Purification, characterization, and functional analysis of a truncated Klebsiella aerogenes UreE urease accessory protein lacking the histidine-rich carboxyl terminus.

Klebsiella aerogenes UreE, one of four accessory proteins involved in urease metallocenter assembly, contains a histidine-rich C terminus (10 of the last 15 residues) that is likely to participate in metal ion coordination by this nickel-binding protein. To study the function of the histidine-rich region in urease activation, ureE in the urease gene cluster was mutated to result in synthesis of a truncated peptide, H144* UreE, lacking the final 15 residues. Urease activity in cells containing H144* UreE approached the activities for cells possessing the wild-type protein at nickel ion concentrations ranging from 0 to 1 mM in both nutrient-rich and minimal media. In contrast, clear reductions in urease activities were observed when two ureE deletion mutant strains were examined, especially at lower nickel ion concentrations. Surprisingly, the H144* UreE, like the wild-type protein, was readily purified with a nickel-nitrilotriacetic acid resin. Denaturing polyacrylamide gel electrophoretic analysis and N-terminal sequencing confirmed that the protein was a truncated UreE. Size exclusion chromatography indicated that the H144* UreE peptide associated into a homodimer, as known for the wild-type protein. The truncated protein was shown to cooperatively bind 1.9 +/- 0.2 Ni(II) ions as assessed by equilibrium dialysis measurements, compared with the 6.05 +/- 0.25 Ni ions per dimer reported previously for the native protein. These results demonstrate that the histidine-rich motif is not essential to UreE function and is not solely responsible for UreE nickel-binding ability. Rather, we propose that internal nickel binding sites of UreE participate in urease metallocenter assembly.     

In vivo and in vitro kinetics of metal transfer by the Klebsiella aerogenes urease nickel metallochaperone, UreE

The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to deliver Ni(II) to the urease apoprotein during enzyme activation. Native UreE possesses a histidine-rich region at its carboxyl terminus that binds several equivalents of Ni(2+); however, a truncated form of this protein (H144*UreE) binds only 2 Ni(2+) per dimer and is functionally active (Brayman, T. G., and Hausinger, R. P. (1996) J. Bacteriol. 178, 5410-5416). The urease activation kinetics were studied in vivo by monitoring the development of urease activity upon adding Ni(2+) to spectinomycin-treated Escherichia coli cells that expressed the complete K. aerogenes urease gene cluster with altered forms of ureE. Site-specific alterations of H144*UreE decrease the rate of in vivo urease activation, with the most dramatic changes observed for the H96A, H110A, D111A, and H112A substitutions. Notably, urease activity in cells producing H96A/H144*UreE was lower than cells containing a ureE deletion. Prior studies had shown that H110A and H112A variants each bound a single Ni(2+) per dimer with elevated K(d) values compared with control H144*UreE, whereas the H96A and D111A variants bound 2 Ni(2+) per dimer with unperturbed K(d) values (Colpas, G. J., Brayman, T. G., Ming, L.-J., and Hausinger, R. P. (1999) Biochemistry 38, 4078-4088). To understand why cells containing the latter two proteins showed reduced rates of urease activation, we characterized their metal binding/dissociation kinetics and compared the results to those obtained for H144*UreE. The truncated protein was shown to sequentially bind two Ni(2+) with k(1) approximately 18 and k(2) approximately 100 M(-1) s(-1), and with dissociation rates k(-1) approximately 3 x 10(-3) and k(-2) approximately 10(-4) s(-1). Similar apparent rates of binding and dissociation were noted for the two mutant proteins, suggesting that altered H144*UreE interactions with Ni(2+) do not account for the changes in cellular urease activation. These conclusions are further supported by in vitro experiments demonstrating that addition of H144*UreE to urease apoprotein activation mixtures inhibited the rate and extent of urease formation. Our results highlight the importance of other urease accessory proteins in assisting UreE-dependent urease maturation.     

Spectroscopic characterization of metal binding by Klebsiella aerogenes UreE urease accessory protein

 The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to function in Ni(II) delivery to the urease apoprotein. Wild-type UreE contains a histidine-rich region at its carboxyl terminus and binds 5–6 Ni per dimer, whereas the functionally active but truncated H144*UreE lacks the histidine-rich motif and binds only two Ni per dimer [Brayman TG, Hausinger RP (1996) J Bacteriol 178 : 5410-5416]. For both proteins, Cu(II), Co(II), and Zn(II) ions compete for the Ni-binding sites. In order to characterize the coordination environments of bound metals, especially features that are unique to Ni, the Ni-, Cu-, and Co-bound forms of H144*UreE were studied by a combination of EPR, ESEEM, hyperfine-shifted ¹H-NMR, XAS, and RR spectroscopic methods. For each metal ion, the two binding sites per homodimer were spectroscopically distinguishable. For example, the two Ni-binding sites each have pseudo-octahedral geometry in an N/O coordination environment, but differ in their number of histidine donors. The two Cu-binding sites have tetragonal geometry with two histidine donors each; however, the second Cu ion is bound by at least one cysteine donor in addition to the N/O-type donors found for the first Cu ion. Two Co ions are bound to H144*UreE in pseudo-octahedral geometry with N/O coordination, but the sites differ in the number of histidine donors that can be observed by NMR. The differences in coordination for each type of metal ion are relevant to the proposed function of UreE to selectively facilitate Ni insertion into urease in vivo. Received: 8 October 1997 / Accepted: 30 December 1997     

Crystal structure of Klebsiella aerogenes UreE, a nickel-binding metallochaperone for urease activation

UreE is proposed to be a metallochaperone that delivers nickel ions to urease during activation of this bacterial virulence factor. Wild-type Klebsiella aerogenes UreE binds approximately six nickel ions per homodimer, whereas H144*UreE (a functional C-terminal truncated variant) was previously reported to bind two. We determined the structure of H144*UreE by multi-wavelength anomalous diffraction and refined it to 1.5 A resolution. The present structure reveals an Hsp40-like peptide-binding domain, an Atx1-like metal-binding domain, and a flexible C terminus. Three metal-binding sites per dimer, defined by structural analysis of Cu-H144*UreE, are on the opposite face of the Atx1-like domain than observed in the copper metallochaperone. One metal bridges the two subunits via the pair of His-96 residues, whereas the other two sites involve metal coordination by His-110 and His-112 within each subunit. In contrast to the copper metallochaperone mechanism involving thiol ligand exchanges between structurally similar chaperones and target proteins, we propose that the Hsp40-like module interacts with urease apoprotein and/or other urease accessory proteins, while the Atx1-like domain delivers histidyl-bound nickel to the urease active site.     

Identification of metal-binding residues in the Klebsiella aerogenes urease nickel metallochaperone, UreE

The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to bind intracellular Ni(II) for transfer to urease apoprotein. While native UreE possesses a histidine-rich region at its carboxyl terminus that binds several equivalents of Ni, the Ni-binding sites associated with urease activation are internal to the protein as shown by studies involving truncated H144UreE [Brayman and Hausinger (1996) J. Bacteriol. 178, 5410-5416]. Nine potential Ni-binding residues (five His, two Cys, one Asp, and one Tyr) within H144UreE were independently substituted by mutagenesis to determine their roles in metal binding and urease activation. In vivo effects of these substitutions on urease activity were measured in Escherichia coli strains containing the K. aerogenes urease gene cluster with the mutated ureE genes. Several mutational changes led to reductions in specific activity, with substitution of His96 producing urease activity below the level obtained from a ureE deletion mutant. The metal-binding properties of purified variant UreE proteins were characterized by a combination of equilibrium dialysis and UV/visible, EPR, and hyperfine-shifted 1H NMR spectroscopic methods. Ni binding was unaffected for most H144UreE variants, but mutant proteins substituted at His110 or His112 exhibited greatly reduced affinity for Ni and bound one, rather than two, metal ions per dimer. Cys79 was identified as the Cu ligand responsible for the previously observed charge-transfer transition at 370 nm, and His112 also was shown to be associated with this chromophoric site. NMR spectroscopy provided clear evidence that His96 and His110 serve as ligands to Ni or Co. The results from these and other studies, in combination with prior spectroscopic findings for metal-substituted UreE [Colpas et al. (1998) J. Biol. Inorg. Chem. 3, 150-160], allow us to propose that the homodimeric protein possesses two nonidentical metal-binding sites, each symmetrically located at the dimer interface. The first equivalent of added Ni or Co binds via His96 and His112 residues from each subunit of the dimer, and two other N or O donors. Asp111 either functions as a ligand or may affect this site by secondary interactions. The second equivalent of Ni or Co binds via the symmetric pair of His110 residues as well as four other N or O donors. In contrast, the first equivalent of Cu binds via the His110 pair and two other N/O donors, while the second equivalent of Cu binds via the His112 pair and at least one Cys79 residue. UreE sequence comparisons among urease-containing microorganisms reveal that residues His96 and Asp111, associated with the first site of Ni binding, are highly conserved, while the other targeted residues are missing in many cases. Our data are most compatible with one Ni-binding site per dimer being critical for UreE's function as a metallochaperone.     

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.     

Selectivity of Ni(II) and Zn(II) binding to Sporosarcina pasteurii UreE, a metallochaperone in the urease assembly: a calorimetric and crystallographic study

Urease is a nickel-dependent enzyme that plays a critical role in the biogeochemical nitrogen cycle by catalyzing the hydrolysis of urea to ammonia and carbamate. This enzyme, initially synthesized in the apo form, needs to be activated by incorporation of two nickel ions into the active site, a process driven by the dimeric metallochaperone UreE. Previous studies reported that this protein can bind different metal ions in vitro, beside the cognate Ni(II). This study explores the metal selectivity and affinity of UreE from Sporosarcina pasteurii (Sp, formerly known as Bacillus pasteurii) for cognate [Ni(II)] and noncognate [Zn(II)] metal ions. In particular, the thermodynamic parameters of SpUreE Ni(II) and Zn(II) binding have been determined using isothermal titration calorimetry. These experiments show that two Ni(II) ions bind to the protein dimer with positive cooperativity. The high-affinity site involves the conserved solvent-exposed His¹⁰⁰ and the C-terminal His¹⁴⁵, whereas the low-affinity site comprises also the C-terminal His¹⁴⁷. Zn(II) binding to the protein, occurring in the same protein regions and with similar affinity as compared to Ni(II), causes metal-driven dimerization of the protein dimer. The crystal structure of the protein obtained in the presence of equimolar amounts of both metal ions indicates that the high-affinity metal binding site binds Ni(II) preferentially over Zn(II). The ability of the protein to select Ni(II) over Zn(II) was confirmed by competition experiments in solution as well as by analysis of X-ray anomalous dispersion data. Overall, the thermodynamics and structural parameters that modulate the metal ion specificity of the different binding sites on the protein surface of SpUreE have been established.     

Probing copper2+ binding to the prion protein using diamagnetic nickel2+ and 1H NMR: the unstructured N terminus facilitates the coordination of six copper2+ ions at physiological concentrations

The prion protein (PrP) is a Cu2+ binding cell surface glyco-protein. Misfolding of PrP into a beta-sheet rich conformation is associated with transmissible spongiform encephalopathies. Here we use Ni2+ as a diamagnetic probe to further understand Cu2+ binding to PrP. Like Cu2+, Ni2+ preferentially binds to an unstructured region between residues 90 and 126 of PrP, which is a key region for amyloidogenicity and prion propagation. Using both 1H NMR and visible-circular dichroism (CD) spectroscopy, we show that two Ni2+ ions bind to His96 and His111 independently of each other. 1H NMR indicates that both Ni2+ binding sites form square-planar diamagnetic complexes. We have previously shown that Cu2+ forms a paramagnetic square-planar complex in this region, suggesting that Ni2+ could be used as a probe for Cu2+ binding. In addition, competition studies show that two Cu2+ ions can displace Ni2+ from these sites. Upon Ni2+ addition 1H NMR changes in chemical shifts indicate the imidazole ring and amide nitrogen atoms to the N terminus of both His96 and His111 act as coordinating ligands. Use of peptide fragments confirm that PrP(92-96) and PrP(107-111) represent the minimal binding motif for the two Ni2+ binding sites. Analysis of Cu2+ loaded visible-CD spectra show that as with Ni2+, PrP(90-115) binds two Cu2+ ions at His96 and His111 independently of each other. Visible CD studies with PrP(23-231Delta51-90), a construct of PrP(23-231) with the octarepeat region deleted to improve solubility, confirm binding of Ni2+ to His96 and His111 in octarepeat deleted PrP(23-231). The structure of the Cu/Ni complexes is discussed in terms of the implications for prion protein function and disease.     

Nickel trafficking: insights into the fold and function of UreE, a urease metallochaperone

UreE is a metallo-chaperone assisting the incorporation of two adjacent Ni(2+) ions in the active site of urease. This study describes an attempt to distill general information on this protein using a computational post-genomic approach for the understanding of the structural details of the molecular function of UreE in nickel trafficking. The two crystal structures recently determined for UreE from Bacillus pasteurii (BpUreE) and Klebsiella aerogenes (KaUreE) were comparatively analyzed. This analysis provided insights into the protein structural and conformational features. A structural database of UreE proteins from a large number of different genomes was built using homology modeling. All available sequences of UreE were retrieved from protein and cDNA databases, and their structures were modeled on the crystal structures of BpUreE and KaUreE. A self-consistent iterative protocol was devised for multiple sequence alignment optimization involving secondary structure prediction and evaluation of the energy features of the obtained modeled structures. The quality of all models was tested using standard assessment procedures. The final optimized structure-based multiple alignment and the derived model structures provided insightful information on the evolutionary conservation of key residues in the protein sequence and surface patches presumably involved in protein recognition during the urease active site assembly.     

Inhibition of dextransucrase by Zn2+, Ni2+, Co2+, and Tris(hydroxymethyl)aminomethane (Tris)

Initial rate kinetics of polysaccharide formation indicate that Zn2+, Ni2+, and Co2+ inhibit dextransucrase [sucrose: 1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase, EC 2.4.1.5] by binding to two types of metal ion sites. One type consists of a single site and has a low apparent affinity for Ca2+. At the remaining site(s), Ca2+ has a much higher apparent affinity than Zn2+, Ni2+, or Co2+, and prevents inhibition by these metal ions. These findings are consistent with a two-site model previously proposed from studies with Ca2+ and EDTA. Initial rate kinetics also show that Tris is competitive with sucrose, but that, unlike Zn2+, Tris does not bind with significant affinity to a second site. This argues that there is a site which is both the sucrose binding site and a general cation site.     

Molecular characterization of Bacillus pasteurii UreE, a metal-binding chaperone for the assembly of the urease active site

The present study describes the cloning, isolation, and thorough biochemical characterization of UreE from Bacillus pasteurii, a novel protein putatively involved in the transport of Ni in the urease assembly process. A DNA fragment of the B. pasteurii urease operon, containing all four accessory genes (ureE, ureF, ureG, and ureD) required for the incorporation of Ni ions into the active site of urease, was cloned, sequenced, and analyzed. B. pasteurii ureE was cloned, and the UreE protein (BpUreE) was over-expressed and purified to homogeneity. The identity of the recombinant protein was determined by N- and C-terminal sequencing and by mass spectrometry. BpUreE has a chain length of 147 amino acids, and features a p I value of 4.7. As isolated, BpUreE contains one Zn(II) ion per dimer, while no Ni(II) is present, as shown by mass spectrometry and atomic absorption spectroscopy. BpUreE behaves as a dimer independently of the presence of Zn(II), as shown by gel filtration and mass spectrometry. Paramagnetic NMR spectroscopy on concentrated (2 mM) UreE solutions reveals a one Ni atom per tetramer stoichiometry, with the Ni(II) ion bound to histidines in an octahedral coordination environment. BpUreE has a high sequence similarity with UreE proteins isolated from different biological sources, while no sequence homology is observed with proteins belonging to different classes. In particular, BpUreE is most similar to UreE from Bacillus halodurans (55% identity). A multiple sequence alignment reveals the presence of four strictly conserved residues (Leu55, Gly97, Asn98, His100; BpUreE numbering), in addition to position 115, conservatively occupied by an Asp or a Glu residue. Several secondary structure elements, including a betaalphabetabetaalphabeta "ferredoxin-like" motif, are highly conserved throughout the UreE sequences.     

Complexes of arabinogalactan of Pereskia aculeata and Co2+, Cu2+, Mn2+, and Ni2+

The main interest in the biopolymer arabinogalactan is that it is edible. Complementing its high protein percentage, when complexed to essential metal ions, widens the use in food and pharmacology industries and technologies. The binding constants of Co2+, Cu2+, Mn2+ and Ni2+ with arabinogalactan, extracted from the leaves of Pereskia aculeata from Brazil were determined by potentiometric titrations and also the speciation according to pH values. The complexed species proposed by potentiometric titrations and the unique complexing ability of galacturonic acid groups towards Cu2+ and Ni2+ in the tridimensional web structure of arabinogalactan were confirmed by IR and EPR spectroscopies. The thermal stability of the complexed species also varied with the metal ion employed in the complexation when compared to the biopolymer alone. These complexes are new sources of additives for the food and pharmacology industries and carriers of essential metal ions to animal and vegetal biochemistry.     

Structural characterization of the nickel-binding properties of Bacillus pasteurii urease accessory protein (Ure)E in solution

Urease activation is critical to the virulence of many human and animal pathogens. Urease possesses multiple, nickel-containing active sites, and UreE, the only nickel-binding protein among the urease accessory proteins, activates urease by transporting nickel ions. We performed NMR experiments to investigate the solution structure and the nickel-binding properties of Bacillus pasteurii (Bp) UreE. The secondary structures and global folds of BpUreE were determined for its metal-free and nickel-bound forms. The results indicated that no major structural change of BpUreE arises from the nickel binding. In addition to the previously identified nickel-binding site (Gly(97)-Cys(103)), the C-terminal tail region (Lys(141)-His(147)) was confirmed for the first time to be involved in the nickel binding. The C-terminally conserved sequence ((144)GHQH(147)) was confirmed to have an inherent nickel-binding ability. Nickel addition to 1.6 mm subunit, a concentration where BpUreE predominantly forms a tetramer upon the nickel binding, induced a biphasic spectral change consistent with binding of up to at least three nickel ions per tetrameric unit. In contrast, nickel addition to 0.1 mm subunit, a concentration at which the protein is primarily a dimer, caused a monophasic spectral change consistent with more than 1 equivalent per dimeric unit. Combined with the equilibrium dialysis results, which indicated 2.5 nickel equivalents binding per dimer at a micromolar protein concentration, the nickel-binding stoichiometry of BpUreE at a physiological concentration could be three nickel ions per dimer. Altogether, the present results provide the first detailed structural data concerning the nickel-binding properties of intact, wild-type BpUreE in solution.     

Nickel-binding properties of the C-terminal tail peptide of Bacillus pasteurii UreE

Urease activation, which is critical to the virulence of many human and animal pathogens, is mediated by several accessory proteins. UreE, the only nickel-binding protein among the urease accessory proteins, catalyzes the activation of urease by transporting nickel ions into the urease active sites. The nickel-binding properties of UreE are still not clear, particularly for the protein from Bacillus pasteurii (Bp). Since the flexible C-terminal tail of BpUreE possesses two conserved histidines, the nickel-binding properties of the tail peptide were examined by circular dichroism spectroscopy, size-exclusion chromatography, and nuclear magnetic resonance spectroscopy. Specific nickel binding leading to alteration of the peptide backbone geometry was clearly observed. Side-chains of the two conserved histidines were identified as the main ligands for nickel coordination. The peptide became dimerized upon nickel binding and the binding stoichiometry was estimated as 1 equivalent of nickel per peptide dimer. Altogether, it is postulated that the C-terminal tail of BpUreE contributes to the nickel binding of the protein in different ways between the dimeric and tetrameric protein folds.     

Crystallographic and X-ray absorption spectroscopic characterization of Helicobacter pylori UreE bound to Ni²⁺ and Zn²⁺ reveals a role for the disordered C-terminal arm in metal trafficking

The survival and growth of the pathogen Helicobacter pylori in the gastric acidic environment is ensured by the activity of urease, an enzyme containing two essential Ni2? ions in the active site. The metallo-chaperone UreE facilitates in vivo Ni2? insertion into the apoenzyme. Crystals of apo-HpUreE (H. pylori UreE) and its Ni?- and Zn?-bound forms were obtained from protein solutions in the absence and presence of the metal ions. The crystal structures of the homodimeric protein, determined at 2.00 ? (apo), 1.59 ? (Ni2?) and 2.52 ? (Zn2?) resolution, show the conserved proximal and solvent-exposed His1?2 residues from two adjacent monomers invariably involved in metal binding. The C-terminal regions of the apoprotein are disordered in the crystal, but acquire significant ordering in the presence of the metal ions due to the binding of His1?2. The analysis of X-ray absorption spectral data obtained using solutions of Ni2?- and Zn2?-bound HpUreE provided accurate information of the metal-ion environment in the absence of solid-state effects. These results reveal the role of the histidine residues at the protein C-terminus in metal-ion binding, and the mutual influence of protein framework and metal-ion stereo-electronic properties in establishing co-ordination number and geometry leading to metal selectivity.     

Purification and characterization of Klebsiella aerogenes UreE protein: a nickel-binding protein that functions in urease metallocenter assembly.

The Klebsiella aerogenes ureE gene product was previously shown to facilitate assembly of the urease metallocenter (Lee, M.H., et al., 1992, J. Bacteriol. 174, 4324-4330). UreE protein has now been purified and characterized. Although it behaves as a soluble protein, UreE is predicted to possess an amphipathic beta-strand and exhibits unusually tight binding to phenyl-Sepharose resin. Immunogold electron microscopic studies confirm that UreE is a cytoplasmic protein. Each dimeric UreE molecule (M(r) = 35,000) binds 6.05 + 0.25 nickel ions (Kd of 9.6 +/- 1.3 microM) with high specificity according to equilibrium dialysis measurements. The nickel site in UreE was probed by X-ray absorption and variable-temperature magnetic circular dichroism spectroscopies. The data are most consistent with the presence of Ni(II) in pseudo-octahedral geometry with 3-5 histidyl imidazole ligands. The remaining ligands are nitrogen or oxygen donors. UreE apoprotein has been crystallized and analyzed by X-ray diffraction methods. Addition of nickel ion to apoprotein crystals leads to the development of fractures, consistent with a conformational change upon binding nickel ion. We hypothesize that UreE binds intracellular nickel ion and functions as a nickel donor during metallocenter assembly into the urease apoprotein.     

Isothermal titration calorimetry of metal ions binding to proteins: An overview of recent studies

Isothermal titration calorimetry (ITC) is a technique that is capable of quantifying the stoichiometry, equilibrium constants and thermodynamics of molecular binding events. Thus, important information about the interaction of metal ions with biological macromolecules can be obtained with ITC measurements. This review highlights many of the recent studies of metal ions binding to proteins that have used ITC to quantify the thermodynamics of metal-protein interactions.     

Structural basis for Ni(2+) transport and assembly of the urease active site by the metallochaperone UreE from Bacillus pasteurii

Bacillus pasteurii UreE (BpUreE) is a putative chaperone assisting the insertion of Ni(2+) ions in the active site of urease. The x-ray structure of the protein has been determined for two crystal forms, at 1.7 and 1.85 A resolution, using SIRAS phases derived from a Hg(2+)-derivative. BpUreE is composed of distinct N- and C-terminal domains, connected by a short flexible linker. The structure reveals the topology of an elongated homodimer, formed by interaction of the two C-terminal domains through hydrophobic interactions. A single Zn(2+) ion bound to four conserved His-100 residues, one from each monomer, connects two dimers resulting in a tetrameric BpUreE known to be formed in concentrated solutions. The Zn(2+) ion can be replaced by Ni(2+) as shown by anomalous difference maps obtained on a crystal of BpUreE soaked in a solution containing NiCl(2). A large hydrophobic patch surrounding the metal ion site is surface-exposed in the biologically relevant dimer. The BpUreE structure represents the first for this class of proteins and suggests a possible role for UreE in the urease nickel-center assembly.     

NMR study of Ni2+ binding to the H-N-H endonuclease domain of colicin E9.

Ni2+ affinity columns are widely used for protein purification, but they carry the risk that Ni2+ ions may bind to the protein, either adventitiously or at a physiologically important site. Dialysis against ethylenediaminetetraacetic acid (EDTA) is normally used to remove metal ions bound adventitiously to proteins; however, this approach does not always work. Here we report that a bacterial endonuclease, the DNase domain of colicin E9, binds Ni2+ acquired from Ni2+ affinity columns, and appears to bind [Ni(EDTA)(H2O)n]2- at low ionic strength. NMR was used to detect the presence of both Ni2+ coordinated to amino acid side chains and [Ni(EDTA)(H2O)N]2-. Dialysis against > or =0.2 M NaCl was required to remove the [Ni(EDTA)(H2O)n]2-. The NMR procedure we have used to characterize the presence of Ni2+ and [Ni(EDTA)(H2O)n]2- should be applicable to other proteins where there is the possibility of binding paramagnetic metal ions that are present to expedite protein purification. In the present case, the binding of Ni2+ seems likely to be physiologically relevant, and the NMR data complement recent X-ray crystallographic evidence concerning the number of histidine ligands to bound Ni2+.     

Stoichiometry and dynamic interaction of metal ion activators with calcineurin phosphatase

Calcineurin, a calmodulin-regulated phosphatase, is composed of two distinct subunits (A and B) and requires certain metal ions for activity. The binding of the two most potent activators, Ni2+ and Mn2+, to calcineurin and its subunits has been studied. Incubation of the protein with 63Ni2+ (or 54Mn2+) followed by gel filtration to separate free and protein-bound ions indicated that calcineurin could maximally bind 2 mol/mol of Ni2+ or Mn2+. While isolated A subunit also bound 2 mol/mol of Ni2+, no Mn2+ binding was demonstrated for either isolated A or B subunit. When bindings were monitored by nitrocellulose filter assay, only 1 mol/mol bound Ni2+ or Mn2+ was detected, suggesting that the two Ni2+ (or Mn2+) binding sites had different relative affinities and that only metal ions bound at the higher affinity sites were detected by the filter assay. Preincubation of calcineurin with Mn2+ (or Ni2+) decreased the filter assay-measured Ni2+ (or Mn2+) binding by only 30%. Preincubation of the protein with Zn2+ decreased the filter assay-measured Ni2+ or Mn2+ binding by 90 or 17%, respectively. The results suggest that the higher affinity sites are a Ni2+-specific site and a distinct Mn2+-specific site. Preincubation of calcineurin with Mn2+ (or Ni2+) decreased the gel filtration-determined Ni2+ (or Mn2+) binding from 2 to 1 mol/mol suggesting that calcineurin also contains a site which binds either metal ion. The time course of Ni2+ (or Mn2+) binding was correlated with that of the enzyme activation, and the extent of deactivation of the Ni2+-activated calcineurin by EDTA or by incubation with Ca2+ and calmodulin (Pallen, C. J., and Wang, J. H. (1984) J. Biol. Chem. 259, 6134-6141) was correlated with the release of the bound ions, thus suggesting that the bound ion is directly responsible for enzyme activation.