Homing in on the role of transition metals in the HNH motif of colicin endonucleases.

The cytotoxic domain of the bacteriocin colicin E9 (the E9 DNase) is a nonspecific endonuclease that must traverse two membranes to reach its cellular target, bacterial DNA. Recent structural studies revealed that the active site of colicin DNases encompasses the HNH motif found in homing endonucleases, and bound within this motif a single transition metal ion (either Zn(2+) or Ni(2+)) the role of which is unknown. In the present work we find that neither Zn(2+) nor Ni(2+) is required for DNase activity, which instead requires Mg(2+) ions, but binding transition metals to the E9 DNase causes subtle changes to both secondary and tertiary structure. Spectroscopic, proteolytic, and calorimetric data show that, accompanying the binding of 1 eq of Zn(2+), Ni(2+), or Co(2+), the thermodynamic stability of the domain increased substantially, and that the equilibrium dissociation constant for Zn(2+) was less than or equal to nanomolar, while that for Co(2+) and Ni (2+) was micromolar. Our data demonstrate that the transition metal is not essential for colicin DNase activity but rather serves a structural role. We speculate that the HNH motif has been adapted for use by endonuclease colicins because of its involvement in DNA recognition and because removal of the bound metal ion destabilizes the DNase domain, a likely prerequisite for its translocation across bacterial membranes.     

Catalytic mechanism of endonuclease v: a catalytic and regulatory two-metal model

The enzyme endonuclease V initiates repair of deaminated DNA bases by making an endonucleolytic incision on the 3' side one nucleotide from a base lesion. In this study, we have used site-directed mutagenesis to characterize the role of the highly conserved residues D43, E89, D110, and H214 in Thermotoga maritima endonuclease V catalysis. DNA cleavage and Mn(2+)-rescue analysis suggest that amino acid substitutions at D43 impede the enzymatic activity severely while mutations at E89 and D110 may be tolerated. Mutations at H214 yield enzyme that maintains significant DNA cleavage activity. The H214D mutant exhibits little change in substrate specificity or DNA cleavage kinetics, suggesting the exchangeability between His and Asp at this site. DNA binding analysis implicates the involvement of the four residues in metal binding. Mn(2+)-mediated cleavage of inosine-containing DNA is stimulated by the addition of Ca(2+), a metal ion that does not support catalysis. The effects of Mn(2+) on Mg(2+)-mediated DNA cleavage show a complexed initial stimulatory and later inhibitory pattern. The data obtained from the dual metal ion analyses lead to the notion that two metal ions are involved in endonuclease V-mediated catalysis. A catalytic and regulatory two-metal model is proposed.     

Metal ion binding to human hemopexin

Binding of divalent metal ions to human hemopexin (Hx) purified by a new protocol has been characterized by metal ion affinity chromatography and potentiometric titration in the presence and absence of bound protoheme IX. ApoHx was retained by variously charged metal affinity chelate resins in the following order: Ni(2+) > Cu(2+) > Co(2+) > Zn(2+) > Mn(2+). The Hx-heme complex exhibited similar behavior except the order of retention of the complex on Zn(2+)- and Co(2+)-charged columns was reversed. One-dimensional (1)H NMR of apoHx in the presence of Ni(2+) implicates at least two His residues and possibly an Asp, Glu, or Met residue in Ni(2+) binding. Potentiometric titrations establish that apoHx possesses more than two metal ion binding sites and that the capacity and/or affinity for metal ion binding is diminished when heme binds. For most metal ions that have been studied, potentiometric data did not fit to binding isotherms that assume one or two independent binding sites. For Mn(2+), however, these data were consistent with a high-affinity site [K(A) = (15 +/- 3) x 10(6) M(-)(1)] and a low-affinity site (K(A) 相似文献   

Mechanism of the phosphatase component of Clostridium thermocellum polynucleotide kinase-phosphatase

Polynucleotide kinase-phosphatase (Pnkp) from Clostridium thermocellum catalyzes ATP-dependent phosphorylation of 5'-OH termini of DNA or RNA polynucleotides and Ni(2+)/Mn(2+)-dependent dephosphorylation of 2',3' cyclic phosphate, 2'-phosphate, and 3'-phosphate ribonucleotides. CthPnkp is an 870-amino-acid polypeptide composed of three domains: an N-terminal module similar to bacteriophage T4 polynucleotide kinase, a central module that resembles the dinuclear metallo-phosphoesterase superfamily, and a C-terminal ligase-like adenylyltransferase domain. Here we conducted a mutational analysis of CthPnkp that identified 11 residues required for Ni(2+)-dependent phosphatase activity with 2'-AMP and 3'-AMP. Eight of the 11 CthPnkp side chains were also required for Ni(2+)-dependent hydrolysis of p-nitrophenyl phosphate. The ensemble of essential side chains includes the conserved counterparts (Asp187, His189, Asp233, Arg237, Asn263, His264, His323, His376, and Asp392 in CthPnkp) of all of the amino acids that form the dinuclear metal-binding site and the phosphate-binding site of bacteriophage lambda phosphatase. Three residues (Asp236, His264, and Arg237) required for activity with 2'-AMP or 3'-AMP were dispensable for Ni(2+)-dependent hydrolysis of p-nitrophenyl phosphate. Our findings, together with available structural information, provide fresh insights to the metallophosphoesterase mechanism, including the roles of His264 and Asp236 in proton donation to the leaving group. Deletion analysis defined an autonomous phosphatase domain, CthPnkp-(171-424).     

NMR studies of metal ion binding to the Zn-finger-like HNH motif of colicin E9

The 134 amino acid DNase domain of colicin E9 contains a zinc-finger-like HNH motif that binds divalent transition metal ions. We have used 1D 1H and 2D 1H-15N NMR methods to characterise the binding of Co2+, Ni2+ and Zn2+ to this protein. Data for the Co2+-substituted and Ni2+-substituted proteins show that the metal ion is coordinated by three histidine residues; and the NMR characteristics of the Ni2+-substituted protein show that two of the histidines are coordinated through their N(epsilon2) atoms and one via its N(delta1). Furthermore, the NMR spectrum of the Ni2+-substituted protein is perturbed by the presence of phosphate, consistent with an X-ray structure showing that phosphate is coordinated to bound Ni2+, and by a change in pH, consistent with an ionisable group at the metal centre with a pKa of 7.9. Binding of an inhibitor protein to the DNase does not perturb the resonances of the metal site, suggesting there is no substantial conformation change of the DNase HNH motif on inhibitor binding. 1H-15N NMR data for the Zn2+-substituted DNase show that this protein, like the metal-free DNase, exists as two conformers with different 1H-15N correlation NMR spectra, and that the binding of Zn2+ does not significantly perturb the spectra, and hence structures, of these conformers beyond the HNH motif region.     

Metal ions binding to NAD-glycohydrolase from the venom of Agkistrodon acutus: regulation of multicatalytic activity

AA-NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA-NADase contains Cu(2+) ions that are essential for its multicatalytic activity. In this study, the interactions between divalent metal ions and AA-NADase and the effects of metal ions on its structure and activity have been investigated by equilibrium dialysis, isothermal titration calorimetry, fluorescence, circular dichroism, dynamic light scattering and HPLC. The results show that AA-NADase has two classes of Cu(2+) binding sites, one activator site with high affinity and approximately six inhibitor sites with low affinity. Cu(2+) ions function as a switch for its NADase activity. In addition, AA-NADase has one Mn(2+) binding site, one Zn(2+) binding site, one strong and two weak Co(2+) binding sites, and two strong and six weak Ni(2+) binding sites. Metal ion binding affinities follow the trend Cu(2+) > Ni(2+) > Mn(2+) > Co(2+) > Zn(2+), which accounts for the existence of one Cu(2+) in the purified AA-NADase. Both NADase and ADPase activities of AA-NADase do not have an absolute requirement for Cu(2+), and all tested metal ions activate its NADase and ADPase activities and the activation capacity follows the trend Zn(2+) > Mn(2+) > Cu(2+) ~Co(2+) > Ni(2+). Metal ions serve as regulators for its multicatalytic activity. Although all tested metal ions have no obvious effects on the global structure of AA-NADase, Cu(2+)- and Zn(2+)-induced conformational changes around some Trp residues have been observed. Interestingly, each tested metal ion has a very similar activation of both NADase and ADPase activities, suggesting that the two different activities probably occur at the same site.     

Domain organization and metal ion requirement of the Type IIS restriction endonuclease MnlI

A two-domain structure of the Type IIS restriction endonuclease MnlI has been identified by limited proteolysis. An N-terminal domain of the enzyme mediates the sequence-specific interaction with DNA, whereas a monomeric C-terminal domain resembles bacterial colicin nucleases in its requirement for alkaline earth as well as transition metal ions for double- and single-stranded DNA cleavage activities. The results indicate that the fusion of the non-specific HNH-type nuclease to the DNA binding domain had transformed MnlI into a Mg(2+)-, Ni(2+)-, Co(2+)-, Mn(2+)-, Zn(2+)-, Ca(2+)-dependent sequence-specific enzyme. Nevertheless, MnlI retains a residual single-stranded DNA cleavage activity controlled by its C-terminal colicin-like nuclease domain.     

The nickel site of Bacillus pasteurii UreE, a urease metallo-chaperone, as revealed by metal-binding studies and X-ray absorption spectroscopy

UreE is a homodimeric metallo-chaperone that assists the insertion of Ni(2+) ions in the active site of urease. The crystal structures of UreE from Bacillus pasteurii and Klebsiella aerogenes have been determined, but the details of the nickel-binding site were not elucidated due to solid-state effects that caused disorder in a key portion of the protein. A complementary approach to this problem is described here. Titrations of wild-type Bacillus pasteurii UreE (BpUreE) with Ni(2+), followed by metal ion quantitative analysis using inductively coupled plasma optical emission spectrometry (ICP-OES), established the binding of 2 Ni(2+) ions to the functional dimer, with an overall dissociation constant K(D) = 35 microM. To establish the nature, the number, and the geometry of the ligands around the Ni(2+) ions in BpUreE-Ni(2), X-ray absorption spectroscopy data were collected and analyzed using an approach that combines ab initio extended X-ray absorption fine structure (EXAFS) calculations with a systematic search of several possible coordination geometries, using the Simplex algorithm. This analysis indicated the presence of Ni(2+) ions in octahedral coordination geometry and an average of two histidine residues and four O/N ligands bound to each metal ion. The fit improved significantly with the incorporation, in the model, of a Ni-O-Ni moiety, suggesting the presence of a hydroxide-bridged dinuclear cluster in the Ni-loaded BpUreE. These results were interpreted using two possible models. One model involves the presence of two identical metal sites binding Ni(2+) with negative cooperativity, with each metal ion bound to the conserved His(100) as well as to either His(145) or His(147) from each monomer, residues found largely conserved at the C-terminal. The alternative model comprises the presence of two different binding sites featuring different affinity for Ni(2+). This latter model would involve the presence of a dinuclear metallic core, with one Ni(2+) ion bound to one His(100) from each monomer, and the second Ni(2+) ion bound to a pair of either His(145) or His(147). The arguments in favor of one model as compared to the other are discussed on the basis of the available biochemical data.     

The functional role of the binuclear metal center in D-aminoacylase: one-metal activation and second-metal attenuation

Our structural comparison of the TIM barrel metal-dependent hydrolase(-like) superfamily suggests a classification of their divergent active sites into four types: alphabeta-binuclear, alpha-mononuclear, beta-mononuclear, and metal-independent subsets. The d-aminoacylase from Alcaligenes faecalis DA1 belongs to the beta-mononuclear subset due to the fact that the catalytically essential Zn(2+) is tightly bound at the beta site with coordination by Cys(96), His(220), and His(250), even though it possesses a binuclear active site with a weak alpha binding site. Additional Zn(2+), Cd(2+), and Cu(2+), but not Ni(2+), Co(2+), Mg(2+), Mn(2+), and Ca(2+), can inhibit enzyme activity. Crystal structures of these metal derivatives show that Zn(2+) and Cd(2+) bind at the alpha(1) subsite ligated by His(67), His(69), and Asp(366), while Cu(2+) at the alpha(2) subsite is chelated by His(67), His(69) and Cys(96). Unexpectedly, the crystal structure of the inactive H220A mutant displays that the endogenous Zn(2+) shifts to the alpha(3) subsite coordinated by His(67), His(69), Cys(96), and Asp(366), revealing that elimination of the beta site changes the coordination geometry of the alpha ion with an enhanced affinity. Kinetic studies of the metal ligand mutants such as C96D indicate the uniqueness of the unusual bridging cysteine and its involvement in catalysis. Therefore, the two metal-binding sites in the d-aminoacylase are interactive with partially mutual exclusion, thus resulting in widely different affinities for the activation/attenuation mechanism, in which the enzyme is activated by the metal ion at the beta site, but inhibited by the subsequent binding of the second ion at the alpha site.     

Molecular simulations to determine the chelating mechanisms of various metal ions to the His-tag motif: a preliminary study

In the present study, molecular simulations were performed to investigate the chelating mechanisms of various metal ions to the His-tag motifs with various His residues. The chelation mostly involved the i and i+2 His residues for Ni(2+), Zn(2+), Cu(2+), and Co(2+), while the cooperation of 3 His residues was necessary when Fe(3+) was involved in chelation with His-tags having more than 4 His residues. Metal ion was best fitted into the pocket formed by the imidazole nitrogens while it was about equally located among these nitrogen atoms. His-tag6 was found to have little effect on the structural integrity while the target protein contains more than 68 amino acid residues. Ni(2+) interacted with the imidazole nitrogen of His3 in the beginning of chelation, and then entered into the pocket formed by His3 and His5 at 4 ns during the 10 ns molecular dynamics simulations. The fast chelating process resulted in successful application of IMAC techniques in efficient protein purification.     

Type II restriction endonuclease R.KpnI is a member of the HNH nuclease superfamily

The restriction endonuclease (REase) R.KpnI is an orthodox Type IIP enzyme, which binds to DNA in the absence of metal ions and cleaves the DNA sequence 5′-GGTAC^C-3′ in the presence of Mg²⁺ as shown generating 3′ four base overhangs. Bioinformatics analysis reveals that R.KpnI contains a ββα-Me-finger fold, which is characteristic of many HNH-superfamily endonucleases, including homing endonuclease I-HmuI, structure-specific T4 endonuclease VII, colicin E9, sequence non-specific Serratia nuclease and sequence-specific homing endonuclease I-PpoI. According to our homology model of R.KpnI, D148, H149 and Q175 correspond to the critical D, H and N or H residues of the HNH nucleases. Substitutions of these three conserved residues lead to the loss of the DNA cleavage activity by R.KpnI, confirming their importance. The mutant Q175E fails to bind DNA at the standard conditions, although the DNA binding and cleavage can be rescued at pH 6.0, indicating a role for Q175 in DNA binding and cleavage. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD…D/EXK superfamily of nucleases, instead is a member of the HNH superfamily.     

The conserved asparagine in the HNH motif serves an important structural role in metal finger endonucleases

The HNH motif is a small nucleic acid binding and cleavage module, widespread in metal finger endonucleases in all life kingdoms. Here we studied a non-specific endonuclease, the nuclease domain of ColE7 (N-ColE7), to decipher the role of the conserved asparagine and histidine residues in the HNH motif. We found, using fluorescence resonance energy transfer (FRET) assays, that the DNA hydrolysis activity of H545 N-ColE7 mutants was completely abolished while activities of N560 and H573 mutants varied from 6.9% to 83.2% of the wild-type activity. The crystal structures of three N-ColE7 mutants in complex with the inhibitor Im7, N560A-Im7, N560D-Im7 and H573A-Im7, were determined at a resolution of 1.9 A to 2.2 A. H573 is responsible for metal ion binding in the wild-type protein, as the zinc ion is still partially associated in the structure of H573A, suggesting that H573 plays a supportive role in metal binding. Both N560A and N560D contain a disordered loop in the HNH motif due to the disruption of the hydrogen bond network surrounding the side-chain of residue 560, and as a result, the imidazole ring of the general base residue H545 is tilted slightly and the scissile phosphate is shifted, leading to the large reductions in hydrolysis activities. These results suggest that the highly conserved asparagine in the HNH motif, in general, plays a structural role in constraining the loop in the metal finger structure and keeping the general base histidine and scissile phosphate in the correct position for DNA hydrolysis.     

Multistep binding of transition metals to the H-N-H endonuclease toxin colicin E9

We report the first stopped-flow fluorescence analysis of transition metal binding (Co(2+), Ni(2+), Cu(2+), and Zn(2+)) to the H-N-H endonuclease motif within colicin E9 (the E9 DNase). The H-N-H consensus forms the active site core of a number of endonuclease groups but is also structurally homologous to the so-called treble-clef motif, a ubiquitous zinc-binding motif found in a wide variety of metalloproteins. We find that all the transition metal ions tested bind via multistep mechanisms. Binding was further dissected for Ni(2+) and Zn(2+) ions through the use of E9 DNase single tryptophan mutants, which demonstrated that most steps reflect conformational rearrangements that occur after the bimolecular collision, many common to the two metals, while one appears specific to zinc. The kinetically derived equilibrium dissociation constants (K(d)) for transition metal binding to the E9 DNase agree with previously determined equilibrium measurements and so confirm the validity of the derived kinetic mechanisms. Zn(2+) binds tightest to the enzyme (K(d) approximately 10(-)(9) M) but does not support endonuclease activity, whereas the other metals (K(d) approximately 10(-)(6) M) are active in endonuclease assays implying that the additional step seen for Zn(2+) traps the enzyme in an inactive but high affinity state. Metal-induced conformational changes are likely to be a conserved feature of H-N-H/treble clef motif proteins since similar Zn(2+)-induced, multistep binding was observed for other colicin DNases. Moreover, they appear to be independent both of the conformational heterogeneity that is naturally present within the E9 DNase at equilibrium, as well as the conformational changes that accompany the binding of its cognate inhibitor protein Im9.     

The Crystal Structure of Bacteriophage HK97 gp6: Defining a Large Family of Head-Tail Connector Proteins

The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 Å crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophage SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly.     

Crystal structures of the EVE-HNH endonuclease VcaM4I in the presence and absence of DNA

Many modification-dependent restriction endonucleases (MDREs) are fusions of a PUA superfamily modification sensor domain and a nuclease catalytic domain. EVE domains belong to the PUA superfamily, and are present in MDREs in combination with HNH nuclease domains. Here, we present a biochemical characterization of the EVE-HNH endonuclease VcaM4I and crystal structures of the protein alone, with EVE domain bound to either 5mC modified dsDNA or to 5mC/5hmC containing ssDNA. The EVE domain is moderately specific for 5mC/5hmC containing DNA according to EMSA experiments. It flips the modified nucleotide, to accommodate it in a hydrophobic pocket of the enzyme, primarily formed by P24, W82 and Y130 residues. In the crystallized conformation, the EVE domain and linker helix between the two domains block DNA binding to the catalytic domain. Removal of the EVE domain and inter-domain linker, but not of the EVE domain alone converts VcaM4I into a non-specific toxic nuclease. The role of the key residues in the EVE and HNH domains of VcaM4I is confirmed by digestion and restriction assays with the enzyme variants that differ from the wild-type by changes to the base binding pocket or to the catalytic residues.     

Function of the N-terminal segment of the RecA-dependent nuclease Ref

The bacteriophage P1 Ref (recombination enhancement function) protein is a RecA-dependent, HNH endonuclease. It can be directed to create targeted double-strand breaks within a displacement loop formed by RecA. The 76 amino acid N-terminal region of Ref is positively charged (25/76 amino acid residues) and inherently unstructured in solution. Our investigation of N-terminal truncation variants shows this region is required for DNA binding, contains a Cys involved in incidental dimerization and is necessary for efficient Ref-mediated DNA cleavage. Specifically, Ref N-terminal truncation variants lacking between 21 and 47 amino acids are more effective RecA-mediated targeting nucleases. We propose a more refined set of options for the Ref-mediated cleavage mechanism, featuring the N-terminal region as an anchor for at least one of the DNA strand cleavage events.     

Defining proximity relationships in the tertiary structure of the dopamine transporter. Identification of a conserved glutamic acid as a third coordinate in the endogenous Zn(2+)-binding site

Recently, we have described a distance constraint in the unknown tertiary structure of the human dopamine transporter (hDAT) by identification of two histidines, His(193) in the second extracellular loop and His(375) at the top of transmembrane (TM) 7, that form two coordinates in an endogenous, high affinity Zn(2+)-binding site. To achieve further insight into the tertiary organization of hDAT, we set out to identify additional residues involved in Zn(2+) binding and subsequently to engineer artificial Zn(2+)-binding sites. Ten aspartic acids and glutamic acids, predicted to be on the extracellular side, were mutated to asparagine and glutamine, respectively. Mutation of Glu(396) (E396Q) at the top of TM 8 increased the IC(50) value for Zn(2+) inhibition of [(3)H]dopamine uptake from 1.1 to 530 microM and eliminated Zn(2+)-induced potentiation of [(3)H]WIN 35,428 binding. These data suggest that Glu(396) is involved in Zn(2+) binding to hDAT. Importantly, Zn(2+) sensitivity was preserved following substitution of Glu(396) with histidine, indicating that the effect of mutating Glu(396) is not an indirect effect because of the removal of a negatively charged residue. The common participation of Glu(396), His(193), and His(375) in binding the small Zn(2+) ion implies their proximity in the unknown tertiary structure of hDAT. The close association between TM 7 and 8 was further established by engineering of a Zn(2+)-binding site between His(375) and a cysteine inserted in position 400 in TM 8. Summarized, our data define an important set of proximity relationships in hDAT that should prove an important template for further exploring the molecular architecture of Na(+)/Cl(-)-dependent neurotransmitter transporters.     

Site-directed mutagenesis of the bacterial metalloamidase UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC). Identification of the zinc binding site

UDP-3-O-(acyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second step in the biosynthesis of lipid A in Gram-negative bacteria. Compounds targeting this enzyme are proposed to chelate the single, essential zinc ion bound to LpxC and have been demonstrated to stop the growth of Escherichia coli. A comparison of LpxC sequences from diverse bacteria identified 10 conserved His, Asp, and Glu residues that might play catalytic roles. Each amino acid was altered in both E. coli and Aquifex aeolicus LpxC and the catalytic activities of the variants were determined. Three His and one Asp residues (H79, H238, D246, and H265) are essential for catalysis based on the low activities (<0.1% of wild-type LpxC) of mutants with alanine substitutions at these positions. H79 and H238 likely coordinate zinc; the Zn(2+) content of the purified variant proteins is low and the specific activity is enhanced by the addition of Zn(2+). The third side chain to coordinate zinc is likely either H265 or D246 and a fourth ligand is likely a water molecule, as indicated by the hydroxamate inhibition, suggesting a His(3)H(2)O or His(2)AspH(2)O Zn(2+)-polyhedron in LpxC. The decreased zinc inhibition of LpxC mutants at E78 suggests that this side chain may coordinate a second, inhibitory Zn(2+) ion. Given the absence of any known Zn(2+) binding motifs, the active site of LpxC may have evolved differently than other well-studied zinc metalloamidases, a feature that should aid in the design of safe antibiotics.     

Individual metal ligands play distinct functional roles in the zinc sensor Staphylococcus aureus CzrA

Recent studies on metalloregulatory proteins suggest that coordination number/geometry and metal ion availability in a host cytosol are key determinants for biological specificity. Here, we investigate the contribution that individual metal ligands of the alpha5 sensing site of Staphylococcus aureus CzrA (Asp84, His86, His97', and His100') make to in vitro metal ion binding affinity, coordination geometry, and allosteric negative regulation of DNA operator/promoter region binding. All ligand substitution mutants exhibit significantly reduced metal ion binding affinity (K(Me)) by > or =10(3) M(-1). Substitutions of Asp84 and His97 give rise to non-native coordination geometries upon metal binding and are non-functional in allosteric coupling of metal and DNA binding (DeltaG(coupling) approximately 0 kcal mol(-1)). In contrast, His86 and His100 could be readily substituted with potentially liganding (Asp, Glu) and poorly liganding (Asn, Gln) residues with significant native-like tetrahedral metal coordination geometry retained in these mutants, leading to strong functional coupling (DeltaG(coupling) > or = +3.0 kcal mol(-1)). 1H-(15)N heteronuclear single quantum coherence (HSQC) spectra of wild-type and mutant CzrAs reveal that all H86 and H100 substitution mutants undergo 4 degrees structural switching on binding Zn(II), while D84N, H97N and H97D CzrAs do not. Thus, only those variant CzrAs that retain some tetrahedral coordination geometry characteristic of wild-type CzrA upon metal binding are capable of driving 4 degrees structural conformational changes linked to allosteric regulation of DNA binding in vitro, irrespective of the magnitude of K(Me).