Ni(II) coordination to mixed sites modulates DNA binding of HpNikR via a long-range effect

Abby L West; Sarah E Evans; Javier M González; Lester G Carter; Hiro Tsuruta; Edwin Pozharski; Sarah L J Michel

doi:10.1073/pnas.1120283109

. 2012 Mar 26;109(15):5633–5638. doi: 10.1073/pnas.1120283109

Ni(II) coordination to mixed sites modulates DNA binding of HpNikR via a long-range effect

Abby L West ^a, Sarah E Evans ^a, Javier M González ^a, Lester G Carter ^b, Hiro Tsuruta ^b, Edwin Pozharski ^a,¹, Sarah L J Michel ^a,¹

PMCID: PMC3326501 PMID: 22451934

Abstract

Helicobacter pylori NikR (HpNikR) is a nickel-dependent transcription factor that regulates multiple genes in the H. pylori pathogen. There are conflicting data regarding the locations of the Ni(II) sites and the role of Ni(II) coordination in DNA recognition. Herein, we report crystal structures of (i) the metal-binding domain (MBD) of HpNikR (3.08 Å) and (ii) a mutant, H74A (2.04 Å), designed to disrupt native Ni(II) coordination. In the MBD structure, four nickel ions are coordinated to two different types of nickel sites (4-coordinate, square planar, and 5/6-coordinate, square pyramidal/octahedral). In the H74A structure, all four nickel ions are coordinated to 4-coordinate square-planar sites. DNA-binding studies reveal tighter binding for target DNA sequences for holo-HpNikR compared with the affinities of Ni(II) reconstituted apo-HpNikR and H74A for these same DNA targets, supporting a role for Ni(II) coordination to 5/6 sites in DNA recognition. Small-angle X-ray scattering studies of holo-HpNikR and H74A reveal a high degree of conformational flexibility centered at the DNA-binding domains of H74A, which is consistent with disorder observed in the crystal structure of the protein. A model of DNA recognition by HpNikR is proposed in which Ni(II) coordination to specific sites in the MBD have a long-range effect on the flexibility of the DNA-binding domains and, consequently, the DNA recognition properties.

Keywords: DNA transcription, metalloregulation

The virulent bacterium, Helicobacter pylori, colonizes the gastric epithelium of humans, leading to gastric ulcers and gastric cancers (1–3). H. pylori encounter a wide range of pH fluctuations during their lifetimes (pH 1.0–8.0) (4). The intracellular cytoplasmic pH for H. pylori during colonization has been reported to vary between ∼5.3 and 7.5, resulting in a broad physiological pH range for this microorganism (5–7). A key protein necessary for H. pylori growth and virulence is the metalloregulatory protein NikR (8–11). HpNikR is a nickel-dependent transcription factor that directly regulates multiple genes, including many that encode for nickel cofactored proteins (12–19). A particularly important protein whose expression is regulated by HpNikR is the urease enzyme, which protects H. pylori from acidic shock at low pH by converting urea to ammonia and bicarbonate (20).

HpNikR is a tetrameric protein that consists of two domains: a central tetrameric metal-binding domain (MBD) and two flanking ribbon–helix–helix DNA-binding domains (DBD; Fig. 1) (21–24). To recognize and target DNA, HpNikR requires four nickel ions coordinated to high-affinity sites located on the MBD (12, 13, 15), in addition to magnesium (or calcium or manganese), which coordinate to low-affinity sites on the DBD (13, 15). Similar nickel ion requirements are observed for the E. coli homolog, EcNikR, for which DNA binding is achieved when four nickel ions are bound to high-affinity sites, and potassium (rather than magnesium, calcium, or manganese for HpNikR) is bound to two low-affinity sites (25–27). The nickel coordination sites in EcNikR are 4-coordinate, square planar with a His₃Cys ligand set (25, 28, 29). Identical nickel coordination geometry is observed in the NikR homolog from Pyrococcus horikoshii (PhNikR) (30), thus HpNikR was expected to coordinate Ni(II) in a similar fashion (12, 13, 15). However, the details of nickel coordination for HpNikR remain controversial. The first published structures of HpNikR, obtained after soaking crystals of apo-protein at pH 4.0 with excess nickel (20–100 mM), had only two nickel ions coordinated at predicted 4-coordinate sites, with additional nickel ions coordinated to “intermediate” and “external” sites (21, 22). Subsequently, the crystal structure of Ni(II) bound HpNikR at pH 5.6 was reported by our laboratories (24). In the structure, four nickel ions per protein tetramer were observed in two distinct coordination geometries. Two nickel ions were bound at 4-coordinate, square-planar sites with a His₃Cys donor set (further referred to as 4-sites), whereas the two other Ni(II) sites were coordinated by His₃(H₂O)_2–3 ligand sets in square pyramidal /octahedral geometries (further referred to as 5/6-sites). The 5/6-sites are analogous to the intermediate sites in the Ni(II)-soaked HpNikR structures (21, 22). The two nickel sites cannot be occupied simultaneously because two of the histidine ligands (H88 and H101) coordinate Ni(II) at both sites. These structures provided compelling evidence that HpNikR uses two different nickel coordination geometries for DNA recognition, and led us to suggest a role for the 5/6-site of HpNikR in modulating the recognition properties (24). Recently, a crystal structure of HpNikR was reported at pH 7.3 (31). Surprisingly, in this structure all four nickel ions were bound to 4-sites, opening up the possibility for multiple nickel coordination modes for HpNikR and a role of pH in nickel coordination.

Fig. 1. — (A) Cartoon of the domain architecture of HpNikR. The tetrameric protein is composed of four MBD (blue) and four DBD (red). (B) Structure of MBD subunit of HpNikR with the two Ni(II) sites highlighted. The Ni(II) 4-sites are colored dark green, and the Ni(II) 5/6-sites are colored light green (PDB ID code 2QSI). (C) Ligands that coordinate Ni(II) at 4-site (sp) and 5/6-site (sqp/oc). His88 and His101 (colored red) serve as ligand for both sites.

Herein, we present a crystal structure of the MBD of HpNikR at pH 7.5 and demonstrate that mixed Ni(II) coordination geometry occurs over the range of pH that is physiologically relevant for H. pylori. In addition, we present the crystal structure of a mutant of HpNikR, H74A, which is designed to force all four nickel ions to bind to 4-sites. By comparing the DNA recognition properties and conformational flexibility of holo-HpNikR with H74A using a combination of fluorescence anisotropy and small-angle X-ray scattering (SAXS), we have obtained compelling evidence that mixed Ni(II) coordination controls the flexibility of HpNikR's DBDs via a long-range effect and consequently affects the protein's DNA recognition properties. In addition, we show that the method of protein isolation is critical to obtain the true holo form of the protein. We discuss these findings in the context of HpNikR's ability to recognize multiple DNA targets.

Results and Discussion

Nomenclature.

HpNikR can be isolated using two protocols: (i) directly in the holo form upon overexpression in E. coli or (ii) by first reconstituting apo-HpNikR (prepared by addition of excess EDTA to holo-HpNikR followed by exhaustive dialysis) with Ni(II) (15, 24, 31). We will refer to HpNikR that is prepared directly as holo-HpNikR and HpNikR that is prepared via addition of Ni(II) to apo-HpNikR as reconstituted HpNikR. The mutants described in this work (MBD and H74A) will be designated similarly. The tetrameric MBD and the dimeric DBD will be referred to as the MBD and the DBD, respectively; individual chains that form these will be referred to as MBD/DBD monomers.

Crystal Structure of the HpNikR MBD at pH 7.5.

As part of our efforts to structurally characterize and identify the Ni(II) sites of HpNikR, we have prepared just the MBD by truncating the protein at position 58 (SI Materials and Methods). Inductively coupled plasma (ICP)-MS analysis confirmed a stoichiometry of four Ni ions per MBD tetramer. Apo-MBD was generated using methods developed to prepare apo-HpNikR and a titration of apo-MBD with Ni(II) monitored by UV-visible spectroscopy saturated at four equivalents of Ni(II) per MBD tetramer (15) (Fig. S1). Holo-MBD was crystallized at pH 7.5 (Fig. 1).

Holo-MBD adopts a ferredoxin-like fold with an antiparallel β-sheet sandwiched against a pair of α-helices forming a characteristic βαββαβ secondary structure pattern. The four chains that form the central MBD subunit of HpNikR and coordinate the nickel ions adopt a “dimer of dimers” architecture, with each nickel ion using ligands from two MBD monomers. The asymmetric unit contains 10 MBD monomers that form three tetramers, with the last subunit assembled by crystal symmetry. The four nickel ions are separated into two sites: two nickel ions bind to 4-sites composed of H88′, H99, H101, and C107; and two nickel ions bind to 5/6-sites composed of H74, H88′, and H101, and up to three putative water molecules (due to the limited resolution, no explicit water ligands were placed in the crystal structure; H88′ is contributed by a different MBD monomer than the rest of the residues in both sites). Thus, mixed Ni(II) coordination can be observed for HpNikR at neutral pH.

No overall changes in the backbone conformation of the MBD are observed compared with the crystal structure of full-length holo-HpNikR at lower pH (24). The maximum-likelihood based estimates of coordinate uncertainty (32) for the holo-HpNikR and MBD structures are 0.29 Å and 0.46 Å, respectively. Accordingly, the differences between the two structures that are ≤0.55 Å are within experimental error. With the exception of the third tetramer, which is formed by crystal symmetry (see below), the baseline difference in atomic positions, as defined by the percentile-based spread (p.b.s.) (33), is 0.52 Å; hence, the two MBDs can be considered identical.

It was recently suggested by Benini et al. (31) that the mixed coordination observed in the pH 5.6 structure of holo-HpNikR was due to the lower pH. However, the pH 7.5 MBD structure reported here clearly demonstrates that mixed coordination for Ni(II) is not abolished at neutral pH. Analysis of the conditions used to generate crystals by Benini et al. (31) suggests that the singular type of nickel coordination observed in their pH 7.3 structure may be attributable to the method of protein isolation, which involved first preparing apo-HpNikR and then reconstituting the protein with Ni(II). We also note that all of the structures of HpNikR, which range in pH from 5.6 to 7.5, are physiologically relevant because the cytoplasmic pH of H. pylori fluctuates between 5.3 and 7.5 (5–7).

Previously published isothermal titration calorimetry (ITC) data for Ni(II) binding to HpNikR identified a two-step Ni(II) binding process (34). The Ni(II) binding affinities differ for each binding step (e.g., ∼12/125 nM at pH 7.0) and correspond to two nickel ions each per protein tetramer. These data are consistent with the mixed nickel coordination environments that we have observed structurally. Although the binding affinities exhibited a slight pH dependence, the difference between the corresponding free energies of binding remained constant at ∼1.5 kcal/mol, supporting a model in which Ni(II) binds to two different sites over a range of pH values.

Ni(II) binds to 5/6-sites and 4-sites in two of the three tetramers present in the asymmetric unit of the holo-MBD crystals. In the third tetramer, generated by crystal symmetry, only two Ni(II) ions, both of which are coordinated to 4-sites, are observed. (SI Materials and Methods and Fig. S2). The 5/6-site is unoccupied, although the corresponding ligands are properly positioned to coordinate Ni(II) in this geometry. There is a higher level of positional variation in this half-occupied form of the protein, compared with the two fully occupied forms. The overall p.b.s. between the two forms is ∼0.9 Å (which substantially exceeds the experimental error) (33). More detailed analysis shows that these differences may be described as a slight opening up of the interface between the two MBDs, because the nickel ions no longer keep the domains together (Fig. S2 and Movie S1). In the context of the ITC data, this crystal form suggests that Ni(II) binds to the 4-site with higher affinity than the 5/6-site (34).

Crystal Structure of an HpNikR Mutant, H74A.

To determine the role of Ni(II) coordination to the 5/6-site in protein function, a mutant protein (H74A) was constructed in which a ligand unique to the 5/6-site (H74) was mutated to a noncoordinating alanine (SI Materials and Methods). This mutant should force all four Ni(II) ions to coordinate 4-sites. H74A was isolated in the holo form and ICP-MS analysis confirmed four Ni ions per H74A tetramer. Apo-H74A was also prepared using protocols developed for HpNikR (15), and a titration of apo-H74A with Ni(II), monitored by UV-visible spectroscopy, showed that the H74A tetramer binds four Ni(II) ions, as predicted (Fig. S3). As evidenced by CD spectroscopy, no changes to the secondary structure were observed upon addition of Ni(II) to apo-H74A or between “reconstituted” H74A and holo-H74A (Fig. S4).

Holo-H74A was crystallized at pH 6.5, and the structure was solved to 2.04 Å resolution (Fig. 2A). As predicted, all four nickel ions are bound to square-planar 4-sites via a His₃Cys ligand set. The asymmetric unit contains two polypeptide chains (the full tetramer is obtained by crystal symmetry) that are related by nearly perfect noncrystallographic twofold rotation. In both positions, nickel is coordinated by H88′, H99, H101, and C107. The two chains are structurally similar and upon superposition are characterized by all atom p.b.s. of 0.38 Å. The main differences are observed upstream from K64 in the loop connecting MBD and DBD, apparently due to differences in the positions of the corresponding DBD helices.

A large portion of the H74A DBD is disordered, with only the α-helix comprising residues 37–54 traceable in the electron density. There is room for the full DBD in the unit cell, as indicated by the Matthews coefficient (35) of ∼2.23 for the complete HpNikR tetramer (∼45% solvent content). An extrapolated positioning of the DBDs, based upon the structure of the wild-type protein and the single α-helix found in the electron density, indicates an absence of potential clashes with symmetry-related copies. We note that a lack of electron density for the most of the DBD is also observed in the crystal structure of reconstituted HpNikR, reported by Benini et al. (31). Inspection of the electron density from that structure (PDB ID code 2Y3Y) shows that the same α-helix can be traced (although the authors of the structure have chosen to model this density as either unexplained undecapeptide or several polyethyleneglycol fragments), which suggests that mixed nickel coordination may be required for proper docking of the DBD and MBD to form an interface.

A global analysis of the domain movement that occurs when H74 is mutated to alanine using the DynDom server (36) shows that the overall global conformational change can be described as ∼15° closure-type rotation. The two domains undergoing the motion each consist of two of four MBD monomers. The four nickel ions in the holo-HpNikR structure form a rectangle with the square-planar sites located on one side (and not in a diagonal arrangement). The hinge region connecting the two moving domains of the molecule is primarily composed of the residues at the section of the interface of the two MBD dimers closest to the square-planar nickel-binding sites. This arrangement is expected because these two metal ions and their coordinating residues may be considered largely unaffected by the transition.

A structural comparison of holo-H74A with holo-HpNikR reveals several notable differences. The most striking observation is the significantly higher degree of flexibility of the DBD in H74A. As a result of this increased flexibility, H74A binds target DNA with weaker affinity (vide infra). The nickel coordination environment triggers certain conformational changes that affect the DBD flexibility. Two helices (α3 and α4) are shifted when Ni(II) is coordinated to all 4-sites in H74A compared with their location when the Ni(II) is coordinated to 5/6-sites in holo-HpNikR (Fig. 2B and Movie S2). The helix α3 contains H88, which serves as a ligand for Ni(II) in both coordination geometries. H88 translates by ∼5 Å upon changes in Ni(II) coordination, inducing a shift of the entire α3 helix. The α4 helix is located between α3 (from the MBD) and α2 (from the DBD), and does not contain any residues directly involved in nickel coordination. It shifts in concert with α3. The interaction between the amphiphilic α3 and α4 helices occurs primarily via hydrophobic interactions. The movement of the α4 helix disrupts hydrogen-bonding interactions of two residues, S117 and Q121, which contribute to the MBD/DBD interface via an interaction with α2 (Fig. 2B). As a result of this disruption to hydrogen bonding, the conformations of two lysines, K64 and K140 from the MBD, which form stacking interactions with W54 from the DBD, are altered. Thus, the switch in Ni(II) coordination from 5/6-sites to 4-sites that occurs in the H74A results in a series of conformational changes (e.g., shifting of helices, disruption of H-bonds, and disruption of the MBD/DBD interface) that likely contribute to the decreased DNA-binding affinities.

When the MBD of H74A is compared with the corresponding portion of holo-HpNikR, each H74A monomer is closer to the conformation in which H88 is part of a square-planar site (p.b.s. of 0.8 Å and 0.6 Å, respectively), as expected. The largest difference in local conformation is observed in the loop connecting the first α-helix of the MBD with its second β-strand, with the largest changes in dihedral angles for residues G91, T92, and H93. T92 is displaced by 4.7 Å, and its side chain shifts from being buried against the hydrophobic patch formed by V94/L113/I120/L123 to being completely solvent exposed. Interestingly, this transformation is observed in both chains, even when the nearest nickel-coordinating residue, H88, retains its position with respect to the wild-type structure.

It has been reported that in the structure of the EcNikR the two nickel ions that are closer to each other are connected via a hydrogen-bonding network that includes residues H87, E97, Y58, Q75, and H89′ (28, 29). This network is not found in holo-HpNikR. This absence can be attributed to the presence of the 5/6-site in which ligands important for the network, Q87 and H101 (Q75 and H89 in EcNikR), have shifted ∼5 Å to accommodate the 5/6-site geometry. In H74A, the distance between corresponding nickel ions is shortened and matches that observed for EcNikR, at 15 Å. Residues Q87/H101 are in symmetrical positions, and the properly positioned water molecule substitutes for the O_η of the missing Y58 (mutated to valine in HpNikR), thus completing the hydrogen-bonding network connecting the two metal ions (shown in Fig. 2C).

HpNikR/DNA-Binding Studies to Define the Contribution of Ni(II) Coordination to DNA Recognition.

Using fluorescence anisotropy, the affinities of holo-HpNikR, holo-H74A, and reconstituted H74A for two DNA targets, P_ureA and P_exbB, were measured. We had previously reported the affinity of reconstituted HpNikR for these two DNA targets, and measured dissociation constants (K_d) of 67 ± 1 nM and 1.20 ± 0.15 μM, respectively (16). Given our discovery that the Ni(II) coordination geometry for reconstituted HpNikR is all 4-coordinate, it was necessary to measure the DNA-binding properties for holo-HpNikR to determine the role(s) of mixed-nickel coordination in protein/DNA recognition. Holo-HpNikR bound P_ureA with a dissociation constant (K_d) of 8 ± 1 nM, and P_exbB with a dissociation constant of 0.7 ± 0.1 μM. The binding is tighter for both DNA targets, and the selectivity (defined here as the ratio of the corresponding dissociation constants) improves from ∼20 to ∼100. Both reconstituted and holo-H74A bound to P_ureA and P_exbB targets with dissociation constants of 62 ± 4 nM and 72 ± 5 nM for the former, and 2.1 ± 0.1 μM and 1.9 ± 0.2 μM for the latter, which are similar selectivities to reconstituted HpNikR (Fig. 3 and Fig. S5). Taken together, these data demonstrate that when Ni(II) is coordinated to mixed sites in “as isolated” holo-HpNikR, the protein binds to its target DNA with higher affinity and greater selectivity than when Ni(II) is coordinated to all 4-sites (i.e., reconstituted HpNikR, holo-H74A, and reconstituted H74A). These findings suggest that the method of isolation for HpNikR is extremely important, and raises the question as to whether mixed Ni(II) sites are present in structurally conserved homologs of NikR, because these homologs have all been studied in the reconstituted form.

Fig. 3. — FA-monitored binding of holo-HpNikR (○), reconstituted HpNikR (□), holo-H74A (●), reconstituted H74A (■), and holo-W54A (▲) with the P_ureA promoter. All FA experiments were performed in 20 mM Hepes, 100 mM NaCl, 20 mM glycine, and 3 mM MgCl₂ at pH 7.5 and 25 °C. Data were fit to a 1:1 binding equilibrium.

W54A Mutation Abrogates DNA Binding.

To further clarify the role of the MBD/DBD interface in nickel-mediated DNA recognition by HpNikR, we mutated W54, a key residue in the α2 helix that is anchored between two lysines from the MBD, K64, and K140, to alanine. Mutation at this site was expected to impact DNA recognition by significantly weakening the interaction between the MBD and DBD domains. W54A was prepared using standard mutagenesis techniques, and the protein was isolated in both the apo and holo forms. ICP-MS and Ni(II) titrations monitored by UV-visible spectroscopy confirmed a stoichiometry of four Ni(II) ions per W54A tetramer, and CD spectroscopy matched data attained for holo-HpNikR as well as H74A (Fig. S4). DNA binding by W54A is completely abrogated, as measured by fluorescence anisotropy (FA; Fig. 3), providing strong evidence that the correct orientation of the DBD with respect to the MBD is essential for nickel-mediated DNA recognition by HpNikR.

SAXS Studies of HpNikR vs. H74A.

As observed in the crystal structures, reconstituted HpNikR (31) and holo-H74A exhibit significant disorder at the DBD, whereas holo-HpNikR orients the DBD in a trans conformation (24). This disorder may be a function of Ni(II) coordination and thus explain the differences in DNA recognition properties. To determine if this disorder is also observed in solution, SAXS studies, described in detail in SI Materials and Methods, were performed with holo-HpNikR and holo-H74A. The radius of gyration, Rg, decreases by ∼5% for H74A compared with holo-HpNikR (Fig. S6); this is comparable to the expected theoretical changes (e.g., the calculated Rg is 27.5 Å for holo-HpNikR and 26.0 Å for H74A with the DBDs in a transconformation). Importantly, the cis conformation models (i.e., the expected conformation for the HpNikR/DNA complex) have a smaller Rg for both proteins, indicating that the reduction of the experimental Rg may be a consequence of the shift of the equilibrium toward cis conformation and the corresponding general increase in the flexibility of the DBDs.

Protein models based upon crystal structures were used to calculate predicted SAXS profiles for holo-HpNikR and H74A, which were then compared with the experimental results. Curiously, neither of the models can be selected as the best model based solely on their fits to the experimental data, which might be attributable to the approximate nature of the crystal structure based models (e.g., the positions of the DBD are projected rather than determined directly) or because the protein exhibits a range of conformations in solution, particularly with the orientation of the DBDs with respect to the MBD.

Ab initio models for the SAXS data were generated as described in SI Materials and Methods. The analysis was performed assuming either no symmetry or twofold internal symmetry of the scattering particle, and the resulting molecular envelopes are shown in Fig. 4 and Fig. S7. The atomic models were manually placed inside the molecular envelope and are presented here purely to provide a guide with respect to the protein size. The ab initio molecular envelope of the holo-HpNikR matches the expected shape of the molecule, with the spherical central (MBD) domain flanked by two somewhat elongated (DBD) domains. The shape of the molecule does not change when a twofold symmetry is imposed during simulated annealing.

Fig. 4. — The ab initio molecular envelopes determined from the SAXS data for (A) holo-HpNikR and (B) H74A were obtained without imposing particle symmetry.

The SAXS profile of the H74A mutant produces an ab initio molecular envelope that appears to lack one of the two DBD domains, which is consistent with an increased flexibility of the DBD domains and disruption of the MBD/DBD interface for the mutant, as seen in the crystal structure. Interestingly, when a twofold symmetry is imposed on H74A data, the resulting envelope resembles the full-length protein that lacks the peripheral part of both DBDs, consistent with the crystal structure. Thus, the SAXS data support a model in which the conformational flexibility in the DBD is significantly increased when Ni(II) is coordinated only to 4-sites.

Conclusions: Role of Nickel Coordination in DNA Recognition by HpNikR.

Two important conclusions can be drawn from the body of structural and functional studies that are presented here. (i) Ni(II) is found coordinated to two different sites: a 4-site and a 5/6-site over the pH range of 5.6–7.5, which spans the range of cytosolic pH encountered by the bacteria (5–7). The singular nickel coordination with all four Ni(II) ions coordinated to 4-sites, as observed by Benini et al. (31), is likely a consequence of the method used to isolate the protein, but also could reflect an equilibrium between the two forms of the protein at elevated pH. (ii) Ni(II) coordination to two different sites promotes tighter and more selective DNA binding (lower K_d) than Ni(II) coordination to a singular site. This DNA recognition effect appears to be related to the conformational flexibility of the DBD, which is modulated by Ni(II) coordination via a long-range effect that includes orientation of specific α-helices on the MBD and hydrogen-bonding interactions at the MBD/DBD interface. When Ni(II) is coordinated to two different types of sites, the DBDs are oriented in a trans conformation, whereas, when Ni(II) is coordinated to a singular site, the DBDs are highly disordered. To bind to DNA, the DBD must reorient from a trans to a cis confirmation and dock to the MBD via an interface centered at W54. Our observation that W54 is essential for DNA recognition substantiates a role of the MBD/DBD interface in DNA recognition.

Scheme 1 shows a proposed mechanism for nickel-mediated DNA recognition by HpNikR that incorporates all of the currently available structural and spectroscopic data. In this mechanism, apo-HpNikR is primed to bind Ni(II) at 5/6-sites. Upon addition of two equivalents of Ni(II), the ligands rearrange to bind two Ni(II) ions in two 4-sites. Addition of two more Ni(II) ions results in mixed Ni(II) coordination geometry [two Ni(II) ions at 4-sites and two Ni(II) ions at 5/6-sites]. At this point, the protein is primed to bind to target DNA and the orientation of the DBD changes from trans to cis, which is an orientation that is also stabilized at the MBD/DBD interface. It remains unresolved whether the form of the protein with the DBD in a trans conformation is in equilibrium with a cis conformation that binds directly to DNA or whether the protein first partially binds to DNA in a trans conformation followed by a rearrangement to a cis conformation to recognize the remainder of the DNA target. Similarly, the fate of the Ni(II) sites upon DNA recognition is not known. The nickel ions may maintain mixed coordination geometry or they may rearrange to bind nickel at singular sites.

The biological role of HpNikR is to regulate multiple genes with different sequences via a direct protein/DNA interaction in response to acid stress and nickel availability (8–10). This function is more complex than that of the well-studied homolog EcNikR, which evolved to regulate a single gene directly (37). The complexity in the nickel sites observed for HpNikR may be a consequence of the protein's biological role. As we continue to unravel the mechanism of nickel-mediated multi-DNA recognition by HpNikR, we will be able to clarify the role(s) of nickel in this process and understand why Ni(II) can coordinate to different sites in the protein.

Materials and Methods

Protein Preparation.

HpNikR-MBD (residues 58–148) and the H74A and W54A mutant proteins were prepared using the standard cloning protocols and purification procedures used to prepare wild-type HpNikR (15). Similarly, the apo forms of the proteins were prepared by incubation with EDTA, and metal-binding titrations were carried out by monitoring the nickel binding spectrophotometrically following protocols established for wild-type HpNikR (15).

Protein Crystallization, X-Ray Data Collection, Processing, and Model Building.

Crystallization trials were carried out with the OryxNano crystallization robot (Douglas Instruments Ltd.) at the X-Ray Crystallography Core Facility located at the University of Maryland. Diffraction-quality crystals appeared in 0.1 M Hepes (pH 7.5), 2% (wt/vol) PEG 400, 2 M ammonium sulfate (HpNikR-MBD), and in 12% (wt/vol) PEG 20000, and 0.1 M Mes (pH 6.5; H74A) after several weeks of incubation at 21 °C. Crystals were cryoprotected by replacing mother liquor with 3.4 M sodium malonate at pH 7.2 (HpNikR-MBD) and by supplementing the mother liquor with 12% (vol/vol) glycerol (H74A). Crystals were flash cooled in liquid nitrogen before data collection.

X-ray diffraction data were collected remotely at the Stanford Synchrotron Radiation Lightsource (SSRL, beamline 7-1) for HpNikR-MBD and using in-house a Rigaku MicroMax-007 rotating anode generator and R-AXIS IV++ image plate detector at 100 K for H74A (X-Ray Crystallography Core Facility, University of Maryland). Diffraction images were processed with DENZO/SCALEPACK. Both structures were solved by molecular replacement using PHASER with the tetrameric assembly of metal-binding domains from the structure of holo-HpNikR (PDB ID code 3LGH). Maximum likelihood-restrained refinement was performed with REFMAC5 (38) implemented in the CCP4 suite (39). Manual building and validation was done with COOT (40). Data processing and refinement statistics are shown in Table 1.

Table 1.

X-ray crystallographic data processing and refinement statistics

	HpNikR MBD	H74A
PDB ID code	3QSI	3PHT
Space group	C2	P4₃2₁2
Cell constants
a, b, c, Å	147.38, 79.40, 122.42	73.2, 73.2, 114.4
β, °	122.28
Unique reflections	21,352	20,487
Solvent content	0.59	0.45
Resolution ranges
Overall, Å	3.08–103.5	2.04–61.7
Outer shell, Å	3.08–3.16	2.04–2.09
R_pim^*	0.13 (0.53)	0.04 (0.43)
Completeness, %^*	95.5 (89.7)	100.0 (100.0)
<I/σ(I)>^*	5.5 (1.1)	25.8 (1.1)
Multiplicity	2.7(2.6)	24.3 (22.1)
R_cryst, all reflections, %	26.2	19.1
R_free, 10/5% of reflections	30.4	23.0
Atoms
Protein	6,104	1,620
Solvent	0	96
Sulfate	25
Nickel	9	2
Deviations from ideality
Rmsd bonds, Å	0.017	0.024
Rmsd angles, °	1.7	2.1
DPI, Å	0.51	0.09
Ramachandran statistics
Preferred	718 (92.7%)	193 (92.0%)
Outliers	21 (2.7%)	2 (1.0%)

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*Outer shell values are in parentheses. DPI, diffraction-component precision index.

Protein–DNA Binding Assay.

A fluorescence anisotropy assay was used to determine the DNA-binding properties of HpNikR and associated mutants. EMSA studies of the proteins with DNA were performed to confirm binding (Fig. S8). Experimental details are provided in SI Materials and Methods.

SAXS.

SAXS data were collected at the SSRL beamline 4-2 and processed using SasTool. The SAXS profiles for both wild-type HpNikR and the H74A mutant were used to determine Rg using CRYSOL and GNOM software (41, 42). Predicted X-ray scattering profiles were calculated using CRYSOL from the crystal structures that were generated as described in SI Materials and Methods. Ab initio modeling of both structures was performed using DAMMIF software (43), with 20 models obtained by simulated annealing and averaged using DAMAVER software (44).

Supplementary Material

Supporting Information

supp_109_15_5633__index.html^{(1.1KB, html)}

Acknowledgments

S.L.J.M. acknowledges NSF CAREER Award CHE0747863.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors reported in this paper have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 3QSI and 3PHT).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1120283109/-/DCSupplemental.

References

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27.Wang SC, Li Y, Robinson CV, Zamble DB. Potassium is critical for the Ni(II)-responsive DNA-binding activity of Escherichia coli NikR. J Am Chem Soc. 2010;132:1506–1507. doi: 10.1021/ja909136h. [DOI] [PubMed] [Google Scholar]
28.Phillips CM, et al. Structural basis of the metal specificity for nickel regulatory protein NikR. Biochemistry. 2008;47:1938–1946. doi: 10.1021/bi702006h. [DOI] [PMC free article] [PubMed] [Google Scholar]
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31.Benini S, Cianci M, Ciurli S. Holo-Ni(2+) Helicobacter pylori NikR contains four square-planar nickel-binding sites at physiological pH. Dalton Trans. 2011;40:7831–7833. doi: 10.1039/c1dt11107h. [DOI] [PubMed] [Google Scholar]
32.Murshudov GN, Vagin AA, Dodson EJ. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr. 1997;53:240–255. doi: 10.1107/S0907444996012255. [DOI] [PubMed] [Google Scholar]
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34.Zambelli B, Bellucci M, Danielli A, Scarlato V, Ciurli S. The Ni2+ binding properties of Helicobacter pylori NikR. Chem Commun (Camb) 2007;(35):3649–3651. doi: 10.1039/b706025d. [DOI] [PubMed] [Google Scholar]
35.Matthews BW. Solvent content of protein crystals. J Mol Biol. 1968;33:491–497. doi: 10.1016/0022-2836(68)90205-2. [DOI] [PubMed] [Google Scholar]
36.Hayward S, Berendsen HJ. Systematic analysis of domain motions in proteins from conformational change: New results on citrate synthase and T4 lysozyme. Proteins. 1998;30:144–154. [PubMed] [Google Scholar]
37.Chivers PT, Sauer RT. Regulation of high affinity nickel uptake in bacteria. Ni2+-Dependent interaction of NikR with wild-type and mutant operator sites. J Biol Chem. 2000;275:19735–19741. doi: 10.1074/jbc.M002232200. [DOI] [PubMed] [Google Scholar]
38.Murshudov GN, et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr. 2011;67:355–367. doi: 10.1107/S0907444911001314. [DOI] [PMC free article] [PubMed] [Google Scholar]
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42.Svergun D, Barberato C, Koch MHJ. CRYSOL—a program to evaluate x-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Cryst. 1995;28:768–773. [Google Scholar]
43.Franke D, Svergun DI. DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J Appl Cryst. 2009;42:342–356. doi: 10.1107/S0021889809000338. [DOI] [PMC free article] [PubMed] [Google Scholar]
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[r1] 1.Marshall BJ, Warren JR. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet. 1984;1:1311–1315. doi: 10.1016/s0140-6736(84)91816-6. [DOI] [PubMed] [Google Scholar]

[r2] 2.Cover TL, Blaser MJ. Helicobacter pylori in health and disease. Gastroenterology. 2009;136:1863–1873. doi: 10.1053/j.gastro.2009.01.073. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r3] 3.Sepulveda AR, Coelho LG. Helicobacter pylori and gastric malignancies. Helicobacter. 2002;7(Suppl 1):37–42. doi: 10.1046/j.1523-5378.7.s1.6.x. [DOI] [PubMed] [Google Scholar]

[r4] 4.Pflock M, Kennard S, Finsterer N, Beier D. Acid-responsive gene regulation in the human pathogen Helicobacter pylori. J Biotechnol. 2006;126:52–60. doi: 10.1016/j.jbiotec.2006.03.045. [DOI] [PubMed] [Google Scholar]

[r5] 5.Wen Y, et al. Acid-adaptive genes of Helicobacter pylori. Infect Immun. 2003;71:5921–5939. doi: 10.1128/IAI.71.10.5921-5939.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r6] 6.Wen Y, Feng J, Scott DR, Marcus EA, Sachs G. The pH-responsive regulon of HP0244 (FlgS), the cytoplasmic histidine kinase of Helicobacter pylori. J Bacteriol. 2009;191:449–460. doi: 10.1128/JB.01219-08. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r7] 7.Stingl K, et al. Prolonged survival and cytoplasmic pH homeostasis of Helicobacter pylori at pH 1. Infect Immun. 2001;69:1178–1180. doi: 10.1128/IAI.69.2.1178-1180.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r8] 8.Bury-Moné S, et al. Responsiveness to acidity via metal ion regulators mediates virulence in the gastric pathogen Helicobacter pylori. Mol Microbiol. 2004;53:623–638. doi: 10.1111/j.1365-2958.2004.04137.x. [DOI] [PubMed] [Google Scholar]

[r9] 9.Contreras M, Thiberge JM, Mandrand-Berthelot MA, Labigne A. Characterization of the roles of NikR, a nickel-responsive pleiotropic autoregulator of Helicobacter pylori. Mol Microbiol. 2003;49:947–963. doi: 10.1046/j.1365-2958.2003.03621.x. [DOI] [PubMed] [Google Scholar]

[r10] 10.Ernst FD, et al. The nickel-responsive regulator NikR controls activation and repression of gene transcription in Helicobacter pylori. Infect Immun. 2005;73:7252–7258. doi: 10.1128/IAI.73.11.7252-7258.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r11] 11.van Vliet AH, et al. NikR mediates nickel-responsive transcriptional induction of urease expression in Helicobacter pylori. Infect Immun. 2002;70:2846–2852. doi: 10.1128/IAI.70.6.2846-2852.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r12] 12.Abraham LO, Li Y, Zamble DB. The metal- and DNA-binding activities of Helicobacter pylori NikR. J Inorg Biochem. 2006;100:1005–1014. doi: 10.1016/j.jinorgbio.2005.10.014. [DOI] [PubMed] [Google Scholar]

[r13] 13.Benanti EL, Chivers PT. The N-terminal arm of the Helicobacter pylori Ni2+-dependent transcription factor NikR is required for specific DNA binding. J Biol Chem. 2007;282:20365–20375. doi: 10.1074/jbc.M702982200. [DOI] [PubMed] [Google Scholar]

[r14] 14.Delany I, et al. In vitro analysis of protein-operator interactions of the NikR and fur metal-responsive regulators of coregulated genes in Helicobacter pylori. J Bacteriol. 2005;187:7703–7715. doi: 10.1128/JB.187.22.7703-7715.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r15] 15.Dosanjh NS, Hammerbacher NA, Michel SL. Characterization of the Helicobacter pylori NikR-P(ureA) DNA interaction: Metal ion requirements and sequence specificity. Biochemistry. 2007;46:2520–2529. doi: 10.1021/bi062092w. [DOI] [PubMed] [Google Scholar]

[r16] 16.Dosanjh NS, West AL, Michel SL. Helicobacter pylori NikR's interaction with DNA: A two-tiered mode of recognition. Biochemistry. 2009;48:527–536. doi: 10.1021/bi801481j. [DOI] [PubMed] [Google Scholar]

[r17] 17.Ernst FD, et al. NikR mediates nickel-responsive transcriptional repression of the Helicobacter pylori outer membrane proteins FecA3 (HP1400) and FrpB4 (HP1512) Infect Immun. 2006;74:6821–6828. doi: 10.1128/IAI.01196-06. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r18] 18.Li Y, Zamble DB. pH-responsive DNA-binding activity of Helicobacter pylori NikR. Biochemistry. 2009;48:2486–2496. doi: 10.1021/bi801742r. [DOI] [PubMed] [Google Scholar]

[r19] 19.Zambelli B, et al. High-affinity Ni2+ binding selectively promotes binding of Helicobacter pylori NikR to its target urease promoter. J Mol Biol. 2008;383:1129–1143. doi: 10.1016/j.jmb.2008.08.066. [DOI] [PubMed] [Google Scholar]

[r20] 20.Carter EL, Flugga N, Boer JL, Mulrooney SB, Hausinger RP. Interplay of metal ions and urease. Metallomics. 2009;1:207–221. doi: 10.1039/b903311d. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r21] 21.Bahlawane C, et al. Structural and mechanistic insights into Helicobacter pylori NikR activation. Nucleic Acids Res. 2010;38:3106–3118. doi: 10.1093/nar/gkp1216. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r22] 22.Dian C, et al. Structural basis of the nickel response in Helicobacter pylori: Crystal structures of HpNikR in Apo and nickel-bound states. J Mol Biol. 2006;361:715–730. doi: 10.1016/j.jmb.2006.06.058. [DOI] [PubMed] [Google Scholar]

[r23] 23.Dosanjh NS, Michel SL. Microbial nickel metalloregulation: NikRs for nickel ions. Curr Opin Chem Biol. 2006;10:123–130. doi: 10.1016/j.cbpa.2006.02.011. [DOI] [PubMed] [Google Scholar]

[r24] 24.West AL, et al. Holo-Ni(II)HpNikR is an asymmetric tetramer containing two different nickel-binding sites. J Am Chem Soc. 2010;132:14447–14456. doi: 10.1021/ja104118r. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r25] 25.Schreiter ER, Wang SC, Zamble DB, Drennan CL. NikR-operator complex structure and the mechanism of repressor activation by metal ions. Proc Natl Acad Sci USA. 2006;103:13676–13681. doi: 10.1073/pnas.0606247103. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r26] 26.Chivers PT, Sauer RT. NikR repressor: High-affinity nickel binding to the C-terminal domain regulates binding to operator DNA. Chem Biol. 2002;9:1141–1148. doi: 10.1016/s1074-5521(02)00241-7. [DOI] [PubMed] [Google Scholar]

[r27] 27.Wang SC, Li Y, Robinson CV, Zamble DB. Potassium is critical for the Ni(II)-responsive DNA-binding activity of Escherichia coli NikR. J Am Chem Soc. 2010;132:1506–1507. doi: 10.1021/ja909136h. [DOI] [PubMed] [Google Scholar]

[r28] 28.Phillips CM, et al. Structural basis of the metal specificity for nickel regulatory protein NikR. Biochemistry. 2008;47:1938–1946. doi: 10.1021/bi702006h. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r29] 29.Schreiter ER, et al. Crystal structure of the nickel-responsive transcription factor NikR. Nat Struct Biol. 2003;10:794–799. doi: 10.1038/nsb985. [DOI] [PubMed] [Google Scholar]

[r30] 30.Chivers PT, Tahirov TH. Structure of Pyrococcus horikoshii NikR: Nickel sensing and implications for the regulation of DNA recognition. J Mol Biol. 2005;348:597–607. doi: 10.1016/j.jmb.2005.03.017. [DOI] [PubMed] [Google Scholar]

[r31] 31.Benini S, Cianci M, Ciurli S. Holo-Ni(2+) Helicobacter pylori NikR contains four square-planar nickel-binding sites at physiological pH. Dalton Trans. 2011;40:7831–7833. doi: 10.1039/c1dt11107h. [DOI] [PubMed] [Google Scholar]

[r32] 32.Murshudov GN, Vagin AA, Dodson EJ. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr. 1997;53:240–255. doi: 10.1107/S0907444996012255. [DOI] [PubMed] [Google Scholar]

[r33] 33.Pozharski E. Percentile-based spread: A more accurate way to compare crystallographic models. Acta Crystallogr D Biol Crystallogr. 2010;66:970–978. doi: 10.1107/S0907444910027927. [DOI] [PubMed] [Google Scholar]

[r34] 34.Zambelli B, Bellucci M, Danielli A, Scarlato V, Ciurli S. The Ni2+ binding properties of Helicobacter pylori NikR. Chem Commun (Camb) 2007;(35):3649–3651. doi: 10.1039/b706025d. [DOI] [PubMed] [Google Scholar]

[r35] 35.Matthews BW. Solvent content of protein crystals. J Mol Biol. 1968;33:491–497. doi: 10.1016/0022-2836(68)90205-2. [DOI] [PubMed] [Google Scholar]

[r36] 36.Hayward S, Berendsen HJ. Systematic analysis of domain motions in proteins from conformational change: New results on citrate synthase and T4 lysozyme. Proteins. 1998;30:144–154. [PubMed] [Google Scholar]

[r37] 37.Chivers PT, Sauer RT. Regulation of high affinity nickel uptake in bacteria. Ni2+-Dependent interaction of NikR with wild-type and mutant operator sites. J Biol Chem. 2000;275:19735–19741. doi: 10.1074/jbc.M002232200. [DOI] [PubMed] [Google Scholar]

[r38] 38.Murshudov GN, et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr. 2011;67:355–367. doi: 10.1107/S0907444911001314. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r39] 39.Winn MD, et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr. 2011;67:235–242. doi: 10.1107/S0907444910045749. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r40] 40.Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010;66:486–501. doi: 10.1107/S0907444910007493. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r41] 41.Svergun D. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Cryst. 1992;25:495–503. [Google Scholar]

[r42] 42.Svergun D, Barberato C, Koch MHJ. CRYSOL—a program to evaluate x-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Cryst. 1995;28:768–773. [Google Scholar]

[r43] 43.Franke D, Svergun DI. DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J Appl Cryst. 2009;42:342–356. doi: 10.1107/S0021889809000338. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r44] 44.Volkov VV, Svergun D. Uniqueness of ab initio shape determination in small-angle scattering. J Appl Cryst. 1992;36(2003):860–864. doi: 10.1107/S0021889809000338. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Ni(II) coordination to mixed sites modulates DNA binding of HpNikR via a long-range effect

Abby L West

Sarah E Evans

Javier M González

Lester G Carter

Hiro Tsuruta

Edwin Pozharski

Sarah L J Michel

Abstract

Fig. 1.