The metal-binding sites of the zinc-transporting P-type ATPase of Escherichia coli. Lys and Asp in the seventh and eighth transmembrane segments of ZntA contribute to the coupling of metal binding and ATPase activity

ZntA is a P-type ATPase which transports Zn²⁺, Pb²⁺ and Cd²⁺ out of the cell. Two cysteine-containing motifs, CAAC near the N-terminus and CPC in transmembrane helix 6, are involved in binding of the translocated metal. We have studied these motifs by mutating the cysteines to serines. The roles of two other possible metal-binding residues, K⁶⁹³ and D⁷¹⁴, in transmembrane helices 7 and 8, were also addressed. The mutation CAAC → SAAS reduces the ATPase activity by 50%. The SAAS mutant is phosphorylated with ATP almost as efficiently as the wild type. However, its phosphorylation with P_i is poorer than that of the wild type and its dephosphorylation rate is faster than that of the wild type ATPase. The CPC → SPS mutant is inactive but residual phosphorylation with ATP could still be observed. The most important findings of this work deal with the prospective metal-binding residues K⁶⁹³ and D⁷¹⁴: the substitution K693N eliminates the Zn²⁺-stimulated ATPase activity completely, although significant Zn²⁺-dependent phosphorylation by ATP remains. The K693N ATPase is hyperphosphorylated by P_i. ZntA carrying the change D714M has strong metal-independent ATPase activity and is very weakly phosphorylated both by ATP and P_i. In conclusion, K⁶⁹³ and D⁷¹⁴ are functionally essential and appear to contribute to the metal specificity of ZntA, most probably by being parts of the metal-binding site made up by the CPC motif.     

Novel metal-binding site of Pseudomonas reinekei MT1 trans-dienelactone hydrolase

Pseudomonasreinekei MT1 is capable of growing on 4- and 5-chlorosalicylate as the sole carbon source involving a pathway with trans-dienelactone hydrolase as the key enzyme. This enzyme transforms 4-chloromuconolactone to maleylacetate and thereby avoids the spontaneous formation of toxic protoanemonin. trans-Dienelactone hydrolase is a Zn²⁺-dependent hydrolase where activity can be modulated by the exchange of Zn²⁺ by Mn²⁺ in at least two of the three metal-binding sites. Site directed variants of conserved residues of the Q₁₀₁XXXQ₁₀₅XD₁₀₇XXXH₁₁₁ motif and of H281 and E294 exhibit a two order of magnitude decrease in activity and a strong decrease in metal-binding capability. As none of the variants exhibited a change in secondary structure, the analyzed amino acid residues can be assumed to be involved in metal binding, forming a novel trinuclear metal-binding motif.     

Cryo-EM structure of a eukaryotic zinc transporter at a low pH suggests its Zn2+-releasing mechanism

Zinc transporter 8 (ZnT8) is mainly expressed in pancreatic islet β cells and is responsible for H⁺-coupled uptake (antiport) of Zn²⁺ into the lumen of insulin secretory granules. Structures of human ZnT8 and its prokaryotic homolog YiiP have provided structural basis for constructing a plausible transport cycle for Zn²⁺. However, the mechanistic role that protons play in the transport process remains unclear. Here we present a lumen-facing cryo-EM structure of ZnT8 from Xenopus tropicalis (xtZnT8) in the presence of Zn²⁺ at a luminal pH (5.5). Compared to a Zn²⁺-bound xtZnT8 structure at a cytosolic pH (7.5), the low-pH structure displays an empty transmembrane Zn²⁺-binding site with a disrupted coordination geometry. Combined with a Zn²⁺-binding assay our data suggest that protons may disrupt Zn²⁺ coordination at the transmembrane Zn²⁺-binding site in the lumen-facing state, thus facilitating Zn²⁺ release from ZnT8 into the lumen.     

New Insights into Histidine Triad Proteins: Solution Structure of a Streptococcus pneumoniae PhtD Domain and Zinc Transfer to AdcAII

Zinc (Zn²⁺) homeostasis is critical for pathogen host colonization and invasion. Polyhistidine triad (Pht) proteins, located at the surface of various streptococci, have been proposed to be involved in Zn²⁺ homeostasis. The phtD gene, coding for a Zn²⁺-binding protein, is organized in an operon with adcAII coding for the extracellular part of a Zn²⁺ transporter. In the present work, we investigate the relationship between PhtD and AdcAII using biochemical and structural biology approaches. Immuno-precipitation experiments on purified membranes of Streptococcus pneumoniae (S. pneumoniae) demonstrate that native PhtD and AdcAII interact in vivo confirming our previous in vitro observations. NMR was used to demonstrate Zn²⁺ transfer from the Zn²⁺-bound form of a 137 amino acid N-terminal domain of PhtD (t-PhtD) to AdcAII. The high resolution NMR structure of t-PhtD shows that Zn²⁺ is bound in a tetrahedral site by histidines 83, 86, and 88 as well as by glutamate 63. Comparison of the NMR parameters measured for apo- and Zn²⁺-t-PhtD shows that the loss of Zn²⁺ leads to a diminished helical propensity at the C-terminus and increases the local dynamics and overall molecular volume. Structural comparison with the crystal structure of a 55-long fragment of PhtA suggests that Pht proteins are built from short repetitive units formed by three β-strands containing the conserved HxxHxH motif. Taken together, these results support a role for S. pneumoniae PhtD as a Zn²⁺ scavenger for later release to the surface transporter AdcAII, leading to Zn²⁺ uptake.     

Divalent metal ion complexes of S100B in the absence and presence of pentamidine

As part of an effort to inhibit S100B, structures of pentamidine (Pnt) bound to Ca²⁺-loaded and Zn²⁺,Ca²⁺-loaded S100B were determined by X-ray crystallography at 2.15 Å (R_free = 0.266) and 1.85 Å (R_free = 0.243) resolution, respectively. These data were compared to X-ray structures solved in the absence of Pnt, including Ca²⁺-loaded S100B and Zn²⁺,Ca²⁺-loaded S100B determined here (1.88 Å; R_free = 0.267). In the presence and absence of Zn²⁺, electron density corresponding to two Pnt molecules per S100B subunit was mapped for both drug-bound structures. One Pnt binding site (site 1) was adjacent to a p53 peptide binding site on S100B (± Zn²⁺), and the second Pnt molecule was mapped to the dimer interface (site 2; ± Zn²⁺) and in a pocket near residues that define the Zn²⁺ binding site on S100B. In addition, a conformational change in S100B was observed upon the addition of Zn²⁺ to Ca²⁺-S100B, which changed the conformation and orientation of Pnt bound to sites 1 and 2 of Pnt-Zn²⁺,Ca²⁺-S100B when compared to Pnt-Ca²⁺-S100B. That Pnt can adapt to this Zn²⁺-dependent conformational change was unexpected and provides a new mode for S100B inhibition by this drug. These data will be useful for developing novel inhibitors of both Ca²⁺- and Ca²⁺,Zn²⁺-bound S100B.     

Crystal structure of the YdjC-family protein TTHB029 from Thermus thermophilus HB8: structural relationship with peptidoglycan N-acetylglucosamine deacetylase

The YdjC-family protein is widely distributed, from human to bacteria, but so far no three-dimensional structure and functional analysis of this family of proteins has been reported. We determined the three-dimensional structure of the YdjC homolog TTHB029 at a resolution of 2.9 Å. The overall structure of the monomer consists of (βα)-barrel fold forming a homodimer. Asp21, His60, and His127 residues coordinate to Mg²⁺ as a possible active site. TTHB029 shows structural similarity to the peptidoglycan N-acetylglucosamine deacetylase from Streptococcus pneumoniae (SpPgdA). The active site groove of SpPgdA includes the Zn²⁺ coordinated to Asp276, His326, and His330. Despite the low sequence identity, metal-binding residues of Asp-His-His were conserved among the two enzymes. There were definitive differences, however, in that one of the histidines of the metal-binding site was substituted for the other histidine located on the other loop. Moreover, these important metal-binding residues and the residues of the presumed active site are fully conserved in YdjC-family protein.     

Structural and kinetic bases for the metal preference of the M18 aminopeptidase from Pseudomonas aeruginosa

The peptidases in clan MH are known as cocatalytic zinc peptidases that have two zinc ions in the active site, but their metal preference has not been rigorously investigated. In this study, the molecular basis for metal preference is provided from the structural and biochemical analyses. Kinetic studies of Pseudomonas aeruginosa aspartyl aminopeptidase (PaAP) which belongs to peptidase family M18 in clan MH revealed that its peptidase activity is dependent on Co²⁺ rather than Zn²⁺: the k_cat (s⁻¹) values of PaAP were 0.006, 5.10 and 0.43 in no-metal, Co²⁺, and Zn²⁺ conditions, respectively. Consistently, addition of low concentrations of Co²⁺ to PaAP previously saturated with Zn²⁺ greatly enhanced the enzymatic activity, suggesting that Co²⁺ may be the physiologically relevant cocatalytic metal ion of PaAP. The crystal structures of PaAP complexes with Co²⁺ or Zn²⁺ commonly showed two metal ions in the active site coordinated with three conserved residues and a bicarbonate ion in a tetragonal geometry. However, Co²⁺- and Zn²⁺-bound structures showed no noticeable alterations relevant to differential effects of metal species, except the relative orientation of Glu-265, a general base in the active site. The characterization of mutant PaAP revealed that the first metal binding site is primarily responsible for metal preference. Similar to PaAP, Streptococcus pneumonia glutamyl aminopeptidase (SpGP), belonging to aminopeptidase family M42 in clan MH, also showed requirement for Co²⁺ for maximum activity. These results proposed that clan MH peptidases might be a cocatalytic cobalt peptidase rather than a zinc-dependent peptidase.     

A tetrahedral coordination of Zinc during transmembrane transport by P-type Zn2+-ATPases

Zn²⁺ is an essential transition metal required in trace amounts by all living organisms. However, metal excess is cytotoxic and leads to cell damage. Cells rely on transmembrane transporters, with the assistance of other proteins, to establish and maintain Zn²⁺ homeostasis. Metal coordination during transport is key to specific transport and unidirectional translocation without the backward release of free metal. The coordination details of Zn²⁺ at the transmembrane metal binding site responsible for transport have now been established. Escherichia coli ZntA is a well-characterized Zn²⁺-ATPase responsible for intracellular Zn²⁺ efflux. A truncated form of the protein lacking regulatory metal sites and retaining the transport site was constructed. Metrical parameters of the metal–ligand coordination geometry for the zinc bound isolated form were characterized using x-ray absorption spectroscopy (XAS). Our data support a nearest neighbor ligand environment of (O/N)₂S₂ that is compatible with the proposed invariant metal coordinating residues present in the transmembrane region. This ligand identification and the calculated bond lengths support a tetrahedral coordination geometry for Zn²⁺ bound to the TM-MBS of P-type ATPase transporters.     

Functional Significance of Calcium Binding to Tissue-Nonspecific Alkaline Phosphatase

The conserved active site of alkaline phosphatases (AP) contains catalytically important Zn²⁺ (M1 and M2) and Mg²⁺-sites (M3) and a fourth peripheral Ca²⁺ site (M4) of unknown significance. We have studied Ca²⁺ binding to M1-4 of tissue-nonspecific AP (TNAP), an enzyme crucial for skeletal mineralization, using recombinant TNAP and a series of M4 mutants. Ca²⁺ could substitute for Mg²⁺ at M3, with maximal activity for Ca²⁺/Zn²⁺-TNAP around 40% that of Mg²⁺/Zn²⁺-TNAP at pH 9.8 and 7.4. At pH 7.4, allosteric TNAP-activation at M3 by Ca²⁺ occurred faster than by Mg²⁺. Several TNAP M4 mutations eradicated TNAP activity, while others mildly influenced the affinity of Ca²⁺ and Mg²⁺ for M3 similarly, excluding a catalytic role for Ca²⁺ in the TNAP M4 site. At pH 9.8, Ca²⁺ competed with soluble Zn²⁺ for binding to M1 and M2 up to 1 mM and at higher concentrations, it even displaced M1- and M2-bound Zn²⁺, forming Ca²⁺/Ca²⁺-TNAP with a catalytic activity only 4–6% that of Mg²⁺/Zn²⁺-TNAP. At pH 7.4, competition with Zn²⁺ and its displacement from M1 and M2 required >10-fold higher Ca²⁺ concentrations, to generate weakly active Ca²⁺/Ca²⁺-TNAP. Thus, in a Ca²⁺-rich environment, such as during skeletal mineralization at pH 7.4, Ca²⁺ adequately activates Zn²⁺-TNAP at M3, but very high Ca²⁺ concentrations compete with available Zn²⁺ for binding to M1 and M2 and ultimately displace Zn²⁺ from the active site, virtually inactivating TNAP. Those ALPL mutations that substitute critical TNAP amino acids involved in coordinating Ca²⁺ to M4 cause hypophosphatasia because of their 3D-structural impact, but M4-bound Ca²⁺ is catalytically inactive. In conclusion, during skeletal mineralization, the building Ca²⁺ gradient first activates TNAP, but gradually inactivates it at high Ca²⁺ concentrations, toward completion of mineralization.     

Helicase binding to DnaI exposes a cryptic DNA-binding site during helicase loading in Bacillus subtilis

The Bacillus subtilis DnaI, DnaB and DnaD proteins load the replicative ring helicase DnaC onto DNA during priming of DNA replication. Here we show that DnaI consists of a C-terminal domain (Cd) with ATPase and DNA-binding activities and an N-terminal domain (Nd) that interacts with the replicative ring helicase. A Zn²⁺-binding module mediates the interaction with the helicase and C67, C70 and H84 are involved in the coordination of the Zn²⁺. DnaI binds ATP and exhibits ATPase activity that is not stimulated by ssDNA, because the DNA-binding site on Cd is masked by Nd. The ATPase activity resides on the Cd domain and when detached from the Nd domain, it becomes sensitive to stimulation by ssDNA because its cryptic DNA-binding site is exposed. Therefore, Nd acts as a molecular ‘switch’ regulating access to the ssDNA binding site on Cd, in response to binding of the helicase. DnaI is sufficient to load the replicative helicase from a complex with six DnaI molecules, so there is no requirement for a dual helicase loader system.     

A Zinc Site in the C-Terminal Domain of RAG1 Is Essential for DNA Cleavage Activity

The recombination-activating protein, RAG1, a key component of the V(D)J recombinase, binds multiple Zn²⁺ ions in its catalytically required core region. However, the role of zinc in the DNA cleavage activity of RAG1 is not well resolved. To address this issue, we determined the stoichiometry of Zn²⁺ ions bound to the catalytically active core region of RAG1 under various conditions. Using metal quantitation methods, we determined that core RAG1 can bind up to four Zn²⁺ ions. Stripping the full complement of bound Zn²⁺ ions to produce apoprotein abrogated DNA cleavage activity. Moreover, even partial removal of zinc-binding equivalents resulted in a significant diminishment of DNA cleavage activity, as compared to holo-Zn²⁺ core RAG1. Mutants of the intact core RAG1 and the isolated core RAG1 domains were studied to identify the location of zinc-binding sites. Significantly, the C-terminal domain in core RAG1 binds at least two Zn²⁺ ions, with one zinc-binding site containing C902 and C907 as ligands (termed the CC zinc site) and H937 and H942 coordinating a Zn²⁺ ion in a separate site (HH zinc site). The latter zinc-binding site is essential for DNA cleavage activity, given that the H937A and H942A mutants were defective in both in vitro DNA cleavage assays and cellular recombination assays. Furthermore, as mutation of the active-site residue E962 reduces Zn²⁺ coordination, we propose that the HH zinc site is located in close proximity to the DDE active site. Overall, these results demonstrate that Zn²⁺ serves an important auxiliary role for RAG1 DNA cleavage activity. Furthermore, we propose that one of the zinc-binding sites is linked to the active site of core RAG1 directly or indirectly by E962.     

Crystal structure of glycoside hydrolase family 127 β-l-arabinofuranosidase from Bifidobacterium longum

Enzymes acting on β-linked arabinofuranosides have been unknown until recently, in spite of wide distribution of β-l-arabinofuranosyl oligosaccharides in plant cells. Recently, a β-l-arabinofuranosidase from the glycoside hydrolase family 127 (HypBA1) was discovered in the newly characterized degradation system of hydroxyproline-linked β-l-arabinooligosaccharides in the bacterium Bifidobacterium longum. Here, we report the crystal structure of HypBA1 in the ligand-free and β-l-arabinofuranose complex forms. The structure of HypBA1 consists of a catalytic barrel domain and two additional β-sandwich domains, with one β-sandwich domain involved in the formation of a dimer. Interestingly, there is an unprecedented metal-binding motif with Zn²⁺ coordinated by glutamate and three cysteines in the active site. The glutamate residue is located far from the anomeric carbon of the β-l-arabinofuranose ligand, but one cysteine residue is appropriately located for nucleophilic attack for glycosidic bond cleavage. The residues around the active site are highly conserved among GH127 members. Based on biochemical experiments and quantum mechanical calculations, a possible reaction mechanism involving cysteine as the nucleophile is proposed.     

Dual role of Zn in maintaining structural integrity and suppressing deacetylase activity of SIRT1

Zn²⁺ directly participates in catalysis of histone deacetylase (HDAC) Classes I, II, IV enzymes while its role in HDAC Class III activity is not well established. Herein we investigated the effects of Zn²⁺ on the deacetylase activity of sirtuin 1 (silent mating type information regulation 2 homolog 1, SIRT1). We found that the inherent Zn²⁺ at the zinc-finger motif of SIRT1 is essential for the structural integrity and the deacetylase activity of SIRT1, whereas the exogenous Zn²⁺ strongly inhibits the deacetylase activity with an IC₅₀ of 0.82 μM for Zn(Gly)₂. SIRT1 activity suppressed by the exogenous Zn²⁺ can be fully recovered by the metal chelator EDTA but not by the activator resveratrol. We also identified Zn²⁺ as a noncompetitive inhibitor for the substrates of NAD⁺ and the acetyl peptide P53-AMC. The 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence titration experiments and site-directed mutagenesis study suggested that the exogenous Zn²⁺ binds to SIRT1 but not at the zinc-finger motif. These results indicate that Zn²⁺ plays a dual role in SIRT1 activity. Inherent Zn²⁺ at the zinc-finger motif is structurally related and essential for SIRT1 activity. On the other hand, Zn²⁺ may also bind to another site different from the zinc-finger motif or the binding sites for the substrates or resveratrol and act as a potent inhibitor of SIRT1.     

Biophysical and Bioinformatic Analyses Implicate the Treponema pallidum Tp34 Lipoprotein (Tp0971) in Transition Metal Homeostasis

Metal ion homeostasis is a critical function of many integral and peripheral membrane proteins. The genome of the etiologic agent of syphilis, Treponema pallidum, is compact and devoid of many metabolic enzyme genes. Nevertheless, it harbors genes coding for homologs of several enzymes that typically require either iron or zinc. The product of the tp0971 gene of T. pallidum, designated Tp34, is a periplasmic lipoprotein that is thought to be tethered to the inner membrane of this organism. Previous work on a water-soluble (nonacylated) recombinant version of Tp34 established that this protein binds to Zn²⁺, which, like other transition metal ions, stabilizes the dimeric form of the protein. In this study, we employed analytical ultracentrifugation to establish that four transition metal ions (Ni²⁺, Co²⁺, Cu²⁺, and Zn²⁺) readily induce the dimerization of Tp34; Cu²⁺ (50% effective concentration [EC₅₀] = 1.7 μM) and Zn²⁺ (EC₅₀ = 6.2 μM) were the most efficacious of these ions. Mutations of the crystallographically identified metal-binding residues hindered the ability of Tp34 to dimerize. X-ray crystallography performed on crystals of Tp34 that had been incubated with metal ions indicated that the binding site could accommodate the metals examined. The findings presented herein, coupled with bioinformatic analyses of related proteins, point to Tp34''s likely role in metal ion homeostasis in T. pallidum.     

Extracellular Zinc Ion Inhibits ClC-0 Chloride Channels by Facilitating Slow Gating

Extracellular Zn²⁺ was found to reversibly inhibit the ClC-0 Cl⁻ channel. The apparent on and off rates of the inhibition were highly temperature sensitive, suggesting an effect of Zn²⁺ on the slow gating (or inactivation) of ClC-0. In the absence of Zn²⁺, the rate of the slow-gating relaxation increased with temperature, with a Q₁₀ of ∼37. Extracellular Zn²⁺ facilitated the slow-gating process at all temperatures, but the Q₁₀ did not change. Further analysis of the rate constants of the slow-gating process indicates that the effect of Zn²⁺ is mostly on the forward rate (the rate of inactivation) rather than the backward rate (the rate of recovery from inactivation) of the slow gating. When ClC-0 is bound with Zn²⁺, the equilibrium constant of the slow-gating process is increased by ∼30-fold, reflecting a 30-fold higher Zn²⁺ affinity in the inactivated channel than in the open-state channel. As examined through a wide range of membrane potentials, Zn²⁺ inhibits the opening of the slow gate with equal potency at all voltages, suggesting that a two-state model is inadequate to describe the slow-gating transition. Following a model originally proposed by Pusch and co-workers (Pusch, M., U. Ludewig, and T.J. Jentsch. 1997. J. Gen. Physiol. 109:105–116), the effect of Zn²⁺ on the activation curve of the slow gate can be well described by adding two constraints: (a) the dissociation constant for Zn²⁺ binding to the open channel is 30 μM, and (b) the difference in entropy between the open state and the transition state of the slow-gating process is increased by 27 J/ mol/°K for the Zn²⁺-bound channel. These results together indicate that extracellular Zn²⁺ inhibits ClC-0 by facilitating the slow-gating process.     

Effects of Zn2+ on the activity and binding of the mitochondrial ATPase inhibitor protein,IF1

Zn²⁺ caused a noninhibitory binding of IF₁ to mitochondrial membranes in both rabbit heart SMP and intact rabbit heart mitochondria. This Zn²⁺-induced IF₁ binding required the presence of at least trace amounts of MgATP and was essentially independent of pH between 6.2 and 8.2. Addition of Zn²⁺ after the formation of fully inhibited IF₁-ATPase complexes very slowly reversed IF₁-mediated ATPase inhibition without causing significant IF₁ release from the membranes. When Zn²⁺ was added during the state 4 energization of ischemic mitochondria in which IF₁ was already functionally bound, it slowed somewhat energy-driven ATPase activation. This slowing was probably due to the fairly large depressing effect Zn²⁺ had upon membrane potential development, but Zn²⁺ did not decrease the degree of ATPase activation eventually reached at 20 min of state 4 incubation. Zn²⁺ also preempted normal IF₁ release from the membranes, causing what little inhibitor that was released to rebind to the enzyme in noninhibitory IF₁-ATPase complexes. The data suggest that IF₁ can interact with the ATPase in two ways or through two kinds of sites: (a) a noninhibitory interaction involving a noninhibitory IF₁ conformation and/or and IF₁ docking site on the enzyme and (b) an inhibitory interaction involving an inhibitory IF₁ conformation and/or a distinct ATPase activity regulatory site. Zn²⁺ appears to have the dual effect of stabilizing the noninhibitory IF₁-ATPase interaction and possibily a noninhibitory IF₁ conformation while concomitantly preventing the formation of an inhibitory IF₁-ATPase interaction and possibly an inhibitory IF₁ conformation, regardless of pH. While the data do not rule out direct effects of Zn²⁺ on either free IF₁ or the free enzyme, they suggest that Zn²⁺ cannot interact readily with either the inhibitor or the enzyme once functional IF₁-ATPase complexes are formed.     

The crystal structure of TRPM2 MHR1/2 domain reveals a conserved Zn2+‐binding domain essential for structural integrity and channel activity

Transient receptor potential melastatin 2 (TRPM2) is a Ca²⁺‐permeable, nonselective cation channel involved in diverse physiological processes such as immune response, apoptosis, and body temperature sensing. TRPM2 is activated by ADP‐ribose (ADPR) and 2′‐deoxy‐ADPR in a Ca²⁺‐dependent manner. While two distinct binding sites exist for ADPR that exert different functions dependent on the species, the involvement of either binding site regarding the superagonistic effect of 2′‐deoxy‐ADPR is not clear yet. Here, we report the crystal structure of the MHR1/2 domain of TRPM2 from zebrafish (Danio rerio), and show that both ligands bind to this domain and activate the channel. We identified a so far unrecognized Zn²⁺‐binding domain that was not resolved in previous cryo‐EM structures and that is conserved in most TRPM channels. In combination with patch clamp experiments we comprehensively characterize the effect of the Zn²⁺‐binding domain on TRPM2 activation. Our results provide insight into a conserved motif essential for structural integrity and channel activity.     

Hints for Metal-Preference Protein Sequence Determinants: Different Metal Binding Features of the Five Tetrahymena thermophila Metallothioneins

The metal binding preference of metallothioneins (MTs) groups them in two extreme subsets, the Zn/Cd- and the Cu-thioneins. Ciliates harbor the largest MT gene/protein family reported so far, including 5 paralogs that exhibit relatively low sequence similarity, excepting MTT2 and MTT4. In Tetrahymena thermophila, three MTs (MTT1, MTT3 and MTT5) were considered Cd-thioneins and two (MTT2 and MTT4) Cu-thioneins, according to gene expression inducibility and phylogenetic analysis. In this study, the metal-binding abilities of the five MTT proteins were characterized, to obtain information about the folding and stability of their cognate- and non-cognate metal complexes, and to characterize the T. thermophila MT system at protein level. Hence, the five MTTs were recombinantly synthesized as Zn²⁺-, Cd²⁺- or Cu⁺-complexes, which were analyzed by electrospray mass spectrometry (ESI-MS), circular dichroism (CD), and UV-vis spectrophotometry. Among the Cd-thioneins, MTT1 and MTT5 were optimal for Cd²⁺ coordination, yielding unique Cd₁₇- and Cd₈- complexes, respectively. When binding Zn²⁺, they rendered a mixture of Zn-species. Only MTT5 was capable to coordinate Cu⁺, although yielding heteronuclear Zn-, Cu-species or highly unstable Cu-homometallic species. MTT3 exhibited poor binding abilities both for Cd²⁺ and for Cu⁺, and although not optimally, it yielded the best result when coordinating Zn²⁺. The two Cu-thioneins, MTT2 and MTT4 isoforms formed homometallic Cu-complexes (major Cu₂₀-MTT) upon synthesis in Cu-supplemented hosts. Contrarily, they were unable to fold into stable Cd-complexes, while Zn-MTT species were only recovered for MTT4 (major Zn₁₀-MTT4). Thus, the metal binding preferences of the five T. thermophila MTs correlate well with their previous classification as Cd- and Cu-thioneins, and globally, they can be classified from Zn/Cd- to Cu-thioneins according to the gradation: MTT1>MTT5>MTT3>MTT4>MTT2. The main mechanisms underlying the evolution and specialization of the MTT metal binding preferences may have been internal tandem duplications, presence of doublet and triplet Cys patterns in Zn/Cd-thioneins, and optimization of site specific amino acid determinants (Lys for Zn/Cd- and Asn for Cu-coordination).