Overall Fold and Characterization of Zinc Binding Sites

The structure of E. coli Zur (EcZur) protein in complex with a 31 bp duplex derived from the znuABC operator (P_znuABC) was determined by X-ray crystallography using multiwavelength anomalous dispersion (MAD) data collected at both high energy and the zinc absorption edge and refined to 2.50 Å (Table 1). Each EcZur monomer includes two domains: an N-terminal DNA-binding domain and a C-terminal dimerization domain. The DNA-binding domain (α2–α4) contains a helix-turn-helix (HTH) motif and is connected to the C-terminal domain (α5/β5) using a characteristic winged-helix motif. The three dimensional model of the complex shows the central portion of the operator DNA surrounded by four Zur monomers forming a dimer of dimers. Analysis of the complex reveals the HTH motif interacts with the major and minor grooves of the DNA. Figure 1 shows the final model which includes amino acid residues 5–152 of four protein monomer chains (designated chains A, B, C, D), eight zinc ions, and two 31 bp strands of DNA (designated chains Y and Z) in the asymmetric unit. The nature of the dimer of dimers ((Zur₂)₂) structure indicated that there were two possible orientations for the DNA to adopt in the crystal. Using DNA brominated at asymmetric points indicated that the crystal has an equal mixture of both DNA orientations. Given the pseudo-palindromic nature of the DNA sequence none of the protein-DNA contact sites were affected by this lack of directionality; however, several bases represent an average over the two conformations. For clarity in the discussion only one DNA orientation is considered.

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Figure 1. Structure of (Zur₂)₂-DNA complex and specific interactions with DNA.

(A) Overall structure of Zn-Zur-33mer DNA complex. The four protein subunits are labeled A–D: dimer 1 contains monomers A and D (green); dimer 2 consists of chains B and C (purple). The DNA axis was generated by Curves+ [58] and is shown in grey. (B) 2D representation of Zur-P_znuABC promoter contacts. Amino acid residues of Zur contacting the DNA are colored by dimer, green for dimer 1 and purple for dimer 2. The subscript of each amino acid refers to the monomer chain involved in binding. The extended −10 RNA polymerase binding site is portrayed in a grey outline. The 2-fold axis is shown between bases 15 and 16. Hydrogen bonds between protein and DNA are shown in red. Hydrophobic interactions are shown in blue and lastly electrostatic interactions are shown in orange. Interactions were obtained with the program Monster [92]. (C) Structure based alignment of EcZur and the known structures of the Fur family. The secondary structure elements of the Zur crystal structure are shown above the corresponding sequence of the Fur proteins. Highlighted in yellow are conserved DNA-binding residues. Highlighted in blue are the conserved cooperativity linker salt bridge residues. Highlighted in red are Tyr45 and the conserved Arg65, which make hydrogen bonds to the DNA bases.

https://doi.org/10.1371/journal.pbio.1001987.g001

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Table 1. Data collection and refinement statistics.

https://doi.org/10.1371/journal.pbio.1001987.t001

Anomalous difference data collected at the Zn edge reveal two distinct Zn²⁺ binding sites in each of the four Zur monomers: the Zn²⁺ in site A is bound to four sulfur atoms from Cys103, Cys106, Cys143, and Cys146 and the Zn²⁺ in site B is bound by residues His77, Cys88, His96, and Glu111 (Figure 2). These results are consistent with earlier extended X-ray absorption fine structure (EXAFS) experiments demonstrating the presence of two Zn-binding sites per monomer: a tighter binding site that is dominated by sulfur coordination and a weaker site that is richer in nitrogen/oxygen atoms [30]. Intriguingly, neither the site A nor the site B zinc coordination in EcZur is observed in other structurally characterized members of the Zur regulon (i.e., ZnuA, L31p, and ZinT) [31]–[37].

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Figure 2. Zinc coordination environments used by E. coli Zur.

(A) Site A ligand coordination for the sulfur-rich zinc site (Zn shown in red). (B) Site B ligand coordination for the nitrogen/oxygen-rich zinc site (Zn shown in blue). (C) Structure based alignment highlighting the zinc-coordinating amino acid side chains in known Zur and Fur structures. The sulfur-rich, nitrogen/oxygen-rich, and so-called “third zinc” binding site ligands are shown in red, blue, and yellow, respectively. Note the third zinc binding site is observed in less than half of the structurally characterized family members. This alignment shows a high degree of conservation of the tight-binding sulfur-rich zinc site, while the other two zinc binding sites vary significantly amongst known Fur family structures.

https://doi.org/10.1371/journal.pbio.1001987.g002

Structure-based alignment of E. coli Zur with sequences of structurally characterized Fur-family proteins shows some conservation of zinc-binding residues. The most highly conserved of these are the two C-X-X-C motifs of the sulfur-rich site A (Figure 2C). Amongst the Zur sub-family members C88 is the most conserved residue in the B site (note: MtFurB is a Zur protein that controls regulation of zinc uptake genes [38]). To test whether zinc occupancy in these two sites is important for repressor function of EcZur, we mutated a conserved residue in each site to serine and compared the ability of these variants to compliment Zur activity in the zur-null strain. Mutations in either site A (C103S) or site B (C88S) lead to a complete loss of Zur-regulated transcription in vivo (Figure 3). Both mutant proteins were stably expressed, isolated, and evaluated in metal-binding, DNA-binding, and dimerization assays. Analysis of zinc stoichiometry by inductively coupled plasma mass spectrometry (ICP-MS) reveals that these mutant proteins can be loaded in the presence of excess zinc, but to a lesser extent than wild-type Zur (WTZur) (Figure 3). To provide a rigorous test of zinc binding activity, i.e., one that mimics better conditions inside the cell, we dialyzed zinc-loaded proteins against a stringent Zn²⁺-chelating buffer solution that contains 25 mM EDTA. Under these zinc-limiting conditions, the site A mutant does not bind Zn²⁺ to any significant degree, whereas the site B mutant binds zinc to a similar extent as WTZur (0.5–0.7 zinc/monomer). To test whether the DNA binding activity of WTZur requires Zn²⁺ occupancy at these sites, we titrated these zinc-loaded Zur proteins in native gel electrophoretic mobility shift assays. We find saturation of DNA binding at ca. 3 nM dimer for wild-type (WT) protein. However, neither the site A (C103S) nor the site B (C88S) mutant exhibits any DNA binding activity at the highest protein concentrations examined (Figure 3B). When these assays are repeated in the presence of excess Zn²⁺ a significant weakening of affinity is still observed (Figure S1). Thus DNA binding activity of EcZur is severely limited when either of the zinc binding sites is compromised. Intriguingly we find that the site A mutant protein does not form a stable dimer, whereas the site B mutant does so under these conditions (Figure 3C). These findings underscore the idea that Zn²⁺ occupancy of both site A and site B is important for inducing the DNA binding conformation of Zur and furthermore that Zn²⁺ occupancy of site A can influence dimer formation. These results for EcZur corroborate studies of the role of analogous zinc binding sites in Zur proteins from S. coelicolor (ScZur) and B. subtilis (BsZur) [39],[40]. Intriguingly, the EcZur site A Zn²⁺ ions superimpose well with both open and closed structures of other dimer Fur proteins (Table 2), whereas the site B zinc ions do not. These similarities in the A site and the differences in the B site provide a structure-based perspective for how zinc occupancy of each sites leads to stabilization of the high-affinity DNA binding conformation of Zur. For example, zinc occupancy at site A involves binding to two C-X-X-C motifs that directly flank the dimerization domain (α5/β5) and these serve to stabilize the dimer interface. The site B zinc binding pocket contains β-strands β1–β3, and is linked by a short 4 amino acid loop to one of two of the principal DNA-recognition helixes, namely α4. We anticipate that zinc occupancy of this site adds significantly to the stabilization of the closed DNA-binding conformation of Zur.

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Figure 3. Characterization of WTZur Zn-binding with A-site (C103S) and B-site (C88S) mutant.

(A) Metal contents of Zur measured by inductively coupled plasma mass spectrometry (ICP-MS). Analysis of both purified and EDTA-treated proteins were measured in triplicate. (B) DNA binding activity of WT and mutant Zur proteins analyzed by EMSA gel shifts of the znuABC operator. Using these qualitative gel shift experiments it is apparent that a single site-directed mutation in site A or site B have a dramatic effect on DNA-binding affinity. (C) Analytical gel filtration chromatograms of WT Zur, site A mutant C103S, and site B mutant C88S. The dotted lines in (B) and (C) indicate the position of the elution volume (V_e) of WT protein as a reference (10.9 ml). These experiments demonstrate that site A residues are critical for Zur dimerization. (D) In vivo complementation assay measurement of L31p and zinT expression demonstrate that mutating either site A or site B removes the ability of Zur to repress transcription. See Data S1 for the raw data used to generate each panel.

https://doi.org/10.1371/journal.pbio.1001987.g003

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Table 2. Superposition of E. coli Zur with known structures of Fur family members.

https://doi.org/10.1371/journal.pbio.1001987.t002

Nearly half of the 13 different crystal structures of Fur family members determined in the absence of DNA are found in the open conformation and the remaining structures are described as closed. When compared to EcZur bound to DNA, both the open and closed conformations show good agreement across the C-terminal dimerization domains, but differ significantly in the relative positioning of the N-terminal DNA binding domains. The open conformation (B. subtilis Apo-PerR-Zn [41], B. subtilis. PerR-Oxo [42], M. tuberculosis FurB [38], Helicobacter pylori Fur [43], Streptococcus pyogenes PerR [44],[45], and Campylobacter jejuni Fur [46]) has been postulated to have a low affinity for DNA, as the DNA-binding domains are too far apart to interact with a DNA molecule. Structures identified in the closed conformation (Streptomyces coelicolor Zur [40], B. subtilis PerR-Zn-Mn [47], S. coelicoler Nur [48], E. coli Fur [49], Vibrio cholera Fur [50], and P. aeruginosa Fur [51]) have been proposed to both correspond to the fully metal loaded form of the repressor and have a high affinity for DNA. Use of the structural alignment program SuperPose [52] indicates that superposition of the E. coli Zur dimers with the known Fur proteins gives the strongest agreement (<1.6 Å rmsd for the α-carbons) with the closed crystal structures. Superpositions with the open form of the Fur proteins show significantly poorer agreement (>3.0 Å rmsd) (Table 2), particularly in the orientation of the DNA-binding domains. The Zur-DNA complex provides the first structural evidence that the previously identified closed conformations of the Fur proteins correspond to the conformation capable of binding DNA, and further reveals the molecular basis of protein-DNA recognition in the unusually long operator sequence.

Tyr45 and Arg65 Interactions with Zur-Box Purines Define the Recognition Motif

Over 100 contacts between protein and nucleic acid atoms are observed in the (Zur₂)₂-DNA complex, and the majority of these are between conserved amino acid side chains and the phosphate backbone. Interestingly, each of the four monomers of E. coli Zur makes two hydrogen bond contacts to specific purine DNA bases for a total of eight direct interactions with the P_znuABC operator (Figure 1B). The majority of these contacts are in the major groove and only two types of functional groups are involved in hydrogen bonding. Each monomer A through D has two hydrogen bond donors, Tyr45 and Arg65, that interact with the N7 nitrogen of bases G7, A11′, A12, A15, A16′, G19′, A20, G24′ (where ′ denotes non-coding strand) (Figures 1A, 1B, and S2). Conservation of Arg65 among both Zur and Fur family members along with previous mutational analysis suggested that this amino acid is critical for binding to DNA by many proteins of the Fur family [53]. Unexpectedly we find that each monomer uses Tyr45 in addition to Arg65 in direct readout of the DNA sequence element or ‘Zur box.’ Tyr45 is not conserved across all Fur homologs, but is unique to gram negative Zur protein-DNA recognition, and likely provides discrimination between Zur- and Fur-regulated promoters (Table S1). Each of the four monomers recognizes two purines on opposite strands of DNA separated by three base pairs in an R-N-N-N-Y motif. In this motif Arg65 hydrogen bonds to the first purine (most frequently a G) followed by Tyr45 hydrogen bonding to the purine compliment of residue Y on the opposite strand (Figure 1B). In addition to these base specific interactions, the backbone shape or indirect readout is also observed between each Zur monomer and the dozens of electrostatic and hydrophobic contacts with the DNA backbone. We first examined whether binding of both dimers to DNA is required to establish a stable repressor complex in solution in order to understand the key elements within our Zur-DNA recognition and thus provide the best molecular definition of a Zur box. We further examined the solution data to evaluate contributions of several of the contacted bases to the thermodynamics of specific protein-DNA complex formation.

Zur-DNA Interaction Is Highly Cooperative at the P_znuABC Operator

Native gel shift experiments were used to evaluate binding of the Zur dimer to the znuABC promoter in solution. Titrations using samples of WTZur consistently reveal a highly cooperative binding that results in complexes that have two dimers bound to DNA under saturating conditions (Figure 4A). Using the method developed by Orchard and May [54] we find that the protein-DNA stoichiometry in solution is the same as that observed in the crystal structure, namely two protein dimers to one duplex DNA (Figure S3). The absence of an intermediate complex (i.e., one dimer per DNA) coupled with Hill coefficients that are significantly greater than unity (i.e., α_H>1) (Figure S4) indicate that formation of the dimer of dimers complex is a highly cooperative process. Therefore, the binding of E. coli Zur and DNA can be described as a two-step mechanism shown in scheme 1a and 1b:Given the highly cooperative binding of two dimers to the operator, it is not possible to determine microscopic site constants K_d1 and K_d2 for each dimer (reactions 1a and 1b or Equation 2a); however, the apparent macroscopic dissociation constant (K_d-app) for reaction 1c is readily determined by fitting the fraction of DNA bound as a function of total Zur dimer concentration using Equation 2b [55] (Scheme S1):(2a)(2b)Equation 2b is identical to the derived thermodynamic expression used to model a highly cooperative two-to-one equilibrium [56] such as the binding of diphtheria toxin repressor (DtxR) to DNA [29]. Fitting the Zur-DNA titration data with equation 2b (Figure 4) yields an apparent macroscopic dissociation constant K_d-app for the cooperative binding of Zur to the znuABC promoter of 8.2 (±0.7)×10⁻¹⁸ M².

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Figure 4. Affinity determination of WT Zur titrations of P_znuABC by EMSA.

(A) Representative gel of the Zur affinity for the znuABC promoter. A Cy5 labeled DNA fluorescent probe was used to monitor the formation of a DNA-protein complex. Each lane represents a different reaction between protein and DNA, where the DNA and Zn²⁺ concentrations are kept constant (≤45 pM and 50 µM, respectively) as increasing concentrations of protein are added to the sample. The mobility of the shifted species corresponds to an apparent molecular weight of 110 kDa, which corresponds to (Zur₂)₂-DNA (see Figure S3). Note the absence of any bands corresponding to the single dimer Zur₂-DNA intermediate species. (B) Graphical representation of the percentage of bound DNA versus the concentration of Zur protein. The data points presented in this graph are representative of three separate gel shift experiments. A binding isotherm fit to Equation 2b gives a protein-DNA dissociation constant of K_d-app = 8.2 (±0.7)×10⁻¹⁸ M². Hill plots identify a Hill coefficient of α_H≥2.0 indicating that protein-DNA binding is highly cooperative (see Figure S4 and Data S2 for the raw data used to generate each plot.).

https://doi.org/10.1371/journal.pbio.1001987.g004

An Asymmetric Pair of Salt Bridges Is Critical for the Cooperative Binding of Two Dimers to DNA

We next addressed whether the two dimers communicate with each other through direct contacts or through modulation of local DNA structure. The structure of the (Zur₂)₂-DNA complex reveals a subtle but direct communication from one dimer to the other. Two salt bridges between adjacent dimers were observed, between Asp49 from monomer A to Arg52 of monomer B, and vice versa (Figure 5A). Mutant forms of Zur where the salt bridge is removed (D49A or R52A) retain both zinc binding (2 mol zinc per mol Zur monomer) and tight DNA binding activity, but do not exhibit the highly cooperative binding observed for the WT protein. Instead, gel shift assays reveal an intermediary species that is not seen in assays using the WT protein and this species persists throughout the central portion of the titration (Figure 5B). Using gels with varying acrylamide percentages, it was determined that the intermediary protein-DNA species has a stoichiometry of one Zur dimer to one DNA molecule (Figure S3) [57]. In this case, the stepwise binding constants K_d1 and K_d2 can be simultaneously estimated by fitting analytical forms of the equilibrium expressions 1a and 1b to the DNA binding data (Equation 2a; Scheme S1). We find the binding of one mutant dimer to the P_znuABC promoter site is significantly favored (K_d1 = 2.1–2.6 nM) over the binding of a second mutant dimer to an adjacent site (K_d2 = 65–220 nM) (Figures 5C and S5). The product of these microscopic dissociation constants estimate that the macroscopic K_d-app for the overall binding is between 140 and 570×10⁻¹⁸ M². When compared to WT protein, the free energy penalty (ΔΔG) for mutating either salt bridge linker is ∼2 kcal mol⁻¹. Based on these findings, we conclude that the cooperativity observed in binding of the WT dimers to the DNA surface arises from the two salt bridge contacts between the dimers. Interestingly, the amino acid residues that form this pair of salt bridges are highly conserved within the gram-negative subset of Zur proteins (Table S1), but not in other members of the Fur family. We propose that these pairs of salt bridges act as a “cooperativity linker” and play a central role in the physiology of Zur repression.

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Figure 5. Identification of cooperativity linker and effect on protein-DNA binding.

(A) Salt bridge formation between monomers A and B. The image illustrates the communication between the A and B monomers across the dimer-dimer interface. The equivalent interaction is not formed in the other dimer-dimer interface (not shown) (B) Native gel shifts demonstrate the isolation of a single dimer-DNA intermediate in mutant protein D49A unseen in the WT Zur gel shifts. Shown here is a representative gel-shift for Zur(D49A)₂ titration of P_znuABC. (C) Two-site binding isotherms modeled for the equilibrium for Zur(D49A)₂ binding corresponding to K_d1 = 2.1 nM (orange) and K_d2 = 65 nM (blue). See Data S3 for the raw data used to generate each plot.

https://doi.org/10.1371/journal.pbio.1001987.g005

Central Purines Are Key to Dimer-Dimer Binding in Zur Regulon Promoters

In order to elucidate the relative importance of specific Zur/DNA contacts in the Zur-znuABC structure, every base involved in hydrogen-bond interactions with Zur was mutated and the affinity of each individual mutant operator DNA was analyzed by titration with WT protein (Figure 6). In all cases, the protein-DNA binding was cooperative; no intermediate species were observed (i.e., corresponding to one Zur dimer per DNA), and the overall DNA-binding affinity was weakened. The thermodynamic analysis of the protein-mutant DNA binding reveals that the central-most bases contacted by Zur, namely A15 and A16′ have the most significant effect on stability of the (Zur₂)₂-DNA complex (Figure 6). The importance of the inner most bases relative to the outer bases has been observed previously, and now has structural support [22]. In the Zur-znuABC structure, the majority of deviations from ideal B-form DNA behavior occur at the contacted central bases A15 and A16′. Whereas the overall shape of the crystallized DNA is not greatly distorted from ideal B-form behavior (the bend of the znuABC DNA is ∼15°), two of the largest major groove widths occur at bases 15 (18.3 Å) and 16 (16.8 Å), significantly larger than 11.4 Å for B-form DNA (Figure S6). In order to accomplish this major groove expansion there must be significant overall unwinding of the DNA. This unwinding is readily quantified using the Curves+ program twist measurements [58], which calculated the znuABC DNA twist as 32.6°, nearly 4° smaller than in ideal DNA. In addition, the analysis indicates that the center of znuABC DNA has negative roll angles and narrow minor grooves widths of 2.5 Å (base 15) and 2.8 Å (base 20), both significantly narrower than for B-form DNA (5.9 Å). This observation that negative roll angles precede narrow minor grooves has been seen in prior analysis of protein-DNA complexes [59]. However, unlike other protein-DNA complexes, the Zur-DNA structure unwinding occurs at the center of the DNA. Closer examination of the Zur-DNA structure reveals that the inner bases (TATA) are contacted by Tyr45. This inner TATA sequence is unique to E. coli Zur and not present in the consensus E. coli Fur box [60]. We speculate that the Tyr45 forms a unique set of bonds with the central TATA nucleotides to provide the key difference between E. coli Zur/Fur recognition.

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Figure 6. Affinity determinations of znuABC purine mutations by EMSA.

(A) Sequence of the znuABC with corresponding purines of Dimer 1 recognition (green) and Dimer 2 (purple) recognition sites. (B) Representative binding isotherms between the WT znu promoter and a single mutation, in this case A15T in the center of the DNA sequence, highlighting the difference in binding affinity. (C) Table summarizes the effect of mutating each purine individually and the relative weakening on the Zur-DNA affinity. In all cases Hill plots indicate that DNA-binding occurred in a cooperative manner α_H≥1 (See Data S4 for the raw data used to generate each panel.).

https://doi.org/10.1371/journal.pbio.1001987.g006

Pattern of Contacts in the (Zur₂)₂-P_znuABC Structure Lead to a Novel Zur Regulated Gene

Using a structure-based molecular recognition analysis of the (Zur₂)₂-P_znuABC complex, we asked if the two other promoters shown to be regulated by E. coli Zur (P_zinT and P_L31p [25]) accommodate the same dimer of dimers pattern of base-specific readout. Thermodynamic analysis using gel-shift assays of WT Zur binding to each of these promoters reveals that all three bind Zur in a highly cooperative “all-or-none” manner to form a dimer of dimer complex with DNA. Furthermore, each promoter has multiple interrupted purine-N-N-N-pyrimidine binding motifs, which are recognized by the Zur monomers in the structurally characterized complex with P_znuABC DNA. Comparison of these three sequences allows us to test the elements necessary for establishing an energetically validated Zur box. In each promoter we find two purine-N-N-N-pyrimidine motifs separated by three residues to give RNNNYxxxRNNNY, which serves as the core recognition element for one Zur dimer. The binding of two dimers at overlapping RNNNY sites seen in the structure of the ZnuABC gives rise to a skeletal structure-based dimer-dimer recognition motif: RNNNYRNNRYNNYRNNNY. A summary of this is graphically depicted in terms of colored arrows in Figure 7A where we have superimposed the base pair occurrence in the three known promoters in a sequence walker format. A more specific pattern for a two dimer recognition site for Zur can be described using IUPAC code: TGWNAYRWTATAWYRTNWCA. When this structure based E. coli Zur box is compared with the Zur box from other organisms it is clear that the central portion of the sequence is key to the recognition of E. coli Zur [19],[20],[40],[61]. This pattern was then used as a search filter of E. coli (K12, strain MG1655) promoter regions to identify possible novel Zur-regulated genes using the program Fitbar [62]. The search identified four promoters, including all three of the known Zur regulated genes, and predicted a single novel Zur binding site 34 bases upstream of the periplasmic lysozyme inhibitor pliG gene. Alignment of the promoter sequence of the pliG gene to the other three known promoters showed favorable agreement with the structure-based Zur recognition profile. Previous microarray studies had demonstrated that pliG expression was affected by changes in Zn²⁺ concentration, however Zur regulation had not been hypothesized [34],[63]. To test whether pliG was in fact regulated by Zur, pliG transcripts were measured by reverse transcription (RT)-PCR. The presence of WTZur in vivo, under growth in rich media (where zinc is present in moderate to high concentrations) leads to significant repression at the pliG promoter (P_pliG) relative to the Δzur strain (Table 3). Taken together with the in vitro P_pliG DNA-binding data (Figure 7C), these results clearly establish the pliG gene as part of the E. coli Zur regulon.

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Figure 7. Structure-based pattern for DNA recognition by E. coli Zur dimers.

(A) Sequence logo representation of the template strand in Zur-DNA recognition based on the four known Zur operators (see text for details). Each number corresponds to the base number in the Zur-DNA crystal structure. The bases in the motif recognized by dimer 1 are highlighted in green, while the dimer 2 recognized bases are highlighted in purple. The purine-N-N-N-pyrimidine (i.e., R-N-N-N-Y) motif is conserved within all four of the operators regulated by Zur. Overlap of the green and purple recognition motifs at positions 15 and 16 highlight the importance of the central AT bases. (B) Purine and pyrimidine pattern of the two dimer DNA recognition of E. coli Zur. The sequence dyad is shown with dotted lines. (C) Representative gel for the Zur titration of P_pliG. The presence of the single dimer intermediate provides the ability to calculate individual macroscopic binding constants. For Zur-PliG binding isotherms see Figure S6.

https://doi.org/10.1371/journal.pbio.1001987.g007

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Table 3. Comparison of in vitro and in vivo DNA binding experiments for all four Zur promoters.

https://doi.org/10.1371/journal.pbio.1001987.t003

The Amplitude of In Vivo Zur Repression Correlates with Protein-DNA Binding Thermodynamics

We next addressed whether the degree of repression at each promoter P_znuABC, P_zinT, P_L31p, and P_pliG correlates with the affinity of Zur for the operators. Measuring the mRNA levels of the Zur regulated genes in both WT and Δzur strains by RT-PCR, we find that the degree of Zur-responsive regulation varies significantly from promoter to promoter, with the most pronounced Zur-regulation seen for the ribosomal subunits and the periplasmic zinc trafficking protein. We find a clear hierarchy: Zur exhibits between 980- to 560-fold repression on zinT and L31p expression, and 7- and 8-fold repression on the znuC and pliG promoters, respectively (Table 3). The trend in these in vivo results correlates strongly with the relative order of in vitro protein-DNA affinity experiments. The binding of the two Zur dimers to form a stable DNA complex is at least 2 orders of magnitude stronger for the zinT and L31p operators than for the znuC and pliG promoters. Intriguingly, in the case of the three known Zur operators the DNA-binding occurs in a highly cooperative fashion. However, the P_pliG operator seemed to interact with Zur in a different manner than the other three promoters (Figures 7C and S7). In the case of P_pliG we observe a single dimer-DNA intermediate, as seen earlier in the mutant protein (R52A/D49A) titrations of P_znuABC. Given that a single dimer of Zur can bind to the pliG operator and the precedence for single dimer Fur repression [64], it is possible that future studies will identify additional Zur operators, including those that contain only a single dimer binding site.

Structure-Based Mechanism of Fur Family Repressors

To date there are 13 structurally characterized members of the Fur family of proteins across gram negative and gram positive bacteria. Each subset of the Fur family including the iron sensor proteins (Fur proteins), the nickel sensor proteins (Nur proteins), and the iron cofactor peroxide sensor proteins (PerR proteins) have been crystallized with zinc, despite the fact that they do not respond to changes in Zn²⁺ ion concentration under physiological conditions [43],[45],[47],[49]–[51],[65]. While the precise metal binding characteristics of Fur proteins remains controversial, this work provides the first structural insight into the DNA-binding characteristics for this family of proteins. The dimer of dimers structure of the Zur-znuABC complex explains the extended DNA footprint observed in previous experiments [26],[53]. Each Zur dimer docks on opposite sides of the DNA as predicted by previous Fur-DNA models [51],[57],[66]. Our structural characterization indicates that each dimer binds such that two successive HTH motifs contact each major groove of the DNA. Previous analysis and modeling of the winged helix motif suggested that the fourth α-helix was a “recognition helix” in the DNA-binding domain [51],[53]. Whereas our crystal structure confirms that the α4-helix (residues Pro60 to Glu72) plays a critical role in DNA-binding, residues 44–72 of the HTH motif provide the majority of the protein-DNA contacts (Figure 1B and 1C). This HTH motif includes the cooperativity linker needed for double dimerization and also Tyr45/Arg65, which make the key hydrogen bonds to the specific purine bases. In light of the (Zur₂)₂-DNA structure, these specific hydrogen bonds of Tyr45 and Arg65 to the purine N7 atoms can be classified as part of the Zur protein base readout mechanism of recognition [67]. Conservation of DNA backbone-contacting ligands within the Fur family, namely: Arg23, Thr25, Gln27, Arg28, and Tyr64 highlight the shape readout mechanism of backbone conformations observed for each monomer [67]. The general pattern of recognition involves several minor groove sugar and phosphate backbone contacts immediately upstream of the canonical 5′ purine and a few additional backbone interactions from the major groove at the downstream purine on the opposite strand.

A cooperative dimer-dimer repression mechanism has been described before for other metalloregulatory repressors, namely the DtxR family of repressor proteins. The prototypical member, DtxR, responds to fluctuations in Fe²⁺ concentrations in gram-positive C. diphtheriae, but exhibits little sequence similarity to the Fur family (<20%) [29],[68]–[71]. The similarities between the Zur and DtxR protein-DNA complexes are intriguing. Previous work highlighted the conservation of thymine bases that are critical to the recognition of the DtxR family of proteins [29],[57],[72]. Mutation of the outermost thymines of the DtxR binding site of the DNA leads to large changes in the DNA binding capability of the protein [29]. Mutation of similar thymines in the B. subtilis Fur protein binding sites also had a large effect on DNA binding [73]. Our structural characterization and DNA binding assays indicate that the critical contact sites are the adenines on the opposite strand, not the thymines, in the E. coli Zur and Fur proteins (Figure S2). In Zur, the central nucleotides play a critical role in recognition, not the outer bases as in DtxR (Figure 6C).

Native gel shift experiments reveal that the metal-saturated states of Zur and DtxR form complexes containing two dimers bound to their respective operators in a highly cooperative manner. In the case of DtxR, an intermediate containing a single dimer/DNA complex was never observed nor were any protein-protein interactions between the two dimers [29]. This leaves the molecular mode of communication between the dimers in the DtxR system as an unanswered question, including a possible role for local DNA distortion [29]. In the case of E. coli Zur, we have identified a pair of salt-bridges, between Asp49 and Arg52, as the key contributors to the cooperative binding of two dimers to the operator (Figure 5). This pair of acidic and basic residues is a highly conserved motif in Zur family members, but is not conserved in the Fur family overall (Table S1). Salt bridges are commonly used in protein folding and have been frequently observed as the critical junction in cooperative binding. Two noteworthy examples include the sequential oxygen binding events in hemoglobin [74],[75] and bacterial histone-like HU proteins, highlighting the prevalence of this protein-protein mode of communication [76],[77]. Whereas there are no reported protein-protein contacts between dimers or within the DNA-binding region of a Fur family member in the literature to date, two groups have identified instances where a hydrogen bond network plays a key role in Fur protein-DNA binding [43],[51]. It is likely that each subfamily of Fur proteins has some unique networks, whether hydrogen bonding or specific salt bridges to stabilize their specific protein-DNA complex.

Structure based alignments of the Zur dimers in this EcZur protein-DNA complex with several Fur family members provide strong support for a mechanism wherein metal-induced allosteric changes stimulate conversion of the wing-helix DNA-binding domain from an “open” to a “closed” conformation that is capable of binding DNA with high affinity [38],[40]–[51]. Our analysis reveals that the active DNA-binding conformation requires Zn²⁺ occupancy of both site A and site B in order to lock the key HTH motifs into the “closed” or DNA binding conformation. The effects of the mutations on the two Zn-binding sites differ. Mutational analysis of each zinc binding site demonstrates that changing even a single site A or site B amino acid dramatically reduces zinc occupancy and DNA binding affinity of EcZur (Figure 3). A mutation in site B (C88S) produces a protein capable of binding a single zinc, while a site A (C103S) mutant is unable to form stable dimers or to bind zinc under Zn²⁺-limiting conditions. These findings are in agreement with site-directed mutagenesis in vivo experiments using BsZur and ScZur [39],[40]. Intriguingly, in the latter two cases a third zinc binding site is observed. Previous studies of S. coelicolor Zur suggests that the different responses of a series of Zur regulated promoters is the result of subtle modulation of Zur-DNA binding affinities at each promoter in response to zinc availability and implicate site B and the third zinc binding sites in ScZur [40]. Our results reveal a layer of regulatory tuning based on differential affinities of zinc-saturated Zur dimers for the various promoters in this regulon. However, we cannot rule out the potential for the disordered C-terminal tail of EcZur to bind a third zinc ion thus leaving any role for this site in EcZur as an open question. This intrinsic variation, as well as the variability in the degree of cooperativity and in the number of repressor molecules binding to DNA, may contribute to the heterotropic cooperativity and differential responses in the Zur regulons of other organisms.

All Known Promoters in the E. coli Zur Regulon Use a Purine-N-N-N-Pyrimidine Sequence

In the absence of Zur-DNA structures, several groups have compared Zur regulated promoters seeking consensus protein-DNA recognition patterns that could be used to define Zur regulons. This approach has been quite successful in γ and β-proteobacteria organisms, identifying as many as 23 Zur regulated genes in the case of C. Anabaena [25],[78],[79]. Previously the “E. coli Zur box” was characterized as a 23 base pair pseudo-palindromic sequence that made up the proteobacteria (gram negative) Zur regulon GAAATGTTATA-N-TATAACATTTC [25]. For the gram positive B. subtilis, DNA binding experiments estimated that in order for Zur to repress a promoter sequence there needed to be agreement with a similar inverted repeat 9-1-9 Zur box [80]. Using the crystallographic and biochemical data we find that the simplest motif to represent the E. coli Zur recognition elements involves precise purine-N-N-N-pyrimidine elements, where the monomer contacts the 5′ purine in these double strand elements. Our structure and biochemical data reveal that each Zur dimer recognizes this palindromic element with a three base spacer (RNNNYxxxRNNNY). While the number of operators is quite limited, we used this skeletal sequence to develop the sequence logo shown in Figure 7A and used it to search the E. coli genome and identify a novel E. coli Zur regulated gene, pliG.

This family of four operators provides a minimal basis for statistical analysis of how palindromic features of the operator sequences correlate with Zur-DNA affinity. Search programs, like SignalX, correctly identified three of the E. coli Zur regulated promoters. However, the positional weighted matrix ranked znuABC with the highest score [25]. Upon measuring the DNA-binding affinity of each of the four promoters we were surprised to find that the Zur affinity for znuABC is among the weakest (Table 3). The core symmetry of a Zur box is best considered as having two distinct but overlapping binding sites within the overall DNA element. Overlaying the two dimer recognition motifs using the offset seen in the crystal structure, reveals a conserved 18 bp inverted repeat RxxxYRxxR*YxxYRxxxY motif, with the * indicating the center of the palindrome (Figure 7B). When we consider the palindromic nature of the variable residues (x) and score the four known operators against the 18 bp inverted repeat we obtain the following hierarchy: L31P>ZinT≈ZnuC>>PliG. While this predicts the general pattern seen in the thermodynamic analysis, we anticipate that a more quantitative algorithm may reveal additional subtle patterns in the purine-pyrimidine repeats, which may give rise to more quantitative predictions. Furthermore, the relative number and affinity of competitive pseudosites in these AT-rich binding sites can decrease the binding free energy for the functional complex [81], and this may provide another basis for the gradation in affinities among the family of Zur regulated promoters.

Thermodynamics of Protein-DNA Interactions Directly Correlates with Physiological Response

We find a surprisingly broad range of DNA-binding affinities (over 10,000-fold) for the known promoters in the Zur regulon (summarized in Table 3). These in vitro thermodynamic properties were also shown to control the functional distribution of Zn-loaded Zur over these DNA-binding sites in vivo (Table 3). Here the repressor-DNA affinity strongly correlates with the fold repression across the four known Zur regulated promoters in E. coli. These results provide strong support for the idea that the thermodynamics of an ensemble of protein-DNA interactions play a dominant role in the physiological control of gene regulation networks [81],[82]. These findings open the door to understanding how zinc availability and partial metal occupancy at a subset of the eight zinc binding sites in the repressor complex can fine tune stress responsive gene expression across a range of promoters [39],[40],[64]. Deconvolution of these factors is important for understanding how Zur and related nutrient-responsive regulons operate in pathogens that survive major shifts in local zinc concentrations induced by the host.

Preparation of E. coli Zur Proteins

WT and mutant Zur proteins were purified from E. coli (BL21 DE3) cells containing pET24d-based recombinant plasmid, as previously described with one minor modifications [30]. Site-directed mutagenesis was performed QuikChange mutagenesis kit and mutagenic primers (Table S2). In order to improve the purity of the Zur proteins a HiPrep Heparin fast flow column equilibrated with buffer A: 20 mM Tris-HCl (pH 8.0) and 5 mM DTT and eluted with a linear salt gradient using buffer A+500 mM NaCl. This purification step replaced of the ammonium sulfate purification step described earlier in [30]. The molecular weights of purified WT and mutant Zur proteins were confirmed by electrospray ionization mass spectrometry (ESI-MS).

Protein-DNA Complex Crystallization and Data Collection

Zur protein and the designed DNA were mixed together to a final concentration of [Zur] = 117 µM, [DNA] = 108 µM, with [DTT] = 5 mM and [ZnSO₄] = 150 µM. The optimal DNA for crystallography included 31 nucleotides known to be protected in footprinting assays [26] with a 3′ overhang of two bases to help stabilize the crystal packing, referred to as 31mer2bpOH. Over 40 sequences of DNA were tested using hanging drop vapor diffusion and 31mer2bpOH led to the most reproducible high quality diffraction data. Crystals were grown by the hanging drop vapor diffusion method, mixing 1 µl of protein/DNA mixture with 1 µl of crystallization buffer (1.5 M ammonium sulfate, 0.1 M citrate [pH 5.5]). Crystal trays were set up and incubated at 4°C. Crystals grew to full size in 2 weeks. The crystals were cryoprotected with mother liquor supplemented with 15% ethylene glycol and flash-cooled in liquid nitrogen. Data were collected at 100 K at the Advanced Photon Source (APS) Life Sciences Collaborative Access Team (LS-CAT) and diffracted to 2.5 Å, crystallographic statistics are summarized in Table 1.

Structure Determination

X-ray diffraction data were processed using the programs XDS and SCALA to 2.5 Å resolution with a C2 space group and a unit cell a = 193.4, b = 80.5, c = 98.8, and α = γ = 90.0 and β = 120.2. The structure was solved by using multiwavelength anomalous dispersion from data collected at the zinc absorption edge (9.7 keV) and a higher energy dataset (12.7 keV). The positions of eight zinc atoms were found and refined using SHARP/autoSHARP [83],[84]. The derived experimental phases were improved by solvent flattening and revealed clear density for DNA and protein regions. The model was built with Coot and refined using REFMAC5 [85],[86] and Phenix [87]. The final model validated by MOLPROBITY has good geometry and most of the residues are in favorable regions in the Ramachandran plot [88]. All images from the Zur crystal structure were rendered using PYMOL [89].

The dimer of dimers nature of protein-DNA interaction made it impossible to assign the proper directionality of the DNA electron density de novo. In order to ascertain whether the Zur protein recognized a single DNA direction over the other, the Zur-DNA protein complex was crystallized with brominated DNA. The coding strand of 31mer2bpOH brominated at positions 8 and 22 of the DNA was ordered commercially in HPLC purified grade from IDT. The brominated DNA pellet was resuspended with MiliQ H₂O (Millipore) annealed with non-brominated HPLC purified 31mer2bpOH non-coding strand. The protein-DNA complex was prepared by mixing the following components to a final concentrations of [Zur] = 120 µM, [Br-31mer2bp OH] = 120 µM, [ZnSO₄] = 150 µM, and DTT = 5 mM. Crystals were grown in the same reservoir solution as non-brominated DNA (1.5 M ammonium sulfate, 0.1 M citrate pH 5.5) and 15% ethylene glycol was used a cryoprotectant. Brominated crystal diffraction data were collected at 13.5 keV (Bromine K edge) and diffracted to 3.5 Å.

Preparation of Fluorescently Labeled DNA

Fluorescently labeled DNA was isolated using PCR amplification of 51 bases from the ZnuC footprinting plasmid pUC19-Znufoot [26]. Two primers Cy5-Znu-forward and Cy5-Znu-reverse (ordered from IDT [Integrated DNA Technologies]) was used to amplify only a portion of this plasmid. The annotation “/5Cy5/” corresponds to the single Cy5 fluorescent probe covalently attached to the 5′ end of the DNA. The PCR reaction was carried out in 25 µl reaction volume with 20–50 ng of plasmid, 1× Thermopol buffer (New England Biolabs [NEB]), 10 µM of each primer, and 200 µM of each dNTP and 1 unit of DeepVent DNA polymerase (NEB). After 15 cycles of amplification the reaction was run on a 2% w/v agarose gel at 130 volts for 1.5 h. The DNA bands were visualized using UV excitation of the ethidium bromide mixed into the gel and the 51 bp fluorescent product was excised from the agarose gel using a razor blade. The DNA was solubilized using a QIAEX II Gel Extraction kit (Qiagen). Once the DNA was isolated, the concentration was estimated by running a small sample (5 µl) of the newly isolated DNA product, which was run on a new 2% agarose gel along with 5 dilutions of the 100 bp DNA ladder (NEB). The 100 bp ladder contained established DNA concentrations and the intensity of these bands was compared to that of the newly isolated DNA product. Typical PCR reactions lead to approximately 40 µl of ∼0.2 µM fluorescently labeled DNA.

In order to test the effect of mutating the protein contact sites on the znuABC promoter, site directed mutagenesis (Stratagene) was performed on the pUC19-Znufoot plasmid using mutagenic primers (Table S2). This plasmid had previously been used to generate the 340 bp sequence for Zur footprinting experiments [26]. Each of the mutations was confirmed by DNA sequencing using universal M13 primers. Once the mutation was confirmed fluorescently labeled mutant znuABC promoter sequences were isolated in the same manner as the WT sequence described above.

Electrophoretic Mobility Shift Assays

All electrophoretic mobility shift assay (EMSA) experiments were run on a 10% polyacrylamide gel containing 0.1 M Tris (pH 8.3) 0.1 M boric acid, and 50 µM ZnSO₄. For EMSA experiments the DNase I footprinting buffer used by Outten and colleagues [26] was modified in order to weaken the interaction between protein and DNA. Weakening the affinity between protein and DNA allowed us to use DNA concentrations high enough to obtain a reasonable signal, while ensuring that [DNA]<<K_d-app. Both protein and DNA were diluted in the EMSA binding buffer, which contained 10 mM Tris (pH 8.0), 10 mM NaCl, 100 mM L-potassium glutamic acid, 1.5 mM MgCl₂, 1 mM CaCl₂, 0.1 µg/ml bovine serum albumin (BSA), 1 mM dithiothreitol (DTT), 2 µg/ml sonicated salmon sperm DNA, 50 µM ZnSO₄, and 167 mM KCl. DNA fragments (≤45 pM) were mixed together with varying concentrations of Zur protein at 22°C for 30 min, and 85 µl of the 90 µl total volume was loaded directly onto a running gel (constant 145 volts) for 3 h.

Using a Typhoon 9410 Variable Mode imager (GE) each EMSA gel was excited at a wavelength of 633 nm and the intensity of the fluorescent emission at 690 nm was used to identify the presence of the labeled DNA. Apparent affinities were measured by comparing the ratio of bound (shifted species) to that of the free fluorescently labeled DNA within the gel. The ratios of free versus bound DNA were determined by using the imaging software ImageJ. Using the ImageJ profile of each lane in the EMSA gel, the fraction of shifted DNA species was plotted against the concentration of the protein sample. In order to calculate the apparent dissociation constants from the gel-shift data, the models of equilibrium binding in Equations 2a and 2b were fit to the data from two independent experiments using a non-linear least squares regression in MATLAB [90]. The fmincon optimization engine in MATLAB was used with the Interior Point minimization algorithm with default parameters. The standard error of the best fit parameters were calculated using the asymptotic standard error of the best fit determined from GraphPad Prism using the models in Equations 2a and 2b mentioned in the Results section.

RT-PCR assays

RT-PCR was conducted to measure mRNA level changes in E. coli MG1655 WT and Δzur cells. Cells were grown in LB medium into mid-log phase, and collected by centrifugation. Total RNA was purified by Qiagen RNA miRNeasy kit. RNA concentrations and quality were determined by NanoDrop Lite spectrophotometer (Thermal Scientific) and by 2100 Bioanalyzer (Agilent Technologies). The primers used for RT-PCR were ordered from Integrated DNA Technologies (IDT). The complete list of the primers and their sequences is shown below: zinT18_up (5′- CAAACTGGCTGTTGCTTTAGG-3′), zinT104_dn (5′-TCTGTTAAGGGTTTGCCGTG-3′), pliG7_up(5′-ATCAAGAGCATCAGGAAGGC-3′), pliG85_dn(5′-CATTGACATTCTTACCCGCAG-3′), znuC220_up (5′-CAGAAGCTGTATCTCGACACC-3′), znuC297_dn (5′-TTCTTTATGTGTACCAGGGCG-3′), ykgM90_up (5′-AACAGACCGTGAGATTGAGC-3′), ykgM204_dn (5′-TCCTTCTGATGCCACTGTTC-3′). The efficiency of all primer pairs were tested to be within 95% to 105%. The reagent for RT-PCR was the iTaq Universal SYBR Green One-Step kit with SYBR green (Bio-Rad), and the instrument used was the iQ5 (Bio-Rad). Each reaction contained 10 ng of total RNA and 300 nM of each primer.

Data Deposition

Data pertaining to the experiments in this article have been deposited in the Dryad repository at http://dx.doi.org/10.5061/dryad.vn6dv [91].

Accession Numbers

Protein Data Bank (http://www.rcsb.org/pdb): coordinates and structure factors for the Zur-DNA-Zn complex have been deposited in the Protein Data Bank with accession codes: 4MTD (Orientation 1) and 4MTE (both orientations).