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.     

Structural analysis of ABC-family periplasmic zinc binding protein provides new insights into mechanism of ligand uptake and release

ATP-binding cassette superfamily of periplasmic metal transporters are known to be vital for maintaining ion homeostasis in several pathogenic and non-pathogenic bacteria. We have determined crystal structure of the high-affinity zinc transporter ZnuA from Escherichia coli at 1.8 A resolution. This structure represents the first native (non-recombinant) protein structure of a periplasmic metal binding protein. ZnuA reveals numerous conformational features, which occur either in Zn(2+) or in Mn(2+) transporters, and presents a unique conformational state. A comprehensive comparison of ZnuA with other periplasmic ligand binding protein structures suggests vital mechanistic differences between bound and release states of metal transporters. The key new attributes in ZnuA include a C-domain disulfide bond, an extra alpha-helix proximal to the highly charged metal chelating mobile loop region, alternate conformations of secondary shell stabilizing residues at the metal binding site, and domain movements potentially controlled by salt bridges. Based on in-depth structural analyses of five metal binding transporters, we present here a mechanistic model termed as "partial domain slippage" for binding and release of Zn(2+).     

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.     

Structural studies of metal ions in family II pyrophosphatases: the requirement for a Janus ion

Family II inorganic pyrophosphatases (PPases) constitute a new evolutionary group of PPases, with a different fold and mechanism than the common family I enzyme; they are related to the "DHH" family of phosphoesterases. Biochemical studies have shown that Mn(2+) and Co(2+) preferentially activate family II PPases; Mg(2+) partially activates; and Zn(2+) can either activate or inhibit (Zyryanov et al., Biochemistry, 43, 14395-14402, accompanying paper in this issue). The three solved family II PPase structures did not explain the differences between the PPase families nor the metal ion differences described above. We therefore solved three new family II PPase structures: Bacillus subtilis PPase (Bs-PPase) dimer core bound to Mn(2+) at 1.3 A resolution, and, at 2.05 A resolution, metal-free Bs-PPase and Streptococcus gordonii (Sg-PPase) containing sulfate and Zn(2+). Comparison of the new and old structures of various family II PPases demonstrates why the family II enzyme prefers Mn(2+) or Co(2+), as an activator rather than Mg(2+). Both M1 and M2 undergo significant changes upon substrate binding, changing from five-coordinate to octahedral geometry. Mn(2+) and Co(2+), which readily adopt different coordination states and geometries, are thus favored. Combining our structures with biochemical data, we identified M2 as the high-affinity metal site. Zn(2+) activates in the M1 site, where octahedral geometry is not essential for catalysis, but inhibits in the M2 site, because it is unable to assume octahedral geometry but remains trigonal bipyramidal. Finally, we propose that Lys205-Gln81-Gln80 form a hydrophilic channel to speed product release from the active site.     

Identification of the N-terminal region of TjZNT2, a Zrt/Irt-like protein family metal transporter, as a novel functional region involved in metal ion selectivity

The Zrt/Irt-like protein (ZIP) family of transporter proteins is involved in the uptake of essential metal elements in plants. Two homologous ZIP genes from Thlaspi japonicum, TjZNT1 and TjZNT2, encode products that share high amino acid sequence similarity except at the N-terminus and the cytoplasmic loop between transmembrane domains III and IV, and that have been shown to be Zn(2+) and Mn(2+) transporters, respectively. To identify the region that determines the ion selectivity of these transporters, we constructed a series of TjZNT1 and TjZNT2 chimeric genes and assayed for the Zn(2+) uptake of yeast cells expressing them. As a result, the extracellular N-terminal ends were identified as regions involved in Zn(2+) selectivity. TjZNT2 possesses a 36 amino acid hydrophilic extension at its N-terminus that is absent in native TjZNT1, and a mutant TjZNT2 lacking the N-terminal extension was shown to possess Zn(2+) uptake activity. This suggests that the extended N-terminal region inhibits Zn(2+) transport by TjZNT2. Further studies showed that it is the first 25 amino acid region of the N-terminus that is important for the inhibition of Zn(2+) transport. Furthermore, the N-terminal truncated TjZNT2 lacked Mn(2+) uptake activity. These findings suggest that the N-terminal region is a novel substrate selector in the ZIP family of transporters.     

Selective metal binding to a membrane-embedded aspartate in the Escherichia coli metal transporter YiiP (FieF)

The cation diffusion facilitators (CDF) are a ubiquitous family of metal transporters that play important roles in homeostasis of a wide range of divalent metal cations. Molecular identities of substrate-binding sites and their metal selectivity in the CDF family are thus far unknown. By using isothermal titration calorimetry and stopped-flow spectrofluorometry, we directly examined metal binding to a highly conserved aspartate in the Escherichia coli CDF transporter YiiP (FieF). A D157A mutation abolished a Cd2+-binding site and impaired the corresponding Cd2+ transport. In contrast, substitution of Asp-157 with a cysteinyl coordination residue resulted in intact Cd2+ binding as well as full transport activity. A similar correlation was found for Zn2+ binding and transport, suggesting that Asp-157 is a metal coordination residue required for binding and transport of Cd2+ and Zn2+. The location of Asp-157 was mapped topologically to the hydrophobic core of transmembrane segment 5 (TM-5) where D157C was found partially accessible to thiol-specific labeling of maleimide polyethylene-oxide biotin. Binding of Zn2+ and Cd2+, but not Fe2+, Hg2+, Co2+, Ni2+, Mn2+, Ca2+, and Mg2+, protected D157C from maleimide polyethylene-oxide biotin labeling in a concentration-dependent manner. Furthermore, isothermal titration calorimetry analysis of YiiP(D157A) showed no detectable change in Fe2+ and Hg2+ calorimetric titrations, indicating that Asp-157 is not a coordination residue for Fe2+ and Hg2+ binding. Our results provided direct evidence for selective binding of Zn2+ and Cd2+ for to the highly conserved Asp-157 and defined its functional role in metal transport.     

Function of His185 in Aquifex aeolicus 3-deoxy-D-manno-octulosonate 8-phosphate synthase

Aquifex aeolicus 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) by favoring the activation of a water molecule coordinated to the active-site metal ion. Cys11, His185, Glu222 and Asp233 are the other metal ligands. Wild-type KDO8PS is purified with Zn(2+) or Fe(2+) in the active site, but maximal activity in vitro is achieved when the endogenous metal is replaced with Cd(2+). The H185G enzyme retains 8% of the wild-type activity. ICP mass spectrometry analysis indicates that loss of His185 decreases the enzyme affinity for Fe(2+), but not for Zn(2+). However, maximal activity is again achieved by substitution of the endogenous metal with Cd(2+). We have determined the X-ray structures of the Cd(2+) H185G enzyme in its substrate-free form, and in complex with PEP, and PEP plus A5P. These structures show a normal amount of Cd(2+) bound, suggesting that coordination by His185 is not essential to retain Cd(2+) in the active site. Nonetheless, there are significant changes in the coordination sphere of Cd(2+) with respect to the wild-type enzyme, as the carboxylate moiety of PEP binds directly to the metal ion and replaces water and His185 as ligands. These observations indicate that the primary function of His185 in A.aeolicus KDO8PS is to orient PEP in the active site of the enzyme in such a way that a water molecule on the sinister (si) side of PEP can be activated by direct coordination to the metal ion.     

Multiple scattering x-ray absorption studies of Zn2+ binding sites in bacterial photosynthetic reaction centers

Binding of transition metal ions to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides has been previously shown to slow light-induced electron and proton transfer to the secondary quinone acceptor molecule, Q(B). On the basis of x-ray diffraction at 2.5 angstroms resolution a site, formed by AspH124, HisH126, and HisH128, has been identified at the protein surface which binds Cd(2+) or Zn(2+). Using Zn K-edge x-ray absorption fine structure spectroscopy we report here on the local structure of Zn(2+) ions bound to purified RC complexes embedded into polyvinyl alcohol films. X-ray absorption fine structure data were analyzed by combining ab initio simulations and multiparameter fitting; structural contributions up to the fourth coordination shell and multiple scattering paths (involving three atoms) have been included. Results for complexes characterized by a Zn to RC stoichiometry close to one indicate that Zn(2+) binds two O and two N atoms in the first coordination shell. Higher shell contributions are consistent with a binding cluster formed by two His, one Asp residue, and a water molecule. Analysis of complexes characterized by approximately 2 Zn ions per RC reveals a second structurally distinct binding site, involving one O and three N atoms, not belonging to a His residue. The local structure obtained for the higher affinity site nicely fits the coordination geometry proposed on the basis of x-ray diffraction data, but detects a significant contraction of the first shell. Two possible locations of the second new binding site at the cytoplasmic surface of the RC are proposed.     

Structural influence of hydrophobic core residues on metal binding and specificity in carbonic anhydrase II

Aromatic residues in the hydrophobic core of human carbonic anhydrase II (CAII) influence metal ion binding in the active site. Residues F93, F95, and W97 are contained in a beta-strand that also contains two zinc ligands, H94 and H96. The aromatic amino acids contribute to the high zinc affinity and slow zinc dissociation rate constant of CAII [Hunt, J. A., and Fierke, C. A. (1997) J. Biol. Chem. 272, 20364-20372]. Substitution of these aromatic amino acids with smaller side chains enhances Cu(2+) affinity while decreasing Co(2+) and Zn(2+) affinity [Hunt, J. A., Mahiuddin, A., & Fierke, C. A. (1999) Biochemistry 38, 9054-9062]. Here, X-ray crystal structures of zinc-bound F93I/F95M/W97V and F93S/F95L/W97M CAIIs reveal the introduction of new cavities in the hydrophobic core, compensatory movements of surrounding side chains, and the incorporation of buried water molecules; nevertheless, the enzyme maintains tetrahedral zinc coordination geometry. However, a conformational change of direct metal ligand H94 as well as indirect (i.e., "second-shell") ligand Q92 accompanies metal release in both F93I/F95M/W97V and F93S/F95L/W97M CAIIs, thereby eliminating preorientation of the histidine ligands with tetrahedral geometry in the apoenzyme. Only one cobalt-bound variant, F93I/F95M/W97V CAII, maintains tetrahedral metal coordination geometry; F93S/F95L/W97M CAII binds Co(2+) with trigonal bipyramidal coordination geometry due to the addition of azide anion to the metal coordination polyhedron. The copper-bound variants exhibit either square pyramidal or trigonal bipyramidal metal coordination geometry due to the addition of a second solvent molecule to the metal coordination polyhedron. The key finding of this work is that aromatic core residues serve as anchors that help to preorient direct and second-shell ligands to optimize zinc binding geometry and destabilize alternative geometries. These geometrical constraints are likely a main determinant of the enhanced zinc/copper specificity of CAII as compared to small molecule chelators.     

Amino acid screening based on structural modeling identifies critical residues for the function, ion selectivity and structure of Arabidopsis MTP1

Arabidopsis thaliana MTP1 is a vacuolar membrane Zn(2+)/H(+) antiporter of the cation diffusion facilitator family. Here we present a structure-function analysis of AtMTP1-mediated transport and its remarkable Zn(2+) selectivity by functional complementation tests of more than 50 mutant variants in metal-sensitive yeast strains. This was combined with homology modeling of AtMTP1 based on the crystal structure of the Escherichia coli broad-specificity divalent cation transporter YiiP. The Zn(2+)-binding sites of EcYiiP in the cytoplasmic C-terminus, and the pore formed by transmembrane helices TM2 and TM5, are conserved in AtMTP1. Although absent in EcYiiP, Cys31 and Cys36 in the extended N-terminal cytosolic domain of AtMTP1 are necessary for complementation of a Zn-sensitive yeast strain. On the cytosolic side of the active Zn(2+)-binding site inside the transmembrane pore, Ala substitution of either Asn258 in TM5 or Ser101 in TM2 non-selectively enhanced the metal tolerance conferred by AtMTP1. Modeling predicts that these residues obstruct the movement of cytosolic Zn(2+) into the intra-membrane Zn(2+)-binding site of AtMTP1. A conformational change in the immediately preceding His-rich cytosolic loop may displace Asn258 and permit Zn(2+) entry into the pore. This would allow dynamic coupling of Zn(2+) transport to the His-rich loop, thus acting as selectivity filter or sensor of cytoplasmic Zn(2+) levels. Individual mutations at diverse sites within AtMTP1 conferred Co and Cd tolerance in yeast, and included deletions in N-terminal and His-rich intra-molecular cytosolic domains, and mutations of single residues flanking the transmembrane pore or participating in intra- or inter-molecular domain interactions, all of which are not conserved in the non-selective EcYiiP.     

Refinement of a homology model of the mu-opioid receptor using distance constraints from intrinsic and engineered zinc-binding sites

Publication of the rhodopsin X-ray structure has facilitated the development of homology models of other G protein-coupled receptors. However, possible shifts of transmembrane (TM) alpha helices, expected variations in helical distortions, and differences in loop size necessitate experimental verification of these comparative models. To refine a rhodopsin-based homology model of the mu-opioid receptor (MOR), we experimentally determined structural-distance constraints from intrinsic and engineered metal-binding sites in the rat MOR. Investigating the relatively high intrinsic affinity of MOR for Zn(2+) (IC(50) approximately 30microM), we observed that mutation of His(319) (TM7) abolished Zn(2+) inhibition of ligand binding, while mutation of Asp(216) (extracellular loop 2) decreased the effect of Zn(2+), suggesting these residues participate in the intrinsic Zn(2+)-binding center of MOR. To verify the relative orientation of TM5 and TM6 and to examine whether a rhodopsin-like alpha aneurism is present in TM5, we engineered Zn(2+)-binding centers by mutating residues of TM5 and TM6 to Cys or His, making use of the native His(297) in TM6 as an additional Zn(2+)-coordination site. Inhibition of opioid ligand binding by Zn(2+) suggests that residues Ile(234) and Phe(237) in TM5 face the binding-site crevice and form a metal-binding center with His(297) and Val(300) in TM6. This observation is inconsistent with a rhodopsin-like structure, which would locate Ile(234) on the lipid-exposed side of TM5, too distant from other residues making up the Zn(2+)-binding site. Subsequent distance geometry refinement of the MOR model indicates that the rhodopsin-like alpha aneurism is likely absent in TM2 but present in TM5.     

Catalytic zinc site and mechanism of the metalloenzyme PR-AMP cyclohydrolase

The enzyme N(1)-(5'-phosphoribosyl) adenosine-5'-monophosphate cyclohydrolase (PR-AMP cyclohydrolase) is a Zn(2+) metalloprotein encoded by the hisI gene. It catalyzes the third step of histidine biosynthesis, an uncommon ring-opening of a purine heterocycle for use in primary metabolism. A three-dimensional structure of the enzyme from Methanobacterium thermoautotrophicum has revealed that three conserved cysteine residues occur at the dimer interface and likely form the catalytic site. To investigate the functions of these cysteines in the enzyme from Methanococcus vannielii, a series of biochemical studies were pursued to test the basic hypothesis regarding their roles in catalysis. Inactivation of the enzyme activity by methyl methane thiosulfonate (MMTS) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) also compromised the Zn(2+) binding properties of the protein inducing loss of up to 90% of the metal. Overall reaction stoichiometry and the potassium cyanide (KCN) induced cleavage of the protein suggested that all three cysteines were modified in the process. The enzyme was protected from DTNB-induced inactivation by inclusion of the substrate N(1)-(5'-phosphoribosyl)adenosine 5'-monophosphate; (PR-AMP), while Mg(2+), a metal required for catalytic activity, enhanced the rate of inactivation. Site-directed mutations of the conserved C93, C109, C116 and the double mutant C109/C116 were prepared and analyzed for catalytic activity, Zn(2+) content, and reactivity with DTNB. Substitution of alanine for each of the conserved cysteines showed no measurable catalytic activity, and only the C116A was still capable of binding Zn(2+). Reactions of DTNB with the C109A/C116A double mutant showed that C93 is completely modified within 0.5 s. A model consistent with these data involves a DTNB-induced mixed disulfide linkage between C93 and C109 or C116, followed by ejection of the active site Zn(2+) and provides further evidence that the Zn(2+) coordination site involves the three conserved cysteine residues. The C93 reactivity is modulated by the presence of the Zn(2+) and Mg(2+) and substantiates the role of this residue as a metal ligand. In addition, Mg(2+) ligand binding site(s) indicated by the structural analysis were probed by site-directed mutagenesis of three key aspartate residues flanking the conserved C93 which were shown to have a functional impact on catalysis, cysteine activation, and metal (zinc) binding capacity. The unique amino acid sequence, the dynamic properties of the cysteine ligands involved in Zn(2+) coordination, and the requirement for a second metal (Mg(2+)) are discussed in the context of their roles in catalysis. The results are consistent with a Zn(2+)-mediated activation of H(2)O mechanism involving histidine as a general base that has features similar to but distinct from those of previously characterized purine and pyrimidine deaminases.     

Evidence for a metal-thiolate intermediate in alkyl group transfer from epoxypropane to coenzyme M and cooperative metal ion binding in epoxyalkane:CoM transferase

Epoxyalkane:coenzyme M transferase (EaCoMT) catalyzes the nucleophilic addition of coenzyme M (CoM, 2-mercaptoethanesulfonic acid) to epoxypropane forming 2-hydroxypropyl-CoM. The biochemical properties of EaCoMT suggest that the enzyme belongs to the family of alkyltransferase enzymes for which Zn plays a role in activating an organic thiol substrate for nucleophilic attack on an alkyl-donating substrate. The enzyme has a hexameric (alpha(6)) structure with one zinc atom per subunit. In the present work M(2+) binding and the role of Zn(2+) in EaCoMT have been established through a combination of biochemical, calorimetric, and spectroscopic techniques. A variety of metal ions, including Zn(2+), Co(2+), Cd(2+), and Ni(2+), were capable of activating a Zn-deficient "apo" form of EaCoMT, affording enzymes with various levels of activity. Titration of Co(2+) into apo-EaCoMT resulted in UV-visible spectroscopic changes consistent with the formation of a tetrahedral Co(2+) binding site, with coordination of bound Co(2+) to two thiolate ligands. Quantification of UV-visible spectral changes upon Co(2+) titration into apo-EaCoMT demonstrated that EaCoMT binds Co(2+) cooperatively at six interacting sites. Isothermal titration calorimetric studies of Co(2+) and Zn(2+) binding to EaCoMT also showed cooperativity for metal ion binding among six sites. The addition of CoM to Co(2+)-substituted EaCoMT resulted in UV-visible spectral changes indicative of formation of a new thiol-Co(2+) bond. Co(2+)-substituted EaCoMT exhibited a unique Co(2+) EPR spectrum, and this spectrum was perturbed significantly upon addition of CoM. The presence of a divalent metal ion was required for the release of protons from CoM upon binding to EaCoMT, with Zn(2+), Co(2+), and Cd(2+) each facilitating proton release. The divalent metal ion of EaCoMT is proposed to play a key role in the coordination and deprotonation of CoM, possibly through formation of a metal-thiolate that is activated for attack on the epoxide substrate.     

Planar substrate-binding site dictates the specificity of ECF-type nickel/cobalt transporters

The energy-coupling factor (ECF) transporters are multi-subunit protein complexes that mediate uptake of transition-metal ions and vitamins in about 50% of the prokaryotes, including bacteria and archaea. Biological and structural studies have been focused on ECF transporters for vitamins, but the molecular mechanism by which ECF systems transport metal ions from the environment remains unknown. Here we report the first crystal structure of a NikM, TtNikM2, the substrate-binding component (S component) of an ECF-type nickel transporter from Thermoanaerobacter tengcongensis. In contrast to the structures of the vitamin-specific S proteins with six transmembrane segments (TSs), TtNikM2 possesses an additional TS at its N-terminal region, resulting in an extracellular N-terminus. The highly conserved N-terminal loop inserts into the center of TtNikM2 and occludes a region corresponding to the substrate-binding sites of the vitamin-specific S components. Nickel binds to NikM via its coordination to four nitrogen atoms, which are derived from Met1, His2 and His67 residues. These nitrogen atoms form an approximately square-planar geometry, similar to that of the metal ion-binding sites in the amino-terminal Cu²⁺- and Ni²⁺-binding (ATCUN) motif. Replacements of residues in NikM contributing to nickel coordination compromised the Ni-transport activity. Furthermore, systematic quantum chemical investigation indicated that this geometry enables NikM to also selectively recognize Co²⁺. Indeed, the structure of TtNikM2 containing a bound Co²⁺ ion has almost no conformational change compared to the structure that contains a nickel ion. Together, our data reveal an evolutionarily conserved mechanism underlying the metal selectivity of EcfS proteins, and provide insights into the ion-translocation process mediated by ECF transporters.     

Metal selectivity determinants in a family of transition metal transporters

Metal tolerance proteins (MTPs) are plant members of the cation diffusion facilitator (CDF) transporter family involved in cellular metal homeostasis. Members of the CDF family are ubiquitously found in all living entities and show principal selectivity for Zn(2+), Mn(2+), and Fe(2+). Little is known regarding metal selectivity determinants of CDFs. We identified a novel cereal member of CDFs in barley, termed HvMTP1, that localizes to the vacuolar membrane. Unlike its close relative AtMTP1, which is highly selective for Zn(2+), HvMTP1 exhibits selectivity for both Zn(2+) and Co(2+) as assessed by its ability to suppress yeast mutant phenotypes for both metals. Expression of HvMTP1/AtMTP1 chimeras in yeast revealed a five-residue sequence within the AtMTP1 N-segment of the His-rich intracytoplasmic loop that confines specificity to Zn(2+). Furthermore, mutants of AtMTP1 generated through random mutagenesis revealed residues embedded within transmembrane domain 3 that additionally specify the high degree of Zn(2+) selectivity. We propose that the His-rich loop, which might play a role as a zinc chaperone, determines the identity of the metal ions that are transported. The residues within transmembrane domain 3 can also influence metal selectivity, possibly through conformational changes induced at the cation transport site located within the membrane or at the cytoplasmic C-terminal domain.     

Metal transport across biomembranes: emerging models for a distinct chemistry

Transition metals are essential components of important biomolecules, and their homeostasis is central to many life processes. Transmembrane transporters are key elements controlling the distribution of metals in various compartments. However, due to their chemical properties, transition elements require transporters with different structural-functional characteristics from those of alkali and alkali earth ions. Emerging structural information and functional studies have revealed distinctive features of metal transport. Among these are the relevance of multifaceted events involving metal transfer among participating proteins, the importance of coordination geometry at transmembrane transport sites, and the presence of the largely irreversible steps associated with vectorial transport. Here, we discuss how these characteristics shape novel transition metal ion transport models.     

Crystal structures of fructose 1,6-bisphosphatase: mechanism of catalysis and allosteric inhibition revealed in product complexes

Crystal structures of metal-product complexes of fructose 1, 6-bisphosphatase (FBPase) reveal competition between AMP and divalent cations. In the presence of AMP, the Zn(2+)-product and Mg(2+)-product complexes have a divalent cation present only at one of three metal binding sites (site 1). The enzyme is in the T-state conformation with a disordered loop of residues 52-72 (loop 52-72). In the absence of AMP, the enzyme crystallizes in the R-state conformation, with loop 52-72 associated with the active site. In structures without AMP, three metal-binding sites are occupied by Zn(2+) and two of three metal sites (sites 1 and 2) by Mg(2+). Evidently, the association of AMP with FBPase disorders loop 52-72, the consequence of which is the release of cations from two of three metal binding sites. In the Mg(2+) complexes (but not the Zn(2+) complexes), the 1-OH group of fructose 6-phosphate (F6P) coordinates to the metal at site 1 and is oriented for a nucleophilic attack on the bound phosphate molecule. A mechanism is presented for the forward reaction, in which Asp74 and Glu98 together generate a hydroxide anion coordinated to the Mg(2+) at site 2, which then displaces F6P. Development of negative charge on the 1-oxygen of F6P is stabilized by its coordination to the Mg(2+) at site 1.     

Determination of the structure of Escherichia coli glyoxalase I suggests a structural basis for differential metal activation

The metalloenzyme glyoxalase I (GlxI) converts the nonenzymatically produced hemimercaptal of cytotoxic methylglyoxal and glutathione to nontoxic S-D-lactoylglutathione. Human GlxI, for which the structure is known, is active in the presence of Zn(2+). Unexpectedly, the Escherichia coli enzyme is inactive in the presence of Zn(2+) and is maximally active with Ni(2+). To understand this difference in metal activation and also to obtain a representative of the bacterial enzymes, the structure of E. coli Ni(2+)-GlxI has been determined. Structures have also been determined for the apo enzyme as well as complexes with Co(2+), Cd(2+), and Zn(2+). It is found that each of the protein-metal complexes that is catalytically active has octahedral geometry. This includes the complexes of the E. coli enzyme with Ni(2+), Co(2+), and Cd(2+), as well as the structures reported for the human Zn(2+) enzyme. Conversely, the complex of the E. coli enzyme with Zn(2+) has trigonal bipyramidal coordination and is inactive. This mode of coordination includes four protein ligands plus a single water molecule. In contrast, the coordination in the active forms of the enzyme includes two water molecules bound to the metal ion, suggesting that this may be a key feature of the catalytic mechanism. A comparison of the human and E. coli enzymes suggests that there are differences between the active sites that might be exploited for therapeutic use.     

Structural variation in bacterial glyoxalase I enzymes: investigation of the metalloenzyme glyoxalase I from Clostridium acetobutylicum

The glyoxalase system catalyzes the conversion of toxic, metabolically produced α-ketoaldehydes, such as methylglyoxal, into their corresponding nontoxic 2-hydroxycarboxylic acids, leading to detoxification of these cellular metabolites. Previous studies on the first enzyme in the glyoxalase system, glyoxalase I (GlxI), from yeast, protozoa, animals, humans, plants, and Gram-negative bacteria, have suggested two metal activation classes, Zn(2+) and non-Zn(2+) activation. Here, we report a biochemical and structural investigation of the GlxI from Clostridium acetobutylicum, which is the first GlxI enzyme from Gram-positive bacteria that has been fully characterized as to its three-dimensional structure and its detailed metal specificity. It is a Ni(2+)/Co(2+)-activated enzyme, in which the active site geometry forms an octahedral coordination with one metal atom, two water molecules, and four metal-binding ligands, although its inactive Zn(2+)-bound form possesses a trigonal bipyramidal geometry with only one water molecule liganded to the metal center. This enzyme also possesses a unique dimeric molecular structure. Unlike other small homodimeric GlxI where two active sites are located at the dimeric interface, the C. acetobutylicum dimeric GlxI enzyme also forms two active sites but each within single subunits. Interestingly, even though this enzyme possesses a different dimeric structure from previously studied GlxI, its metal activation characteristics are consistent with properties of other GlxI. These findings indicate that metal activation profiles in this class of enzyme hold true across diverse quaternary structure arrangements.