acids involved in DNA binding also in other regions of the 58-aa globular domain suggests a different DNA-binding modality.
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60 aa and is structurally very different from the eukaryotic Cys2His2 zinc-finger domains. In particular, Ros87 shows a globular domain characterized by a conserved extensive 15-residue hydrophobic core, which should play in the fold stabilization a much more relevant role than the zinc coordination. The topology of the region that folds around the zinc ion resembles the structure of the eukaryotic Cys2His2 zinc-finger domain (Fig. 5), but, differently from the eukaryotic counterpart, which clearly folds independent of the rest of the protein, in the Ros DNA-binding domain it is part of a significantly larger globular domain. Nonetheless, the similarity of Ros87 zinc-binding region with the eukaryotic Cys2His2 zinc-finger domain suggests that the two domains could be evolutionarily related. A. tumefaciens is well known for its unique ability to transfer and incorporate foreign DNA into plants; through this mechanism some plant could have acquired from A. tumefaciens or from some other plant-infecting bacterium the region encoding the ros (or a ros homologue) zinc-binding motif, which, in the eukaryotic organisms, could have been modified and mainly used in multiple contiguous copies to recognize DNA sequences. On the contrary, such an event might have taken place in reverse during the course of evolution, and the bacterial genomes may have acquired the region encoding the zinc-finger motif from an eukaryotic source and then used it in a different fashion as part of a larger protein domain. |
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NMR samples typically contained 1 mM 15N Ros87 or 15N-13C Ros87, 20 mM phosphate buffer (pH 6.8), 0.2 M NaCl, and 90% H2O/10% 2H2O or 100% 2H2O. Gel electrophoresis and mass spectrometry were used to verify the identity, purity, and isotopic labeling of the protein.
NMR Spectroscopy.
NMR experiments were acquired at 298 K on four different spectrometers: Bruker Avance 500 MHz with cryoprobe and 800 MHz at the European Magnetic Resonance Center of the University of Florence (Florence, Italy), Varian Unity INOVA 600 MHz at the Institute of Biostructures and Bioimages of Consiglio Nazionale delle Ricerche (Naples, Italy), and Varian Unity INOVA 500 MHz at the Environmental Science Department of the Second University of Naples (Naples, Italy). Triple-resonance NMR experiments including 3D HNCA (23, 24), 3D CBCANH (25), and 3D CBCA(CO)NH (25) were collected to enable sequence-specific backbone and C resonances assignment. The side-chain 1H and 13C NMR signals were assigned from (H)CCH-TOCSY experiments (26). NOE were evaluated from 3D 15N- and 13C-edited NOESY spectra and 2D [1H,1H]-NOESY. All of the NOESY spectra have been acquired with a mixing time of 100 ms. Slowly exchanging amide protons were identified in an 15N-heteronuclear single quantum correlation (HSQC) spectrum recorded immediately after exchanging the protein into a buffer prepared with 2H2O. Vicinal (three-bond) HN-H coupling constants (3JHNH) were evaluated from cross-peak intensities in quantitative J-correlation (HNHA) spectra (27). Residual dipolar couplings (HN-N) were measured by using an in-phase/antiphase HSQC experiment (28) on 15N-13C Ros87 in a liquid crystalline medium of 7% polyacrilamide, 0.1% ammonium persulfate, and 0.5% TEMED. The translation diffusion coefficient (Df) was measured by using the pulsed-field gradient spin-echo DOSY experiment (29). A correction factor was introduced to keep in count of the major viscosity of the solution 90% H2O and 10% 2H2O. The Stokes–Einstein equation was used to calculate the hydrodynamic radius. The hydrodynamic properties were also evaluated by using HYDRO software (30).
NMR experiments were processed by using Varian (VNMR 6.1B) or Bruker (XWIN NMR) software. 1H, 13C, and 15N chemical shifts were calibrated indirectly by using external references. The program XEASY (31) was used to analyze and assign the spectra.
Structure Calculations. NOE-derived distance constraints, coupling constants, and residual dipolar couplings were used to calculate Ros87 structures with the program CYANA (32, 33). The input data for the final structure calculation are reported in Table 1. The zinc ion was not included in the calculations. A total of 100 structures was calculated, and the 20 conformers with the lowest CYANA target function were further refined by means of unrestrained energy minimizations with the program SPDB (34).
The small number of residual constraint violations (Table 1) indicates that the input data represent a self-consistent set and that the constraints are well satisfied in the calculated conformers. The global rmsd value calculated for the backbone atoms of the region 9–66 (Table 1) shows that an overall high precision of the structure determination has been achieved. The structures were visualized and evaluated by using the programs MOLMOL (35) and PROCHECK-NMR (36). The chemical shift assignments are available from the BioMagResBank (accession no. 15373), and the final atomic coordinates are available from the Protein Data Bank (ID code 2JSP).
Relaxation Data Processing and Analysis. The relaxation parameters were evaluated by recording and analyzing the following set of experiments: inversion recovery 1H-15N HSQC for the evaluation of R1; spin echo 1H-15N HSQC for the evaluation of R2; and two 1H-15N HSQCs for the evaluation of the 15N-{1H} steady-state heteronuclear NOE (in one the protons were unsaturated, and in the other the protons were saturated for 3 s). R1 and R2 rates were determined by fitting the peak heights at multiple relaxation delays (37). Uncertainties in R1 and R2 were obtained from the error fit. 15N-{1H} steady-state NOEs were calculated as the ratio of 1H-15N correlation peak heights in the spectra acquired with and without proton saturation, and their uncertainties were set to 5%. S2 values were derived from a model free analysis of the R1, R2, and heteronuclear NOE data using the ModelFree software package (20, 38). An initial estimate of the magnitude and orientation of the diffusion tensor was obtained from the ratios of 15N R2 and R1 values by using the programs QUADRIC_DIFFUSION (39, 40) and R2R1_1.1 (41). Residues with large-amplitude fast internal motions were excluded from the calculation. Among the remaining residues, those with significant conformational exchange on the microsecond to millisecond time scale were also excluded.
Hydrodynamic Properties. Ros87 (100 µl, 1.0 mM) in 20 mM phosphate buffer (pH 6.8) and 0.2 M NaCl solution was loaded onto an S-75 16/60 column (GE Health Biosciences), preequilibrated with the same buffer, and eluted at room temperature at a flow rate of 1 ml/min. The column was connected downstream to a multiangle laser light (690.0 nm) scattering DAWN EOS photometer (Wyatt Technology). Quasi-elastic (dynamic) light scattering data were collected at a 90° angle by using a Wyatt quasi-elastic light scattering device. Data were analyzed by using Astra 4.90.07 software (Wyatt Technology).
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Abbreviations: HSQC, heteronuclear single quantum correlation.
To whom correspondence should be addressed. E-mail: roberto.fattorusso{at}unina2.it
Author contributions: B.D.B., C.I., P.V.P., and R.F. designed research; G.M., L.R., S.E., I.B., L.Z., E.M.P., C.I., P.V.P., and R.F. performed research; G.M., L.R., S.E., I.B., L.Z., E.M.P., B.D.B., C.I., P.V.P., and R.F. analyzed data; and G.M., L.R., C.I., P.V.P., and R.F. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The NMR chemical shifts have been deposited in the BioMagResBank, www.bmrb.wisc.edu (accession no. 15373), and the atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2JSP).
This article contains supporting information online at www.pnas.org/cgi/content/full/0706659104/DC1.
© 2007 by The National Academy of Sciences of the USA
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