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1 Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan 11472, ROC
2 Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 11529, ROC
3 Molecular Biology Institute, University of California at Los Angeles, California 90095-1570, USA
Reprint requests to: Hanna S. Yuan, Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 11529; e-mail: hanna{at}sinica.edu.tw; fax: 886-2-2782-6085.
(RECEIVED June 20, 2002; FINAL REVISION September 13, 2002; ACCEPTED September 13, 2002)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0220602
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Abstract |
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-helix, with a divalent metal ion located in the center. Here we show that the metal-binding sites in the center of the H-N-H motif, for the EDTA-treated and Mg2+-soaked complex crystals, were occupied by water molecules, indicating that an alkaline earth metal ion does not reside in the same position as a transition metal ion in the H-N-H motif. However, a Zn2+ or Mn2+ ions were observed in the center of the H-N-H motif in cases of Zn2+ or Mn2+-soaked crystals, as confirmed in anomalous difference maps. A phosphate ion was found to bridge between the divalent transition metal ion and His545. Based on these structures and structural comparisons with other nucleases, we suggest a functional role for the divalent transition metal ion in the H-N-H motif in stabilizing the phosphoanion in the transition state during hydrolysis. Keywords: Metal binding in proteins; divalent metal ions; magnesium ion; zinc ion; endonuclease; DNase; DNA hydrolysis mechanism
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Introduction |
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. They include the group I homing endonucleases I-HmuI (Goodrich-Blair et al. 1990; Goodrich-Blair 1994; Goodrich-Blair and Shub 1996), I-HmuII (Goodrich-Blair 1994; Goodrich-Blair and Shub 1996), I-HmuIII (Goodrich-Blair 1994), I-TevIII (Eddy and Gold 1991), yosQ (Lazarevic et al. 1998), and ORF253 (Foley et al. 2000); and the group II homing endonucleases Avi (Ferat and Michel 1993), Cpc (Ferat and Michel 1993), and PetD (Kuck 1989).
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The topology and the coordination of the metal ion in the H-N-H motif share several features similar to those of classic zinc finger motifs (Fig. 2). Firstly, both of the structural motifs contain an antiparallel-stranded ß-sheet linked to a C-terminal -helix with a zinc ion situated in the center. Secondly, both motifs have two zinc-coordinated histidines separated by three residues, that is, one helical turn, located in the C-terminal -helix. The major differences in the two motifs are: (1) the relative orientation of ß-strands to -helix; (2) a shorter -helix and a much longer loop between the two ß-strands in the H-N-H motif; and (3) a water molecule instead of a protein residue as the fourth ligand of the zinc ion in the H-N-H motif.
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10-9 M) in comparison to the other transition metal ions (Pommer et al. 1999; Keeble et al. 2002). These results suggest that H-N-H colicin proteins are zinc-dependent enzymes in vivo, and that zinc plays a structural role in stabilizing the enzyme (Pommer et al. 1999). However, structural comparison (Friedhoff et al. 1999b; Miller et al. 1999; Grishin 2001) has revealed that the H-N-H motif has a ßß-Metal fold similar to the active sites of several other nucleases, including the homing endonuclease I-PpoI, Serratia nuclease, and Phage T4 Endonuclease VII (Friedhoff et al. 1999a; Kuhlmann et al. 1999; Raaijmakers et al. 1999). The divalent metal ions in these enzymes have been suggested to play catalytic roles (Lunin et al. 1997; Galburt et al. 1999; Miller et al. 1999), implying that the zinc ion in the H-N-H motif could be involved in DNA hydrolysis. It has been shown that the nuclease domain of ColE9 is inactive in the presence of Zn2+ (Pommer et al. 1999; Keeble et al. 2002); however, our biochemical assays showed that the nuclease domain of ColE7 (referred to as nuclease-ColE7 hereafter) is an active endonuclease in the presence of low concentrations of Zn2+ (Ku et al. 2002). Moreover, the Zn2+-depleted apo-enzyme cannot hydrolyze DNA, although it still binds DNA, indicating that the zinc ion not only plays a structural role but is also involved in catalysis. To further clarify the role of metal ions in the H-N-H motif, we report here the crystal structures of nuclease-ColE7/Im7 in complex with different divalent metal and phosphate ions in two different crystal forms. The divalent metal ions, originally located in the complex, were removed by ETDA, and afterwards different metal ions, including Zn2+, Mn2+, and Mg2+ were soaked into the complex crystals. The metal-binding sites for Zn2+ and Mn2+ were confirmed in anomalous difference maps. The crystal structures demonstrate unambiguously the transition metal ion binding site and phosphate binding site and suggest possible roles of the transition metal ions in the H-N-H motif.
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Results |
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shows that the addition of Zn2+ ions to the zinc-bound holoenzyme inhibited DNase activity; however, the addition of Mg2+ in mM range enhanced the activity (Ku et al. 2002). In the present study, we found that the zinc-holoenzyme was further activated by Mn2+ in mM range.
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radiation from a rotating anode (Table 1). Crystals soaked in solutions containing EDTA, Mn2+, Zn2+, or Mg2+ diffracted from 2.6 to 2.0 Å resolution at low temperature (100 K). The Mg2+-binding site was not found in the Mg2+-soaked crystals, and thus the diffraction statistics for Mg2+-soaked crystals were not included in Table 1.
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. In the P21212 unit cell, there are two complex molecules per asymmetric unit, and they are separated by (0.49, 0.52, 0.50) of unit cell dimensions, but not exactly by (0.5, 0.5, 0.5). Therefore the space group was shifted from the I-center I222 to the primitive P21212; nevertheless, the protein molecules in the two types of crystals packed in a similar way. The average root mean square (rms) difference between the two complex molecules in the asymmetric unit of P21212 cell is only 0.3 Å (for main-chain atoms), and therefore, for clarity only the structure of the first molecule will be used in the following structural comparison.
Comparison between I222 and P21212 structures
The overall folds of the complex in the two crystal forms are identical (see the ribbon model in Figure 4). The His-tags were not observed in either I222 or P21212 structures. Im7 contains four -helices folded in a varied four-helix-bundle structure nearly identical to the previously determined free-form Im7 (Chak et al. 1996). Nuclease-ColE7 has a mixed /ß structure with a metal ion bound in a cleft on the concaved surface. The H-N-H motif (displayed in green in Figure 4) is located at the C-terminus of nuclease-ColE7 containing two ß-strands (ßd and ße) and one -helix (5) with a metal ion situated in the center of the motif.
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Metal ions and phosphate binding in H-N-H motif
The complex crystals were first soaked in EDTA to remove the associated divalent metal ions. X-ray absorption spectra were recorded for the crystals before and after EDTA soaking. The absorption at zinc edge was observed before EDTA soaking and disappeared after ETDA soaking (data not shown). The previously solved complex structure with the zinc ion deleted was used as the initial model for refinement of the EDTA-treated crystal (Ko et al. 1999). A Fourier map (Fig. 5A) revealed the absence of a strong peak in the center of the H-N-H motif, but instead showed two water molecules located in the original metal-binding sites. One water molecule was hydrogen-bonded to His545 (ND1). The second water molecule was hydrogen-bonded to the first water molecule and located approximately at the original metal-binding site. The electron densities of the two histidine residues, His573 and His544, were ill-defined, indicating that the side-chain conformations of the two residues were not fixed by metal ions. This result demonstrated that the divalent metal ions initially associated with the complex had been removed by EDTA.
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, B and C show the Fourier and anomalous difference maps of Zn2+-bound (in P21212) and Mn2+-bound (in I222) structures at the center of the H-N-H motif. The electron densities of a metal ion and a phosphate ion were observed in the Fourier (2Fo-Fc) maps. The locations of metal-binding sites were further confirmed unambiguously by the anomalous difference maps in which Zn2+ has a strong peak, with a peak height of 30 
, and Mn2+ also has a strong peak, with a peak height of 17 . No other metal ion binding sites were identified in the electron density maps for the Zn2+- and Mn2+-bound crystals. This result demonstrated that the transition metal ions of Zn2+ and Mn2+ bind at the same site in the H-N-H motif, coordinating to the three histidine residues (His544, His569, and His573) and one phosphate ion. |
Discussion |
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In our previous study, we found that a water molecule was bound to the zinc ion in the H-N-H motif of nuclease-ColE7 (Ko et al. 1999). In the present study, we show that a phosphate ion replaces the water molecule and forms a bridge between the zinc ion and a histidine residue. The Mn2+ ion binds at the same site and coordinates to the same residues as that of zinc ion. A similar result was reported for Ni2+-bound nuclease-ColE9/Im9 in which a phosphate ion is also bridged between the Ni2+ ion and a histidine residue (Kleanthous et al. 1999). However, we did not find any Mg2+ binding sites in either apo-enzyme or zinc-holoenzyme crystals soaked with Mg2+ ions. The original metal-binding site in the H-N-H motif of the apo-enzyme soaked with Mg2+ ions was occupied with water molecules similar to that of EDTA-treated crystals (data not shown), suggesting that the Mg2+ ion likely does not bind at the center of the H-N-H motif in ColE7. This result was expected, since a magnesium ion does not favor the binding site of a transition metal ion coordinated with three histidine residues (Dudev and Lim 2001).
A previous structural comparison shows that the zinc ion in the H-N-H motif matches well with the Mg2+ ion in active sites of I-PpoI and Serratia nuclease (Kuhlmann et al. 1999). The Mg2+ in I-PpoI and Serratia nuclease are involved in stabilization of the phosphoanion transition state and in providing a proton to the 3' oxygen-leaving group. Therefore we suspect that the zinc ion located in the H-N-H motif has a similar function for the stabilization of the phosphoanion transition state. Because Mg2+ ion further enhanced the nuclease activity of the zinc-bound holoenzyme, it is possible that there is a second metal-binding site for Mg2+ in colicins, but it is likely that Mg2+ is only bound upon DNA binding. It has been proposed that nuclease ColE9 hydrolyses DNA by two distinct catalytic mechanisms in the presence of only Mg2+ or Ni2+ (Pommer et al. 2001; Walker et al. 2002). However, alkaline earth metal ions (Mg2+ in mM) and transition meal ions are both present in cells, and most likely ColE7 is a zinc-enzyme in vivo, because Zn2+ binds colicins with highest affinity and Zn2+ is one of the most abundant transition metal ions in cells. Therefore, we propose here that nuclease-ColE7 functions as a zinc-enzyme and that Mg2+ ions are also involved in DNA hydrolysis in vivo.
H545 is likely the general base
In nuclease-ColE7, there are three histidine residues, H544, H569, and H573, directly bound to the zinc ion. These three residues are not strictly conserved among H-N-H proteins, but the residues that are aligned with these histidines are either polar or charged and capable of binding to metal ions. Therefore, the residues aligned with these histidines are likely to be responsible for metal ion binding. The roles of the two most conserved residues, H545 and N560, in the H-N-H motif, are less clear, however. A structural comparison between the H-N-H motif and the active sites in I-PpoI and Serratia nuclease sheds light on the possible functions of H545 (Kuhlmann et al. 1999). The superimposition of the ßß-fold between nuclease-ColE7 and I-PpoI shows that H545 in ColE7 locates at a similar position as H98 in I-PpoI (Fig. 6A). H98 in I-PpoI serves as a general base to activate a water molecule, which functions as a nucleophile to attack the scissile phosphate group. In our structures, a phosphate ion is bound between H545 and a zinc ion, and the distance between H545 (ND1 atom) and phosphate oxygen is 2.6 Å. Therefore it is very likely that H545 functions as a general base. This could explain the strict requirement for a histidine at this position in the entire H-N-H family of proteins.
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). The ND2 atom makes two hydrogen bonds with the carbonyl groups of G554 and K547, and the OD1 atom also makes two hydrogen bonds with the amino groups of K547 and E546. It appears that N560 has no unoccupied positions in polar atoms to participate in hydrolysis. Therefore, it is likely that N560 only plays a structural role to fix the backbone structure in the loop region so that the general base residue H545, which hydrogen bonds to the carbonyl group of V555, is appropriately held.
A structural model for nuclease-ColE7-bound DNA
A structural model for nuclease-ColE7 bound with DNA was constructed based on the crystal structure of the I-PpoI/DNA complex (Fig. 7). The H-N-H motif in nuclease-ColE7 was superimposed with the two ß-strands and one -helix in the active site of I-PpoI, with an rms difference of only 1.2 Å for the main-chain atoms. Without further adjustment, the nuclease-ColE7 appears to bind DNA snugly. The concave surface of nuclease-ColE7 faces the DNA backbone with the zinc ion positioned close to the phosphate backbone at the minor groove, appropriate for its role in stabilizing the phosphoanion transition state. The -helix, 2, binds DNA at the neighboring major groove, and there are indeed several basic residues (R496, K497, K498, K505) located in this helix.
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Materials and methods |
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The overexpression vector for nuclease-ColE7/Im7 in pQE70 was constructed following a similar procedure (Ko et al. 1999). A 673-bp DNA fragment encoding nuclease-ColE7 and Im7 from the plasmid pHK001 (Chak et al. 1991) was amplified by PCR using a pair of oligonucleotide primers: 5'GCTAAAGGCATGCTAG ATAAGGAGAGTA3' and 5'CATTCATAGATCTGCCC TGTT TAAATCC3'. The cutting sites of SphI and BglII were generated at either end of the PCR-amplified fragment. PCR reactions were carried out in 100-µL volumes using one unit of Taq polymerase with 30 cycles of 94°C for 1 min, 55°C for 30 sec and 72°C for 1 min. The PCR product was then cleaved with SphI and BglII and ligated into the expression vector pQE-70 preincubated with the same restriction enzymes. The resulting recombinant plasmid was transformed into E.coli M15. The overexpressed protein complex contained the nuclease-ColE7 (residues 444 to 576) and a full-length Im7 with a six-histidine tag attached at the C-terminus.
Protein purification
The purification procedures for the two different His-tagged nuclease-ColE7/Im7 complexes were similar to the procedure described previously (Ko et al. 1999). Eight liters of LB M15 cell culture containing either the pOE-30 or pOE-70 expression vectors were incubated and induced by IPTG at A600nm of 0.6 O.D. at 37°C. Crude cell extracts were purified by chromatography on a Ni-NTA resin affinity column (QIAGEN), followed by a CM column (CM Sepharose Fast Flow, Pharmacia). Protein samples were concentrated by Centriplus 3K (Amicon) ultrafiltration and stored at a concentration of 15 mg/mL in H2O, at -70°C. The concentration of the purified protein was determined by DC protein assay (BioRad).
DNase activity assay
A 30-bp oligonucleotide labeled with Hetrachloro-6-carboxyfluorescein and a quencher of BHQ-1TM (DNaseAlertTM QC system DNA Substrate, Ambion) was used to measure the endonuclease activity of nuclease-ColE7. The enzyme was mixed with the oligonucleotide, and the increased fluorescence emission intensity resulting from the DNA cleavage was measured on a NUNCTM black 96-well plate with a Fluoroskan Ascent plate reader. The detailed procedure for the measurement was as described (Ku et al. 2002). The holo-nuclease-ColE7 was obtained by the supplement of zinc ions to the protein samples during the purification processes. Excess zinc ions were removed by a desalting column (Amersham) followed by dialysis of the protein samples against Milli-Q water. The activity assays were carried out in different concentrations of Mn2+ or Mg2+ using the zinc-bound holoenzymes.
Crystallization and X-ray data collection
The two different types of crystals of the nuclease-ColE7/Im7 were obtained by the hanging-drop vapor diffusion method. The protein complex from pQE-30 with the His-tag at the N-terminus of nuclease-ColE7 was crystallized in the I222 space group as described (Ko et al. 1999). The protein complex overexpressed from pQE-70 was crystallized using similar conditions. Drops of a solution containing 20 mg/mL of protein complex, 50 mM phosphate buffer (pH 6.3), 0.35 M ammonium acetate, and 11% PEG4000 were set up against a reservoir of 22.5% PEG4000. Plates of crystal appeared within about 4 d at room temperature. The metal ions originally associated in the complex were removed by soaking the crystals in the mother liquid containing 5–10 mM EDTA for 16 h. After the crystals were washed with phosphate buffer to remove EDTA, different metal ions, including Zn2+, Mn2+, and Mg2+, were soaked into the complex crystals for 16 h–40 h.
Diffraction data were collected at the absorption edge of the correspondent metal ion using synchrotron radiation (Synchrotron Radiation Research Center, BL17B2, Hsin-Chu, Taiwan) or at 1.54 Å using Cu K radiation from a rotating anode. X-ray absorption spectra were measured first to determine the absorption edge for Zn2+ or Mn2+ in crystals. X-ray radiation at inflection points of 
Zn = 1.29004 Å and Mn = 1.89261 Å was used to collect full sets of diffraction data for the Zn2+-soaked or Mn2+-soaked crystals at 100 K. Diffraction data were recorded on a Mac Science image plate (synchrotron data) or on an MSC R-AXIS-IV imaging plate (in-house source). In all cases, data were processed with DENZO and SCALEPACK (Otwinowski and Minor 1997).
Structure determination and refinement
The Zn2+-bound P21212 structure was solved by molecular replacement using the previously determined I222 structure (PDB entry: 7cei) as the searching model. Cross-rotation and translation function [CNS (Brunger et al. 1998)] unambiguously identified two unique complexes in the asymmetric unit using the diffraction data from 15–4.0 Å. Rigid-body refinement gave an R-factor of 38.6%. This model was subjected to manual rebuilding with Turbo-Frodo, alternating with torsional angle-simulated annealing, standard positional and individual isotropic B value refinement, and automatic water placement and deletion with the program CNS (Brunger et al. 1998). Fourier (2Fo-Fc) and anomalous maps in the metal-binding site were calculated at the final stage of refinement. Metal ions and phosphate ions were added into the model before the last cycle of refinement. The final refinement statistics are listed in Table I. The coordinates of the Zn2+-bound P21212 complex structure have been deposited in the Protein Data Bank with the accession code 1MZ8.
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Acknowledgments |
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