Distinct Metal-binding Sites Research Articles

Electron Paramagnetic Resonance, Electronic Ground State, and Electron Spin Relaxation of Seven Lanthanide Ions Bound to Lanmodulin and the Bioinspired Chelator, 3,4,3-LI(1,2-HOPO).

The electron paramagnetic resonance (EPR) spectra of lanthanide(III) ions besides Gd3+ , bound to small-molecule and protein chelators, are uncharacterized. Here, the EPR properties of 7 lanthanide(III) ions bound to the natural lanthanide-binding protein, lanmodulin (LanM), and the synthetic small-molecule chelator, 3,4,3-LI(1,2-HOPO) ("HOPO"), were systematically investigated. Echo-detected pulsed EPR spectra reveal intense signals from ions for which the normal continuous-wave first-derivative spectra are negligibly different from zero. Spectra of Kramers lanthanide ions Ce3+ , Nd3+ , Sm3+ , Er3+ , and Yb3+ , and non-Kramers Tb3+ and Tm3+ , bound to LanM are more similar to the ions in dilute aqueous:ethanol solution than to those coordinated with HOPO. Lanmodulins from two bacteria, with distinct metal-binding sites, had similar spectra for Tb3+ but different spectra for Nd3+ . Spin echo dephasing rates (1/Tm ) are faster for lanthanides than for most transition metals and limited detection of echoes to temperatures below ~6 to 12 K. Dephasing rates were environment dependent and decreased in the order water:ethanol>LanM>HOPO, which is attributed to decreasing librational motion. These results demonstrate that the EPR spectra and relaxation times of lanthanide(III) ions are sensitive to coordination environment, motivating wider application of these methods for characterization of both small-molecule and biomolecule interactions with lanthanides.

A physicochemical investigation on the metal binding properties of TtSmtB, a thermophilic member of the ArsR/SmtB transcription factor family

The transcription factors of the ArsR/SmtB family are widespread within the bacterial and archaeal kingdoms. They are transcriptional repressors able to sense a variety of metals and undergo allosteric conformational changes upon metal binding, resulting in derepression of genes involved in detoxification. So far, the molecular determinants of specificity, selectivity, and metal binding mechanism have been scarcely investigated in thermophilic microorganisms. TtSmtB, the only ArsR/SmtB member present in the genome of Thermus thermophilus HB27, was chosen as a model to shed light into such molecular mechanisms at high temperature. In the present study, using a multidisciplinary approach, a structural and functional characterization of the protein was performed focusing on its metal interaction and chemical-physical stability. Our data demonstrate that TtSmtB has two distinct metal binding sites per monomer and interacts with di-tri-penta-valent ions with different affinity. Detailed knowledge at molecular level of protein-metal interaction is remarkable to design metal binding domains as scaffolds in metal-based therapies as well as in metal biorecovery or biosensing in the environment.

Structural Insights into Divalent Cation Modulations of ATP-Gated P2X Receptor Channels.

P2X receptors are trimeric ATP-gated cation channels involved in physiological processes ranging widely from neurotransmission to pain and taste signal transduction. The modulation of the channel gating, including that by divalent cations, contributes to these diverse physiological functions of P2X receptors. Here, we report the crystal structure of an invertebrate P2X receptor from the Gulf Coast tick Amblyomma maculatum in the presence of ATP and Zn(2+) ion, together with electrophysiological and computational analyses. The structure revealed two distinct metal binding sites, M1 and M2, in the extracellular region. The M1 site, located at the trimer interface, is responsible for Zn(2+) potentiation by facilitating the structural change of the extracellular domain for pore opening. In contrast, the M2 site, coupled with the ATP binding site, might contribute to regulation by Mg(2+). Overall, our work provides structural insights into the divalent cation modulations of P2X receptors.

A DNAzyme requiring two different metal ions at two distinct sites.

Most previously reported RNA-cleaving DNAzymes require only a single divalent metal ion for catalysis. We recently reported a general trivalent lanthanide-dependent DNAzyme named Ce13d. This work shows that Ce13d requires both Na+ and a trivalent lanthanide (e.g. Ce3+), simultaneously. This discovery is facilitated by the sequence similarity between Ce13d and a recently reported Na+-specific DNAzyme, NaA43. The Ce13d cleavage rate linearly depends on the concentration of both metal ions. Sensitized Tb3+ luminescence and DMS footprinting experiments indicate that the guanines in the enzyme loop are important for Na+-binding. The Na+ dissociation constants of Ce13d measured from the cleavage activity assay, Tb3+ luminescence and DMS footprinting are 24.6, 16.3 and 47 mM, respectively. Mutation studies indicate that the role of Ce3+ might be replaced by G23 in NaA43. Ce3+ functions by stabilizing the transition state phosphorane, thus promoting cleavage. G23 competes favorably with low concentration Ce3+ (below 1 μM). The G23-to-hypoxanthine mutation suggests the N1 position of the guanine as a hydrogen bond donor. Together, Ce13d has two distinct metal binding sites, each fulfilling a different role. DNAzymes can be quite sophisticated in utilizing metal ions for catalysis and molecular recognition, similar to protein metalloenzymes.

Identification of Minimal HDV-Like Ribozymes with Unique Divalent Metal Ion Dependence in the Human Microbiome

HDV-like self-cleaving ribozymes have been found in a wide variety of organisms, implicated in diverse biological processes, and their activity typically shows a strong divalent metal dependence, but little metal specificity. Recent studies suggested that very short variants of these ribozymes exist in nature, but their distribution and biochemical properties have not been established. To map out the distribution of small HDV-like ribozymes, the drz-Spur-3 sequence was minimized to yield a core construct for structure-based bioinformatic searches. These searches revealed several microbial ribozymes, particularly in the human microbiome. Kinetic profile of the smallest ribozyme revealed two distinct metal binding sites, only one of which promotes fast catalysis. Furthermore, this ribozyme showed markedly reduced activity in Ca(2+), even in the presence of physiological Mg(2+) concentrations. Our study substantially expands the number of microbial HDV-like ribozymes and provides an example of cleavage regulation by divalent metals.

Mixed-Valent Dicobalt and Iron–Cobalt Complexes with High-Spin Configurations and Short Metal–Metal Bonds

Cobalt-cobalt and iron-cobalt bonds are investigated in coordination complexes with formally mixed-valent [M2](3+) cores. The trigonal dicobalt tris(diphenylformamidinate) compound, Co2(DPhF)3, which was previously reported by Cotton, Murillo, and co-workers (Inorg. Chim. Acta 1996, 249, 9), is shown to have an energetically isolated, high-spin sextet ground-state by magnetic susceptibility and electron paramagnetic resonance (EPR) spectroscopy. A new tris(amidinato)amine ligand platform is introduced. By tethering three amidinate donors to an apical amine, this platform offers two distinct metal-binding sites. Using the phenyl-substituted variant (abbreviated as L(Ph)), the isolation of a dicobalt homobimetallic and an iron-cobalt heterobimetallic are demonstrated. The new [Co2](3+) and [FeCo](3+) cores have high-spin sextet and septet ground states, respectively. Their solid-state structures reveal short metal-metal bond distances of 2.29 Å for Co-Co and 2.18 Å for Fe-Co; the latter is the shortest distance for an iron-cobalt bond to date. To assign the positions of iron and cobalt atoms as well as to determine if Fe/Co mixing is occurring, X-ray anomalous scattering experiments were performed, spanning the Fe and Co absorption energies. These studies show only a minor amount of metal-site mixing in this complex, and that FeCoL(Ph) is more precisely described as (Fe0.94(1)Co0.06(1))(Co0.95(1)Fe0.05(1))L(Ph). The iron-cobalt heterobimetallic has been further characterized by Mössbauer spectroscopy. Its isomer shift of 0.65 mm/s and quadrupole splitting of 0.64 mm/s are comparable to the related diiron complex, Fe2(DPhF)3. On the basis of spectroscopic data and theoretical calculations, it is proposed that the formal [M2](3+) cores are fully delocalized.

Crystal Structure of the Zinc-Dependent MarR Family Transcriptional Regulator AdcR in the Zn(II)-Bound State

Streptococcus pneumoniae adhesin competence regulator (AdcR), the first metal-dependent member of the multiple antibiotic resistance regulator (MarR) family of proteins, represses the transcription of a high-affinity zinc-specific uptake transporter, a group of surface antigen zinc-binding pneumococcal histidine triad proteins (PhtA, PhtB, PhtD, and PhtE), and an AdcA homologue (AdcAII). The 2.0 Å resolution structure of Zn(II)-bound AdcR reveals a highly helical two-fold-symmetric dimer with two distinct metal-binding sites per protomer. Zn(II) is tetrahedrally coordinated by E24, H42, H108, and H112 in what defines the primary sensing site in AdcR. Site 2 is a tetracoordinate site whose function is currently unknown. NMR methyl group perturbation experiments reveal that Zn(II) drives a global change in the structure of apo-AdcR that stabilizes a conformation that is compatible with DNA binding. This co-repression mechanism is unprecedented in MarR transcriptional regulators.

Structure of a Nickel Chaperone, HypA, from Helicobacter pylori Reveals Two Distinct Metal Binding Sites

Metallochaperones bind metals and ensure the safe delivery of metals to the targets. They are required for the activation and maturation of nickel-containing enzymes [Ni,Fe]-hydrogenase and urease. Metallochaperone HypA was found to be essential to facilitate nickel delivery to hydrogenase together with its partner HypB, although the detailed mechanism is not clear. In this study, we have cloned hypA gene from Helicobacter pylori (strain 26695), overexpressed, and purified the protein. The zinc-bound HypA (Zn-HypA) exists as a monomer in solution, and its solution structure was determined by NMR spectroscopy together with molecular dynamics simulated annealing. Zn-HypA folds into two domains, including a zinc domain and a nickel domain with a mixed alpha/beta structure. The former houses a rigid zinc-binding site possibly with the role of structural stabilization, whereas the latter harbors a nickel-binding site at the N-terminus. Zinc binds to the four conserved cysteines tetrahedrally as evidenced by (113)Cd NMR spectroscopy, and nickel coordinates with four nitrogens of the protein probably in a square-planar geometry. Low coordination number of Ni(2+) may allow the metal to be readily transferred to its downstream receptors. Our studies may shed light on how the metallochaperone exerts its functions in intracellular nickel delivery.

Three-dimensional structure of the human copper transporter hCTR1

Copper uptake proteins (CTRs), mediate cellular acquisition of the essential metal copper in all eukaryotes. Here, we report the structure of the human CTR1 protein solved by electron crystallography to an in plane resolution of 7 A. Reminiscent of the design of traditional ion channels, trimeric hCTR1 creates a pore that stretches across the membrane bilayer at the interface between the subunits. Assignment of the helices identifies the second transmembrane helix as the key element lining the pore, and reveals how functionally important residues on this helix could participate in Cu(I)-coordination during transport. Aligned with and sealing both ends of the pore, extracellular and intracellular domains of hCTR1 appear to provide additional metal binding sites. Consistent with the existence of distinct metal binding sites, we demonstrate that hCTR1 stably binds 2 Cu(I)-ions through 3-coordinate Cu-S bonds, and that mutations in one of these putative binding sites results in a change of coordination chemistry.

Translocation of copper within the cavity of cryptands: reversible fluorescence signaling

The movement of a copper ion inside the cavity of cryptands having two distinct metal binding sites at each end is linked to a fluorescence ON-OFF process in a reversible manner, providing a new way of fluorescence switching.

Organization and Assembly of Metal-Thiolate Clusters in Epithelium-specific Metallothionein-4

Mammalian metallothionein-4 (MT-4) was found to be specifically expressed in stratified squamous epithelia where it plays an essential but poorly defined role in regulating zinc or copper metabolism. Here we report on the organization, stability, and the pathway of metal-thiolate cluster assembly in MT-4 reconstituted with Cd(2+) and Co(2+) ions. Both the (113)Cd NMR studies of (113)Cd(7)MT-4 and the spectroscopic characterization of Co(7)MT-4 showed that, similar to the classical MT-1 and MT-2 proteins, metal ions are organized in two independent Cd(4)Cys(11) and Cd(3)Cys(9) clusters with each metal ion tetrahedrally coordinated by terminal and bridging cysteine ligands. Moreover, we have demonstrated that the cluster formation in Cd(7)MT-4 is cooperative and sequential, with the Cd(4)Cys(11) cluster being formed first, and that a distinct single-metal nucleation intermediate Cd(1)MT-4 is required in the cluster formation process. Conversely, the absorption and circular dichroism features of metal-thiolate clusters in Cd(7)MT-4 indicate that marked differences in the cluster geometry exist when compared with those in Cd(7)MT-1/2. The biological implication of our studies as to the role of MT-4 in zinc metabolism of stratified epithelia is discussed.

Sequence of Ligand Binding and Structure Change in the Diphtheria Toxin Repressor upon Activation by Divalent Transition Metals

The diphtheria toxin repressor (DtxR) is an Fe(II)-activated transcriptional regulator of iron homeostatic and virulence genes in Corynebacterium diphtheriae. DtxR is a two-domain protein that contains two structurally and functionally distinct metal binding sites. Here, we investigate the molecular steps associated with activation by Ni(II)Cl(2) and Cd(II)Cl(2). Equilibrium binding energetics for Ni(II) were obtained from isothermal titration calorimetry, indicating apparent metal dissociation constants of 0.2 and 1.7 microM for two independent sites. The binding isotherms for Ni(II) and Cd(II) exhibited a characteristic exothermic-endothermic pattern that was used to infer the metal binding sequence by comparing the wild-type isotherm with those of several binding site mutants. These data were complemented by measuring the distance between specific backbone amide nitrogens and the first equivalent of metal through heteronuclear NMR relaxation measurements. Previous studies indicated that metal binding affects a disordered to ordered transition in the metal binding domain. The coupling between metal binding and structure change was investigated using near-UV circular dichroism spectroscopy. Together, the data show that the first equivalent of metal is bound by the primary metal binding site. This binding orients the DNA binding helices and begins to fold the N-terminal domain. Subsequent binding at the ancillary site completes the folding of this domain and formation of the dimer interface. This model is used to explain the behavior of several mutants.

Heavy metals modulate the activity of the purinergic P2X 4 receptor

To further characterize the nature of the regulatory metal-binding sites of the rat P2X 4 receptor, several transition heavy metals were tested to examine their ability to mimic the facilitator action of zinc or the inhibitory action of copper. cDNA coding for the rat P2X 4 receptor was injected into Xenopus laevis oocytes; the two-electrode voltage-clamp technique was used to measure and quantify the ATP-evoked currents in the absence or presence of the metals. Cadmium facilitated the ATP-gated currents in a reversible and voltage-independent manner; maximal potentiation occurred within less than 1 min. Cadmium displaced leftward, in a concentration-dependent manner, the ATP concentration–response curve. In contrast, mercury reduced the ATP-gated currents in a reversible, time, and concentration manner. Maximal inhibition occurred after about 5 min of metal application. Cobalt also augmented the ATP-evoked currents, but its action was long lasting and did not reverse even after 45 min of metal washout. Other metals such as lead, nickel, manganese, silver, or gallium did not significantly alter the ATP-gated currents. The co-application of cadmium plus zinc or mercury plus copper caused additive effects. Mutation of H140 by alanine (H140A) augmented both the cadmium-induced facilitation and the mercury-induced inhibition. In contrast, the H241A mutant showed characteristics indistinguishable from the wild type. The H286A mutant showed a normal cadmium-induced potentiation, but an increased mercury inhibition. Out of the metals examined, only cadmium mimicked closely the action of zinc, evidencing commonalities. While mercury mimicked the action of copper, both metals apparently interact at distinct metal-binding sites. The present findings allow us to infer that heavy metals modulate the P2X 4 receptor by acting in at least three separate metal-binding sites.

Elucidation of Primary (α 3N) and Vestigial (α 5) Heavy Metal-binding Sites in Staphylococcus aureus pI258 CadC: Evolutionary Implications for Metal Ion Selectivity of ArsR/SmtB Metal Sensor Proteins

Despite a common evolutionary origin, individual members of the ArsR/SmtB family of bacterial metal-responsive transcriptional repressors sense a wide range of heavy-metal ions. The molecular basis for this metal ion selectivity is unclear. Here, we establish that Staphylococcus aureus plasmid pI258 CadC, a Cd(II)/Pb(II)/Bi(III)/Zn(II) sensor, contains two distinct metal-binding sites: a thiolate-rich α 3N site comprised exclusively of cysteine ligands that preferentially binds larger, softer metal ions such as Cd(II), Pb(II) and Bi(III); and a second C-terminal α 5 site, found at the dimer interface, that is devoid of cysteine ligands and preferentially binds smaller, harder metal ions [Co(II) and Zn(II)] concurrently with metal binding to the α 3N site. Optical absorption and X-ray spectroscopies reveal that the α 3N site can adopt distinct coordination geometries in order to accommodate different metal ions, i.e. Cd(II), Bi(III), Co(II) and Zn(II) form distorted tetrahedral S 4 complexes, while Pb(II) adopts a trigonal S 3 complex. Characterization of mutant CadCs reveals that the α 3N site is composed of Cys58 and Cys60 from the α 3 helix of the helix-turn-helix DNA-binding domain and Cys7 and/or Cys11 from the N-terminal “arm” of CadC; Cys11 is excluded from the Pb(II) coordination sphere. Only the thiolate-rich α 3N site is metalloregulatory for repressor binding to a fluorescein-labeled cad O/P oligonucleotide upon coordination to Cd(II), Pb(II), Bi(III), Zn(II), and weakly for Co(II). Substitution of Cys60 and Cys7 with non-ligating residues specifically abrogates metal-dependent negative regulation of cad O/P binding, despite the fact that C60G and C7G CadCs maintain high affinity for metals in altered coordination complexes. These findings reveal that formation of metal coordination bonds to Cys7 and Cys60 play primary roles in transducing the allosteric response in CadC. The evolutionary implications for metal ion selectivity of ArsR/SmtB metal sensor proteins are discussed.

Structures of three diphtheria toxin repressor (DtxR) variants with decreased repressor activity.

The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae regulates the expression of the gene on corynebacteriophages that encodes diphtheria toxin (DT). Other genes regulated by DtxR include those that encode proteins involved in siderophore-mediated iron uptake. DtxR requires activation by divalent metals and holo-DtxR is a dimeric regulator with two distinct metal-binding sites per three-domain monomer. At site 1, three side chains and a sulfate or phosphate anion are involved in metal coordination. In the DtxR-DNA complex this anion is replaced by the side chain of Glu170 provided by the third domain of the repressor. At site 2 the metal ion is coordinated exclusively by constituents of the polypeptide chain. In this paper, five crystal structures of three DtxR variants focusing on residues Glu20, Arg80 and Cys102 are reported. The resolution of these structures ranges from 2.3 to 2.8 A. The side chain of Glu20 provided by the DNA-binding domain forms a salt bridge to Arg80, which in turn interacts with the anion. Replacing either of the salt-bridge partners with an alanine reduces repressor activity substantially and it has been inferred that the salt bridge could possibly control the wedge angle between the DNA-binding domain and the dimerization domain, thereby modulating repressor activity. Cys102 is a key residue of metal site 2 and its substitution into a serine abolishes repressor activity. The crystal structures of Zn-Glu20Ala-DtxR, Zn-Arg80Ala-DtxR, Cd-Cys102Ser-DtxR and apo-Cys102Ser-DtxR in two related space groups reveal that none of these substitutions leads to dramatic rearrangements of the DtxR fold. However, the five crystal structures presented here show significant local changes and a considerable degree of flexibility of the DNA-binding domain with respect to the dimerization domain. Furthermore, all five structures deviate significantly from the structure in the DtxR-DNA complex with respect to overall domain orientation. These results confirm the importance of the hinge motion for repressor activity. Since the third domain has often been invisible in previous crystal structures of DtxR, it is also noteworthy that the SH3-like domain could be traced in four of the five crystal structures.

Electron paramagnetic resonance studies of cobalt-substituted angiotensin I-converting enzyme

Electron paramagnetic resonance (EPR) spectroscopy has been used to study the metal coordination sphere geometry in the cobalt-substituted Zn-protein angiotensin I-converting enzyme (ACE). It has been shown that ACE contains two distinct metal-binding sites. In the presence of the two structurally different inhibitors, captopril and ramiprilat, it is found that the metal binding sites are nearly structurally identical and are separated more than 10 Å from each other. The metal atoms are most likely four- to five-coordinated, and it is argued that the inhibitor binds directly to the metal ion.

Oligopolydentate ligands for helices and metallacycles

Abstract

Investigation of the nature of the two metal-binding sites in 5-aminolaevulinic acid dehydratase from Escherichia coli.

Two distinct metal-binding sites, termed alpha and beta, have been characterized in 5-aminolaevulinic acid dehydratase from Escherichia coli. The alpha-site binds a Zn2+ ion that is essential for catalytic activity. This site can also utilize other metal ions able to function as a Lewis acid in the reaction mechanism, such as Mg2+ or Co2+. The beta-site is exclusively a transition-metal-ion-binding site thought to be involved in protein conformation, although a metal bound at this site only appears to be essential for activity if Mg2+ is to be bound at the alpha-site. The alpha- and beta-sites may be distinguished from one another by their different abilities to bind divalent-metal ions at different pH values. The occupancy of the beta-site with Zn2+ results in a decrease of protein fluorescence at pH 6. Occupancy of the alpha- and beta-sites with Co2+ results in u.v.-visible spectral changes. Spectroscopic studies with Co2+ have tentatively identified three cysteine residues at the beta-site and one at the alpha-site. Reaction with N-ethyl[14C]maleimide preferentially labels cysteine-130 at the alpha-site when Co2+ occupies the beta-site.

TDPAC studies of111mCd in calciproteins: Calmodulin and parvalbumin

TDPAC measurements of the 150–247 keV gamma-ray cascade in111Cd have been performed at room temperature on Calmodulin (CaM) and Parvalbumin (Pa) labelled with radioactive111mCd. The anisotropy of the coincidence counting rate shows a time-dependent behaviour, typical of two different nuclear quadrupole interactions (NQI) in CaM. The NQI parameters indicate the presence of two distinct metal-binding sites, presenting non-equivalent local electric charge structure. In Pa samples, only one NQI is observed and the measured electric field gradient (EFG) value agrees well with the large one obtained in CaM.

Activation of glucose isomerase by divalent cations: evidence for two distinct metal-binding sites

Activity and metal-content measurements have provided evidence for two distinct metal-binding sites in glucose isomerase from Bacillus coagulans. Although each site binds either Mn 2+ or CO 2+, simultaneous addition of Mn 2+ and Co 2+ to the apoenzyme indicated that one site prefers Mn 2+ (site 1) and the other Co 2+ (site 2). Enzyme activity toward fructose was highest when both sites were filled with Co 2+, whereas the activity toward xylose was highest when site 1 was occupied by Mn 2+. The presence of metal in site 2 did not affect the activity toward xylose. Electron paramagnetic resonance (EPR) spectra of Mn 2+ and Co 2+ tightly bound to the enzyme indicated an asymmetric ligand field, whereas the spectrum of each metal more loosely bound was consistent with a symmetric ligand field. In addition, isomerase activity was reduced by oxidizing Co 2+ to Co 3+ with H 2O 2.