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Originally published In Press as doi:10.1074/jbc.M403719200 on June 8, 2004

J. Biol. Chem., Vol. 279, Issue 33, 34763-34769, August 13, 2004
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Structure-based Analysis of the Metal-dependent Mechanism of H-N-H Endonucleases*

María J. Maté and Colin Kleanthous{ddagger}

From the Department of Biology, Area 10, P. O. Box 373, University of York, Heslington YO10 5YW, United Kingdom

Received for publication, April 5, 2004 , and in revised form, June 8, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Controversy surrounds the metal-dependent mechanism of H-N-H endonucleases, enzymes involved in a variety of biological functions, including intron homing and DNA repair. To address this issue we determined the crystal structures for complexes of the H-N-H motif containing bacterial toxin colicin E9 with Zn2+, Zn2+·DNA, and Mg2+·DNA. The structures show that the rigid V-shaped architecture of the active site does not undergo any major conformational changes on binding to the minor groove of DNA and that the same interactions are made to the nucleic acid regardless of which metal ion is bound to the enzyme. The scissile phosphate contacts the single metal ion of the motif through distortion of the DNA brought about by the insertion of the Arg-96-Glu-100 salt bridge into the minor groove and a network of contacts to the DNA phosphate backbone that straddle the metal site. The Mg2+-bound structure reveals an unusual coordination scheme involving two H-N-H histidine residues, His-102 and His-127. The mechanism of DNA cleavage is likely related to that of other single metal ion-dependent endonucleases, such as I-PpoI and Vvn, although in these enzymes the single alkaline earth metal ion is coordinated by oxygen-bearing amino acids. The structures also provide a rationale as to why H-N-H endonucleases are inactive in the presence of Zn2+ but active with other transition metal ions such as Ni2+. This is because of coordination of the Zn2+ ion through a third histidine, His-131. "Active" transition metal ions are those that bind more weakly to the H-N-H motif because of the disengagement of His-131, which we suggest allows a water molecule to complete the catalytic cycle.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Homing endonucleases have played a key role in the evolution of genomes. Normally encoded within introns and inteins, these ubiquitous enzymes promote integration of mobile genetic elements into intronless or inteinless alleles (1). Homing enzymes are classified into four groups based on active site consensus motifs; LAGLIDADG, GIY-YIG, His-Cys box, and H-N-H. Over the last decade, the metal-dependent mechanisms of all but the H-N-H family have been largely resolved (2). Not confined to just homing endonucleases, hundreds of H-N-H enzymes, including DNA repair enzymes and the apoptotic endonuclease CAD, have been identified across the three biological kingdoms (3, 4). We present the first structural analysis of metal binding to an H-N-H motif containing endonuclease in the context of DNA, focusing on the enzyme, colicin E9.

Colicins are plasmid-encoded protein antibiotics made by Escherichia coli during times of stress (5). These potent toxins parasitize nutrient uptake pathways to deliver a cytotoxic domain into the cell by first binding to an outer membrane receptor and then contacting proteins in the periplasm. Together, these associations promote uptake of the toxin into the cell. Colicin E9 is a 61-kDa endonuclease toxin that kills cells through degradation of bacterial DNA. This is accomplished through the action of a C-terminal, 15-kDa DNase domain (the E9 DNase) that has an H-N-H motif as its catalytic core (6).

Homing endonucleases are sequence-specific enzymes that recognize long, degenerate consensus sequences (1). They also tend to be dimeric, cutting both strands of dsDNA1 simultaneously. However, not all enzymes that contain homing endonuclease motifs are involved in homing. The nuclease from Serratia marescens, for example, is a nonspecific endonuclease that shares active site similarity with the His-Cys family of enzymes, such as I-PpoI from Physarum polycephalum (7). Similarly, colicin E9 is a monomeric, nonspecific DNase that hydrolyzes single- and double-stranded DNA and is recognizable as an H-N-H enzyme through a 34-amino acid motif (6). It shares many enzymological characteristics with other H-N-H enzymes, such as a requirement for high concentrations of Mg2+ or Ca2+ ions for activity against dsDNA, the ability to utilize some transition metal ions for catalysis, and cleavage products that have 3'-OH and 5'-phosphate termini (8, 9).

Crystal structures for the related colicins, E7 and E9, have been reported. The structure for the E9 DNase is that of the enzyme bound to its cognate immunity protein, Im9 (6). Immunity proteins are inhibitors of colicin DNases that bind with high affinity to an exosite on the enzyme (10). Structures for the E7 DNase in the free and Im7-bound forms and, more recently, bound to dsDNA have been published, the latter in the absence of divalent cations (11–13).

An inherent assumption of the homing endonuclease field has been that the four recognizable active site motifs have arisen independently and represent distinct architectures. It was a surprise, therefore, when Kühlmann et al. (14) discovered that the H-N-H motif of colicin E9 DNase is structurally related to the His-Cys active sites of I-PpoI and Serratia nuclease. This "{beta}{beta}{alpha}-Me" motif, composed of two {beta}-strands, an {alpha}-helix, and a metal ion, has been found in other endonucleases such as Vvn from Vibrio vulnificus (15) and points to a common nuclease ancestor that has diverged into the distinct families we now recognize as H-N-H and His-Cys enzymes. Indeed, these families have diverged to such an extent that they share only 2 active site residues, a histidine that acts as a general base (His-103 E9 DNase/His-98 I-PpoI) and an arginine that is involved in phosphate binding (Arg-5 E9 DNase/Arg-61 I-PpoI) (14).

The identity of the biologically active metal ion for His-Cys enzymes such as I-PpoI is known to be magnesium (16), whereas this issue remains enigmatic for H-N-H enzymes. This is because of the four histidine residues of the motif, all of which are required for catalytic activity (4). In the absence of DNA, the H-N-H motif binds transition metals directly, with no detectable binding of magnesium (17). This is borne out by crystal structures for the E7 and E9 DNases where Zn2+ and Ni2+, respectively, have been observed bound to the motif histidines (6, 11). Ku et al. (18) have also reported that the E7 enzyme is a zinc-dependent enzyme. By contrast, we have been unable to detect Zn2+ activity for any colicin DNase (E2, E7, E8, or E9), with Zn2+ tending to be inhibitory (8, 9, 17). Nevertheless, colicins are able to utilize some first row transition metal ions such as Co2+ and Ni2+ to cleave DNA (we have speculated that apparent Zn2+ activity may be because of Ni2+ contamination) (19). These differences are related to the kinetics and thermodynamics of transition metal binding. Zn2+ association includes an additional step that traps the enzyme in a high affinity but inactive state (Kd ~ nM) (19). Active transition metals are those that bind with weaker affinities (Kd ~ µM). Through mutagenesis of H-N-H residues, Walker et al. (4) have established that Mg2+ ions are the preferred metal for dsDNA cleavage by the E9 DNase, and this is consistent with the metal requirements for other H-N-H enzymes such as I-CmoeI and LtrA from Chlamydomonas moewussi and Lactococcus lactis, respectively (20, 21).

To understand the mechanism by which these enzymes hydrolyze nucleic acid, in the present work we have used crystallography to address how Mg2+ ions bind to the histidine-rich H-N-H motif of the E9 DNase domain in the context of dsDNA. We have also rationalized why Zn2+ is inactive within the H-N-H motif, whereas other transition metals such as Ni2+ endow the motif with activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
The H103A E9 DNase was expressed and purified as described (4, 8). Apo-H103A E9 DNase was obtained after treatment with 5 mM EDTA and dialysis against 200 mM NaCl followed by dialysis against water and lyophilization (17, 19). The absence of metal in the H-N-H motif of purified protein was monitored routinely by fluorescence spectroscopy as described by Pommer et al. (17). Crystals of the H103A E9 DNase in complex with Zn2+ were obtained by hanging drop vapor diffusion against 26% w/v polyethylene glycol MME 2000, 100 mM KSCN, and 100 mM sodium acetate, pH 5.8, at a protein concentration of 30 mg/ml. Crystals were flash-cooled in Paraton-N and data collection performed at 100 K using the beamline ID14.1 at the European Synchrotron Radiation Facility (Grenoble).

In the preparation of H103A E9 DNase in complex with dsDNA, 8-mer palindromic DNA (5'-GCGATCGC-3') was first denatured at high temperature and then annealed by slow cooling. Bases are numbered in the text as 1–8 for one strand and 9–16 for the complementary strand. A stoichiometric amount of duplex was added to the protein to form the complex (final concentration, 40 mg/ml). Crystals of the Zn2+ complex were grown by hanging drop vapor diffusion against 18% v/v polyethylene glycol 400, 100 mM HEPES buffer, pH 7, and 6 mM ZnCl2, whereas 15% v/v polyethylene glycol 400, 100 mM cacodylate buffer, pH 6.5, containing 100 mM MgCl2 was used for the Mg2+ complex. Crystals were flash-cooled in mother liquor containing 28% v/v polyethylene glycol 400. X-ray diffraction data for both complexes were collected at the European Synchrotron Radiation Facility (Grenoble) beamline ID14.1.

All data sets were integrated and scaled using MOLSFLM (22) and SCALA (23), with the final statistics presented in Table I. Crystals of the H103A E9 DNase·Zn2+ complex belong to the space group P212121 and have two molecules of the complex in the asymmetric unit, with a Matthews coefficient of 2.4. Crystals of complexes with DNA bound belong to the space group C2221 and have four molecules of complex in the asymmetric unit, giving a Matthews coefficient of 2.0. Structures were solved by molecular replacement using the program AmoRe (24), using the E9 DNase domain bound to Im9 (Protein Data Bank code 1EMV [PDB] ) as the search model. All the structures were refined without non-crystallographic symmetry restraints using REFMAC (23), and the rebuilding was done using XtalView (25) and Coot (23). The omit maps for the metal site in the complexes with DNA were calculated independently using CNS (26) with simulated annealing and REFMAC; the residues omitted were the metal, the scissile phosphate, and histidines 102, 127, and 131. The maps calculated with both programs looked the same, and therefore only one is presented.


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  		TABLE I
Data collection and refinement statistics

 
Figures were produced using Molscript (27), Raster3D (28), XtalView (25), and Pymol (29). Atomic coordinates and structure factor amplitudes have been deposited in the Protein Data Bank with accession codes 1V13 [PDB] for H103A E9 DNase·Zn2+, 1V14 [PDB] for H103A E9 DNase·dsDNA·Mg2+, and 1V15 [PDB] for H103A E9 DNase·dsDNA·Zn2+.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Structure Determination and Description—To obtain structures of the E9 DNase bound to metal ions, we focused on an inactive mutant in which His-103, the putative general base, was substituted for alanine. The H103A mutant shows wild type binding of transition metals but is unable to catalyze DNA hydrolysis (4). Structures of three complexes of H103A E9 DNase were obtained in which the H-N-H motif was bound with (i) Zn2+, (ii) Zn2+·dsDNA, and (iii) Mg2+·dsDNA. The dsDNA used in crystallization experiments was an 8-mer palindromic sequence. Hsia et al. (13) used the same DNA duplex in their recent structure of the apo-E7 DNase bound to dsDNA. The three E9 DNase complexes were crystallized and their structures solved by molecular replacement using the coordinates for the wild type E9 DNase (see "Materials and Methods") (6, 30). The resolution for each of the structures of H103A E9 DNase bound to Zn2+, Zn2+·dsDNA, and Mg2+·dsDNA was 2.0, 2.4, and 2.9 Å, respectively. The refinement statistics for each complex are presented in Table I. In the case of the H103A E9 DNase bound to a single Zn2+ ion, two molecules of the metal-bound complex were present in the asymmetric unit, whereas in both DNA·metal complexes the enzyme was bound to a single 8-mer duplex and a single metal ion, with four copies in each asymmetric unit.

The E9 DNase domain is known to exhibit substantial conformational heterogeneity around residues 20–25 and 65–72, regions that are not part of the enzyme active site but are in close proximity to each other. This has been observed by heteronuclear NMR for the unbound protein as two slowly inter-converting conformers (31), with this heterogeneity persisting in the Im9-bound form (32), and for the E9 DNase·Im9 complex crystallographically by virtue of the high B-factors for these regions relative to the rest of the structure (30). The temperature factors for the same regions are also high in the present structures of H103A E9 DNase bound to Zn2+ and the enzyme·DNA complex bound to either Zn2+ or Mg2+. In addition, the N-terminal 3–6 residues of all monomers in both protein·DNA complexes have high B-factors. Moreover, monomer A in the H103A E9 DNase·dsDNA·Zn2+ complex shows higher B-factors than the other monomers, particularly in the conformationally flexible regions. Finally, residues 35–47 and 66–75 were not well defined in the electron density and so were not included in the final model for this monomer only. In summary, the complexes of H103A E9 DNase bound to metal ions and dsDNA display several disordered regions, most of which have been documented previously. These affect the general quality of the model, reflected in the slightly high R factor/Rfree (Table I).

The overall structure for the Mg2+-bound dsDNA complex of H103A E9 DNase is shown in Fig. 1A, with representative electron density for this complex presented in Fig. 1B. As reported previously, the E9 DNase is a mixed {alpha}/{beta} protein, with its 34-amino acid H-N-H motif located at the extreme C terminus of the enzyme (Fig. 1A, dark green) (6, 30). The H-N-H motif (residues 100–134), along with residues 94–100 that are part of a preceding loop, forms the principle contacts with the DNA although other regions of the protein, at positions 5, 51, and 54, also interact with the nucleic acid. The structure for H103A E9 DNase bound to Zn2+ in the present work is very similar to that of the Ni2+-bound wild type enzyme (the root mean square deviation for C{alpha} atoms is 0.7 Å). Binding of dsDNA to the H103A E9 DNase, loaded with Zn2+ or Mg2+ in the H-N-H motif, also only resulted in small structural changes to the enzyme with the root mean square deviations for C{alpha} atoms relative to the Zn2+-bound enzyme being 0.6 and 0.8 Å, respectively. This is in stark contrast to the DNA bound at the active site, the minor groove of which is distorted relative to B-DNA of the same sequence (Fig. 1A).



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  		FIG. 1.
Structure of H103A E9 DNase bound to the minor groove of 8-mer dsDNA and Mg2+. A, the figure shows the protein·DNA complex (E9 DNase in green, dsDNA in yellow ribbon) overlaid with B-DNA of the same sequence (blue ribbon) to illustrate the distortion that results from the binding of the H-N-H motif (dark green) and its associated metal ion (red) to the minor groove. Only nucleotides 3–14 are presented. B, stereo view of the 2Fo - Fc electron density map of the lower resolution H103A E9 DNase·dsDNA·Mg2+ complex. The map is plotted at a contour level of 1{sigma} and shows the region where the salt-bridged residues Arg-96 and Glu-100 (indicated by the dotted lines) are inserted into the DNA minor groove, causing it to widen and bend (see "Results and Discussion" for details). Also shown is the hydrogen bond between Arg-96 and the hydroxyl of one of the DNA bases, T13.

 
In addition to the changes induced in the DNA by binding to the H-N-H motif of the E9 DNase, packing of the protein monomers within the asymmetric unit disrupts the base pairing at one end of the bound DNA duplex (Fig. 2). This results in the flipping-out of the terminal bases of both strands to yield final conformations that are stabilized by contacts with neighboring monomers. These changes, although most likely linked to the distortions induced by binding to the H-N-H motif and the destabilization of the duplex that ensues, are unlikely to be mechanistically relevant.



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  		FIG. 2.
Crystal contacts distort one end of the DNA duplex in the complex of H103A E9 DNase bound to 8-mer dsDNA. In the crystal structure, double-stranded DNA (5'-GCGATCGC-3') undergoes two types of distortion. First and foremost is that associated with minor groove binding, highlighted in Figs. 1 and 3. Secondly, packing of monomers within the asymmetric unit disrupts Watson-Crick base pairing at one end of bound DNA. This is illustrated in the figure for monomer C (green), in which the position of the H-N-H active site motif is denoted by the bound metal ion (buried sphere). The first nucleotide (G1) is not visible in any of the electron densities. The bases at positions 2 and 16 (C2 and C16) are rotated ~180° away from the helical axis, their highly distorted positions stabilized by contacts with neighboring monomers B and D (cream and blue, respectively). Additional stabilization of these distorted conformations is through direct DNA-DNA hydrogen bonds between neighboring complexes (not shown). All monomers in both Zn2+ and Mg2+ complexes make equivalent crystal contacts.

 
Protein-Nucleic Acid Contacts between the Metal-bound H103A E9 DNase and dsDNA—A direct consequence of the unyielding nature of the V-shaped active site is that in order for a scissile phosphate of B-DNA to reach the metal center of the H-N-H motif it must undergo some distortion; this is borne out by the current structures (Fig. 1A). The {alpha}-helix (and accompanying metal ion) of the E9 DNase H-N-H motif is positioned parallel to the helical axis of the DNA minor groove, with residues from the preceding six amino acids inserted into the groove itself. Together, these regions contact predominantly the phosphate backbone and cause a significant bend toward the major groove and widening of the minor groove (from 5.9 to ~9 Å; see Fig. 1A). We note that in the case of the E7 DNase binding to the same DNA sequence (13) this central distortion is essentially equivalent to that induced by the E9 DNase, demonstrating that it is independent of metal ion binding and is an intrinsic property of the H-N-H motif itself. This also emphasizes that the frayed end of the duplex seen in the present structures (but not seen in the E7 structure) is due to crystal packing forces.

One of the striking observations from a comparison of the Zn2+ and Mg2+ structures for dsDNA bound to H103A E9 DNase is the similarity of interactions made by the enzyme to the nucleic acid. In both the Mg2+ and Zn2+ H103A E9 DNase complexes the same scissile phosphate oxygen atom (P6) between T5 and C6 is coordinated by the metal ion, with the same hydrogen-bonding interactions being made toward the surrounding phosphates (Fig. 3). Hence, although Mg2+ ions, but not Zn2+ ions, support DNA cleavage by the E9 DNase the global features of their respective complexes with nucleic acid are essentially identical. However, subtle differences do exist between the two complexes that explain the differing activities of the metal ions, which are largely restricted to the vicinity of the metal ion. We return to this issue below.



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  		FIG. 3.
A hydrogen-bonding network connects DNA distortion to approach of the scissile bond to the H-N-H motif metal center. Stereo representation of the main hydrogen-bonding interactions between the E9 DNase and the DNA minor groove in the Zn2+ complex, with identical interactions observed in the Mg2+-bound complex. The DNA and protein residues are cream and green, respectively, the Zn2+ ion is magenta, and hydrogen bonds are plotted as dotted lines. The scissile bond of the DNA can only approach the metal ion if the DNA is distorted. This is accomplished by the insertion of the Arg-96-Glu-100 salt bridge into the minor groove and stabilization of this configuration through a number of hydrogen-bonding interactions to the DNA phosphates through residues Arg-5, Asp-51, and Arg-54.

 
Given the poorer resolution (2.9 Å) of the data for the Mg2+-bound complex the following is a summary of the main contacts to dsDNA in the Zn2+ complex (2.4 Å). A total of ~700 Å2 of protein surface area is buried on complexation. The distortion of the DNA is because of the insertion of the Arg-96-Glu-100 salt bridge into the minor groove of the DNA, causing it to bend and widen appreciably. This conformation is stabilized by a network of hydrogen bonds between the DNA and residues Arg-5, Arg-54, and Asp-51 that straddle the scissile phosphodiester at P6 (Fig. 3). The E7 DNase makes a similar set of interactions to dsDNA, although interestingly these are centered around a different scissile bond (P4). Arg-5 and Arg-54 make three hydrogen bonds to DNA phosphates P6 and P7. Arg-96 forms a hydrogen bond to the hydroxyl of a base on the opposing strand (T13) in the minor groove (Arg-96 N1 with T13 O2). The equivalent residue in the E7 DNase makes a water-mediated contact with a guanine base (13). Asp-51 of the E9 DNase also makes a base-specific hydrogen bond (Asp-51 OD2 with C8 N4), as well as engaging in a salt bridge with Arg-54 (Fig. 3). The hydrogen bonds either side of the scissile phosphodiester in the E9 DNase complex are linked to each other by virtue of the salt bridge between Arg-96 and Glu-100. In the Zn2+-bound structure, Glu-100 orients His-127, a critical ligand to metal ion, although this interaction is less clear-cut in the Mg2+-bound structure, with the metal in each case contacting the scissile phosphate at P6 (Fig. 3). In summary, a network of bonds from the H-N-H motif of the E9 DNase ensures that deformation of dsDNA is linked to the approach of the scissile phosphodiester to the metal ion embedded within the motif. Consistent with the important roles of Arg-5, Arg-54, Arg-96, and Glu-100 in this deformation, alanine mutants at these positions abolish DNase activity toward dsDNA (4).2

Comparison of Alkaline Earth and Transition Metal Ion Binding Sites in the Colicin E9 H-N-H Motif—The present study demonstrates that in the presence of DNA the H-N-H motif binds a single divalent cation. Single metal coordination has also been observed for the His-Cys enzyme I-PpoI (16), the DNA junction resolving enzyme T4 endonuclease VII (33), and the nonspecific endonuclease Vvn (15). Because all are members of the {beta}{beta}{alpha}-Me superfamily, it is clear that this is the norm for {beta}{beta}{alpha}-Me enzymes and that this contrasts the two-cation mechanisms prevalent in, for example, type II restriction enzymes (2). Based on the properties of the E9 DNase we have proposed that its H-N-H active site is capable of binding Mg2+ or Ca2+ (both can be used by the enzyme) as well as transition metal ions such as Zn2+ (9). The histidine-rich nature of H-N-H enzymes is compatible with their ability to bind transition metal ions. Such a site is not, however, optimal for Mg2+ binding in enzymes, especially endonucleases, where oxygen ligands are generally utilized for the coordination of the metal ion (34).

To ascertain the presence of the individual metal ions in our structures and more critically evaluate the conformation of surrounding residues, omit maps were calculated (Fig. 4). The metal ion within the H103A E9 DNase·dsDNA·Zn2+ complex is held in tetrahedral geometry, with three protein ligands (His-102, His-127, and His-131) and a phosphate oxygen atom from the scissile bond (Fig. 4A). An identical tetrahedral arrangement is seen for Zn2+ bound to the H103A E9 DNase in the absence of DNA, where the phosphate oxygen is replaced by water. The three imidazole nitrogen-Zn2+ distances (2.0–2.1 Å) are consistent with those of other transition metal ions bound to enzyme active sites. By contrast, the distance between the Zn2+ and the phosphate oxygen is shorter, at 1.9 Å. This is the same as that seen for the Zn2+-O bond in the structure of the E7 DNase bound with Zn2+ and phosphate (instead of DNA) at its H-N-H motif (12). Similar Zn2+-O bond distances of ~2 Å have been reported previously, for example in the E. coli repair enzyme endonuclease IV (35).



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  		FIG. 4.
Simulated annealing omit maps around the active site for the complexes of H103A E9 DNase·dsDNA bound to Zn2+ (A) and Mg2+ (B). The metal ion, the scissile phosphate, and histidines 102, 127, and 131 were omitted for the calculation, and the resulting maps were plotted at a contour level of 2.0 {sigma}. The coordination of both metal ions is indicated with dotted lines. The figure highlights how the H-N-H motif is an adaptable metal binding center. It is able to accommodate both the tetrahedral coordination chemistry of a Zn2+ ion (A) and then, through subtle reorientations of the H-N-H histidine residues and the inclusion of DNA phosphodiester oxygen atoms and a water molecule, the octahedral geometry required for Mg2+ ion binding (B). Only five of the possible six coordination sites are discernible for the Mg2+ ion at the current level of resolution.

 
The major differences between the H103A E9 DNase·-dsDNA·Zn2+ and H103A E9 DNase·dsDNA·Mg2+ complexes are localized to the metal centers. Although all four molecules contained metal ion in the DNA·Zn2+ complex, only two of the four copies of the H103A E9 DNase·dsDNA·Mg2+ complex in the asymmetric unit (molecules A and C) contained density consistent with a Mg2+ ion (Fig. 4B). The unambiguous assignment of Mg2+ in endonuclease active sites is often difficult because of the weak nature of the binding. Mg2+ does not bind to the E9 DNase in the absence of DNA, and the high concentrations of Mg2+ required for optimal activity (10–20 mM) (8) further suggest that binding will also be weak even in the presence of DNA. This being the case, we have to consider that the density adjacent to the H-N-H histidines in the Mg2+·DNA complex may be a water molecule. We suggest that this is unlikely for four reasons. First, the H103A E9 DNase·dsDNA·Mg2+ crystals were grown in a high concentration of Mg2+ ions (100 mM). Second, Kuhlmann et al. (30) have determined the structure for apo-E9 DNase, with a water molecule replacing the metal ion in the H-N-H motif. The structure shows that the conformations and hydrogen-bonding interactions of the H-N-H histidines are different from those seen in the current structure. Third, the hydrogen-bonding interactions of the molecule seen in the H-N-H motif are not compatible with those of a water molecule. Fourth, the temperature factors for the Mg2+ ion are of the same order as those of surrounding atoms.

Although the Mg2+ ion in the H103A E9 DNase·dsDNA·Mg2+ complex is present in monomers A and C, the details of its coordination in monomer C are better resolved. The position of this metal ion is displaced by ~1 Å relative to the metal ion in the DNA·Zn2+ structure, which is greater than the root mean square deviation between the two complexes (Fig. 5). The coordination of this metal ion is clearly very different from that of the Zn2+ ion in the H103A E9 DNase·dsDNA·Zn2+ complex. However, at the current level of resolution only five of the anticipated six ligands can be seen to contact the Mg2+ ion. Equatorial positions are taken by two oxygen atoms from the scissile phosphate (O1P and O3') and two nitrogen atoms from the H-N-H histidine residues (His-102 and His-127), with one of the axial positions taken by a water molecule (Fig. 4B). We presume that the sixth position, likely to be another water molecule, is not resolved in the current structure. To our knowledge, this is the first time that the coordination shell of a Mg2+ ion in an enzyme active site has been shown to include two histidine residues, although we note that similar bis-His coordination is seen for manganese ions binding in the DNA repair nuclease Mre11 from Pyrococcus furiosus (36). Intriguingly, His-131, the third ligand in the Zn2+ complex, moves by >2 Å and is replaced by a water molecule that resides midway between it and the Mg2+ ion (Fig. 4B). We note that although only two of the four monomers in the H103A E9 DNase·dsDNA·Mg2+ structure contain magnesium, water does not replace the metal ion in the apparently vacant monomers. Furthermore, the conformations of the three critical histidine residues (His-102, His-127, and His-131) are very similar in all four monomers, especially that of His-131, which is in the same displaced conformation. This suggests that a magnesium ion could be present in all monomers, but its occupancy is either too low or the B-factors too high to be seen in all but two of them.



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  		FIG. 5.
Superposition of H-N-H motif histidine residues from different liganded states of the E9 DNase. Only the phosphate coordinated to the metal ion in each structure is shown. Atoms are color-coded for the different complexes. White, H103A E9 DNase·dsDNA·Zn2+ complex; black, H103A E9 DNase·dsDNA·Mg2+ complex; gray, wild type E9 DNase bound with Ni2+ and a phosphate molecule. The transition metal ions superimpose with each other, whereas the Mg2+ ion is displaced by ~1 Å. His-131 is in a different location in each complex, with the largest change occurring in the Mg2+ complex where a water molecule (not shown) resides midway between the metal ion and the imidazole ring (see Fig. 4B).

 
Although unusual, there is precedent for the ligation of Mg2+ by histidines in enzymes. Dudev and Lim (34) have pointed out that although tetrahedrally oriented histidine active sites ligate alkaline earth metal ions poorly, Mg2+ binding could be accomplished if a negatively charged side chain is incorporated to create a site with octahedral geometry. The scissile phosphate oxygen atoms in the DNA-bound complex provide this negative charge. Such mixed coordination of Mg2+, as well as Ca2+ ions, has been seen in other enzymes, notably xylose isomerase and diisopropylfluorophosphatase (37, 38). In xylose isomerase, one of two Mg2+ ions is coordinated by a histidine residue as well as carboxylate side chains, whereas in diisopropylfluorophosphatase one of two Ca2+ ions is similarly coordinated. The metal-imidazole distances in both enzymes are ~2.6 Å; those for His-102 and His-127 to the Mg2+ in the present structure are 2.5 and 2.2 Å, respectively.

In the original description of the Mg2+-dependent mechanism of the H-N-H class of endonucleases, Pommer et al. (9) postulated that a single histidine residue (His-127) contacted the metal ion, by analogy with the His-Cys/{beta}{beta}{alpha}-Me enzyme I-PpoI that binds a single Mg2+ ion in the presence of DNA (16). This ligand is an asparagine in I-PpoI (Asn-119), and its position is equivalent to His-127 (14). With the recent publication of the structure for Vvn endonuclease (15), it is now evident that {beta}{beta}{alpha}-Me enzymes fall into two subgroups, those utilizing a single protein ligand to the Mg2+ (or Ca2), as seen in I-PpoI and Serratia, and those with two ligands, as seen in Vvn where asparagine and glutamic acid bind the metal ion. We now see that H-N-H enzymes such as the E9 DNase fall into the latter group; the two residues are histidines, with their positions superimposable on the two Mg2+-coordinating amino acids of Vvn. The counterparts of His-102 and His-127 of the E9 DNase are Glu-79 and Asn-127 in Vvn. Interestingly, sequence alignments of H-N-H motif-containing enzymes show that the two metal-coordinating histidines of the E9 DNase are occasionally substituted for oxygen-bearing amino acids, such as asparagine, glutamine, and aspartic acid.

Proposed Mechanism for Mg2+-dependent Cleavage of H-N-H Enzymes and Why Zn2+ Is Inactive—The Mg2+ ion in I-PpoI serves to polarize the scissile phosphate, stabilize the phosphoanion transition state, and activate a water molecule that protonates the leaving group (16). We had proposed previously that for Mg2+-based activity of the E9 DNase the metal ion would serve two of these roles, with that of general acid being taken by His-102 (9). However, it is now clear that this residue, along with His-127, is engaged in Mg2+ coordination. Because there are no other protein side chains within the vicinity of the scissile bond, it seems more likely that this role is taken by a water molecule, possibly that which sits midway between the Mg2+ ion and His-131 (Fig. 4B). His-103 (mutated to alanine in the present study) almost certainly serves as the general base in the activation of the hydrolytic water molecule, the corresponding histidine in I-PpoI being His-98. When His-103, from the Ni2+·phosphate complex of the E9 DNase (6), is superimposed onto the present structures its position is appropriate for such a role (Fig. 5). Moreover, a water molecule is correctly positioned in the E9 DNase·Zn2+·dsDNA complex to act as the attacking nucleophile to the phosphodiester (data not shown).

We now turn to the question of the transition metal-dependent activity of the H-N-H motif within the E9 DNase and the controversy surrounding zinc. We had proposed previously that for activity in the presence of transition metals two non-protein-bound coordination sites at the metal center were required, one for ligation of the scissile phosphate oxygen and the other for the activation of a water molecule for leaving group stabilization (9). The present structure for the Zn2+-bound enzyme shows that this is not possible because three histidine side chains tightly coordinate the metal ion (Fig. 4A). Although we do not have a structure for an active transition metal such as Ni2+ bound to an E9 DNase·dsDNA complex, the structure for the E9 DNase bound with Ni2+ and a phosphate molecule is informative (6). In this case, the third metal-ligating histidine, His-131, disengages from the metal ion, with its distance lying midway between that seen in the Mg2+ and Zn2+ structures of the present study (Fig. 5). This may allow a water molecule to replace the histidine and so serve as a protonating device for DNA hydrolysis, in other words by a similar mechanism to that proposed for Mg2+-based activity.

In conclusion, the current work has provided a structural basis for understanding why high affinity zinc binding inactivates the H-N-H motif and has helped explain why only weakly bound transition metals are active in the motif. It has also provided the first glimpse as to how the motif binds and utilizes Mg2+ ions to cleave dsDNA by a mechanism that likely underpins the action of all known H-N-H/{beta}{beta}{alpha}-Me endonucleases. The current structures of the H-N-H motif support previous work on this class of enzyme that suggested the motif to be an adaptable catalytic center, able to utilize both alkaline earth and transition metal ions. Contrary to expectations, however, the mechanism of DNA cleavage for the different classes of metal ions may be the same.

Note Added in Proof—We note a recent report (Datta, S., Larkin, C., and Schildbach, J. F. (2003) Structure 11, 1369–1379) of the structure of a bacterial relaxase that utilizes a single catalytic Mg2+ ion to cleave single-stranded DNA and that this metal ion is coordinated by histidine residues.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1V13 [PDB] , 1V14 [PDB] , and 1V15 [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was funded by the Biotechnology and Biological Sciences Research Council of the UK. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 44-1904-328820; Fax: 44-1904-328825; E-mail: ck11{at}york.ac.uk.

1 The abbreviations used are: dsDNA, double-stranded DNA; Im, immunity protein. Back

2 A. H. Keeble and C. Kleanthous, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Miguel Ortiz-Lombardia, Eleanor Dodson, Garib Murshudov, Marek Brzozowski, and Fred Anston of the York Structural Biology Laboratory and Geoff Moore (Norwich), Richard James (Nottingham), and members of the Kleanthous laboratory for helpful and stimulating discussions. We also thank Richard James for the gift of the H103A E9 DNase mutant. Finally, we thank the European Synchrotron Radiation Facility for provision of synchrotron radiation and support.



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 RESULTS AND DISCUSSION
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