Conversion of agonist site to metal-ion chelator site in the β2-adrenergic receptor

Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the β2-adrenergic receptor, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III—or a His residue introduced at this position—and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic effect of, for example Cu2+, and in several cases increased the affinity of the ions for the agonistic site. Wash-out experiments and structure–activity analysis indicated, that the high-affinity chelators and the metal ions bind and activate the mutant receptor as metal ion guided ligand complexes. Because of the well-understood binding geometry of the small metal ions, an important distance constraint has here been imposed between TM-III and -VII in the active, signaling conformation of 7TM receptors. It is suggested that atoxic metal-ion chelator complexes could possibly in the future be used as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will.

Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases

The 2.15-Å resolution cocrystal structure of EcoRV endonuclease mutant T93A complexed with DNA and Ca2+ ions reveals two divalent metals bound in one of the active sites. One of these metals is ligated through an inner-sphere water molecule to the phosphate group located 3′ to the scissile phosphate. A second inner-sphere water on this metal is positioned approximately in-line for attack on the scissile phosphate. This structure corroborates the observation that the pro-SP phosphoryl oxygen on the adjacent 3′ phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of EcoRV, EcoRI, PvuII, and BamHI endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous cocrystal structures, these data allow construction of a detailed model for the pretransition state configuration in EcoRV. This model features three divalent metal ions per active site and invokes assistance in the bond-making step by a conserved lysine, which stabilizes the attacking hydroxide ion nucleophile.

Explanation by the double-metal-ion mechanism of catalysis for the differential metal ion effects on the cleavage rates of 5′-oxy and 5′-thio substrates by a hammerhead ribozyme

In a previous examination using natural all-RNA substrates that contained either a 5′-oxy or 5′-thio leaving group at the cleavage site, we demonstrated that (i) the attack by the 2′-oxygen at C17 on the phosphorus atom is the rate-limiting step only for the substrate that contains a 5′-thio group (R11S) and (ii) the departure of the 5′ leaving group is the rate-limiting step for the natural all-RNA substrate (R11O) in both nonenzymatic and hammerhead ribozyme-catalyzed reactions; the energy diagrams for these reactions were provided in our previous publication. In this report we found that the rate of cleavage of R11O by a hammerhead ribozyme was enhanced 14-fold when Mg2+ ions were replaced by Mn2+ ions, whereas the rate of cleavage of R11S was enhanced only 2.2-fold when Mg2+ ions were replaced by Mn2+ ions. This result appears to be exactly the opposite of that predicted from the direct coordination of the metal ion with the leaving 5′-oxygen, because a switch in metal ion specificity was not observed with the 5′-thio substrate. However, our quantitative analyses based on the previously provided energy diagram indicate that this result is in accord with the double-metal-ion mechanism of catalysis.

Two-dimensional protein crystallization via metal-ion coordination by naturally occurring surface histidines.

A powerful and potentially general approach to the targeting and crystallization of proteins on lipid interfaces through coordination of surface histidine residues to lipid-chelated divalent metal ions is presented. This approach, which should be applicable to the crystallization of a wide range of naturally occurring or engineered proteins, is illustrated here by the crystallization of streptavidin on a monolayer of an iminodiacetate-Cu(II) lipid spread at the air-water interface. This method allows control of the protein orientation at interfaces, which is significant for the facile production of highly ordered protein arrays and for electron density mapping in structural analysis of two-dimensional crystals. Binding of native streptavidin to the iminodiacetate-Cu lipids occurs via His-87, located on the protein surface near the biotin binding pocket. The two-dimensional streptavidin crystals show a previously undescribed microscopic shape that differs from that of crystals formed beneath biotinylated lipids.

A method for distance determination in proteins using a designed metal ion binding site and site-directed spin labeling: evaluation with T4 lysozyme.

The use of molecular genetics to introduce both a metal ion binding site and a nitroxide spin label into the same protein opens the use of paramagnetic metalnitroxyl interactions to estimate intramolecular distances in a wide variety of proteins. In this report, a His-Xaa3-His metal ion binding motif was introduced at the N terminus of the long interdomain helix of T4 lysozyme (Lys-65 --> His/Gln-69 --> His) of three mutants, each containing a single nitroxide-labeled cysteine residue at position 71, 76, or 80. The results show that Cu(II)-induced relaxation effects on the nitroxide can be quantitatively analyzed in terms of interspin distance in the range of 10-25 A using Redfield theory, as first suggested by Leigh [Leigh, J.S. (1970) J. Chem. Phys. 52, 2608-2612]. Of particular interest is the observation that distances can be determined both under rigid lattice conditions in frozen solution and in the presence of motion of the spins at room temperature under physiological conditions. The method should be particularly attractive for investigating structure in membrane proteins that are difficult to crystallize. In the accompanying paper, the technique is applied to a polytopic membrane protein, lactose permease.

Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of Escherichia coli.

As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure. To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins. Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV. In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification. After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II). The results demonstrate that positions 103, 111, and 121 are 8, 14, and > 23 A from the metal binding site. These data are consistent with an alpha-helical conformation of transmembrane domain IV of the permease. Application of the technique to determine helix packing in lactose permease is discussed.

Structures of the apo- and the metal ion-activated forms of the diphtheria tox repressor from Corynebacterium diphtheriae.

The diphtheria tox repressor (DtxR) of Corynebacterium diphtheriae plays a critical role in the regulation of diphtheria toxin expression and the control of other iron-sensitive genes. The crystal structures of apo-DtxR and of the metal ion-activated form of the repressor have been solved and used to identify motifs involved in DNA and metal ion binding. Residues involved in binding of the activated repressor to the diphtheria tox operator, glutamine 43, arginine 47, and arginine 50, were located and confirmed by site-directed mutagenesis. Previous biochemical and genetic data can be explained in terms of these structures. Conformational differences between apo- and Ni-DtxR are discussed with regard to the mechanism of action of this repressor.

Transition metal ion activation of DNA binding by the diphtheria tox repressor requires the formation of stable homodimers.

The diphtheria tox repressor (DtxR) is a transition metal ion-dependent regulatory element that controls the expression of diphtheria toxin and several genes involved in the synthesis of siderophores in Corynebacterium diphtheriae. In the presence of transition metal ions apo-DtxR becomes activated and specifically binds to its target DNA sequences. We demonstrate by glutaraldehyde cross-linking that monomeric apo-DtxR is in weak equilibrium with a dimeric form and that upon addition of activating metal ions to the reaction mixture a dimeric complex is stabilized. Addition of the DNA-binding-defective mutant apo-DtxR(delta 1-47) to apo-DtxR in the absence of transition metal ions inhibits conversion of the apo-repressor to its activated DNA-binding form. We also show that the binding of Ni2+ to both apo-DtxR and apo-DtxR(delta 1-47) is cooperative and that upon ion binding there is a conformational change in the environment of the indole ring moiety of Trp-104. For the wild-type repressor the consequences of this conformational change include a shift in equilibrium toward dimer formation and activation of target DNA binding by the repressor. We conclude that the formation of DtxR homodimers is mediated through a protein-protein interaction domain that is also activated on metal ion binding.

Crystallographic evidence for the action of potassium, thallium, and lithium ions on fructose-1,6-bisphosphatase.

Fructose-1,6-bisphosphatase (Fru-1,6-Pase; D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) requires two divalent metal ions to hydrolyze alpha-D-fructose 1,6-bisphosphate. Although not required for catalysis, monovalent cations modify the enzyme activity; K+ and Tl+ ions are activators, whereas Li+ ions are inhibitors. Their mechanisms of action are still unknown. We report here crystallographic structures of pig kidney Fru-1,6-Pase complexed with K+, Tl+, or both Tl+ and Li+. In the T form Fru-1,6-Pase complexed with the substrate analogue 2,5-anhydro-D-glucitol 1,6-bisphosphate (AhG-1,6-P2) and Tl+ or K+ ions, three Tl+ or K+ binding sites are found. Site 1 is defined by Glu-97, Asp-118, Asp-121, Glu-280, and a 1-phosphate oxygen of AhG-1,6-P2; site 2 is defined by Glu-97, Glu-98, Asp-118, and Leu-120. Finally, site 3 is defined by Arg-276, Glu-280, and the 1-phosphate group of AhG-1,6-P2. The Tl+ or K+ ions at sites 1 and 2 are very close to the positions previously identified for the divalent metal ions. Site 3 is specific to K+ or Tl+. In the divalent metal ion complexes, site 3 is occupied by the guanidinium group of Arg-276. These observations suggest that Tl+ or K+ ions can substitute for Arg-276 in the active site and polarize the 1-phosphate group, thus facilitating nucleophilic attack on the phosphorus center. In the T form complexed with both Tl+ and Li+ ions, Li+ replaces Tl+ at metal site 1. Inhibition by lithium very likely occurs as it binds to this site, thus retarding turnover or phosphate release. The present study provides a structural basis for a similar mechanism of inhibition for inositol monophosphatase, one of the potential targets of lithium ions in the treatment of manic depression.

Nicotianamine Chelates Both FeIII and FeII. Implications for Metal Transport in Plants1

Nicotianamine (NA) occurs in all plants and chelates metal cations, including FeII, but reportedly not FeIII. However, a comparison of the FeII and ZnII affinity constants of NA and various FeIII-chelating aminocarboxylates suggested that NA should chelate FeIII. High-voltage electrophoresis of the FeNA complex formed in the presence of FeIII showed that the complex had a net charge of 0, consistent with the hexadentate chelation of FeIII. Measurement of the affinity constant for FeIII yielded a value of 1020.6, which is greater than that for the association of NA with FeII (1012.8). However, capillary electrophoresis showed that in the presence of FeII and FeIII, NA preferentially chelates FeII, indicating that the FeIINA complex is kinetically stable under aerobic conditions. Furthermore, Fe complexes of NA are relatively poor Fenton reagents, as measured by their ability to mediate H2O2-dependent oxidation of deoxyribose. This suggests that NA will have an important role in scavenging Fe and protecting the cell from oxidative damage. The pH dependence of metal ion chelation by NA and a typical phytosiderophore, 2′-deoxymugineic acid, indicated that although both have the ability to chelate Fe, when both are present, 2′-deoxymugineic acid dominates the chelation process at acidic pH values, whereas NA dominates at alkaline pH values. The consequences for the role of NA in the long-distance transport of metals in the xylem and phloem are discussed.

Isolation of a myoglobin molten globule by selective cobalt(III)-induced unfolding

Reaction of the Schiff-base complex [Co(acetylacetonate-ethylenediimine)(NH3)2]+ with metmyoglobin at pH 6.5 yields a partially folded protein containing six Co(III) complexes. Although half of its α-helical secondary structure is retained, absorption and CD spectra indicate that the tertiary structure in both B-F and AGH domains is disrupted in the partially folded protein. In analogy to proton-induced unfolding, it is likely that the loss of tertiary structure is triggered by metal-ion binding to histidines. Cobalt(III)-induced unfolding of myoglobin is unique in its selectivity (other proteins are unaffected) and in allowing the isolation of the partially folded macromolecule (the protein does not refold or aggregate upon removal of free denaturant).

Three metal ions at the active site of the Tetrahymena group I ribozyme

Metal ions are critical for catalysis by many RNA and protein enzymes. To understand how these enzymes use metal ions for catalysis, it is crucial to determine how many metal ions are positioned at the active site. We report here an approach, combining atomic mutagenesis with quantitative determination of metal ion affinities, that allows individual metal ions to be distinguished. Using this approach, we show that at the active site of the Tetrahymena group I ribozyme the previously identified metal ion interactions with three substrate atoms, the 3′-oxygen of the oligonucleotide substrate and the 3′- and 2′-moieties of the guanosine nucleophile, are mediated by three distinct metal ions. This approach provides a general tool for distinguishing active site metal ions and allows the properties and roles of individual metal ions to be probed, even within the sea of metal ions bound to RNA.

Structural origins of the exonuclease resistance of a zwitterionic RNA

Nuclease resistance and RNA affinity are key criteria in the search for optimal antisense nucleic acid modifications, but the origins of the various levels of resistance to nuclease degradation conferred by chemical modification of DNA and RNA are currently not understood. The 2′-O-aminopropyl (AP)-RNA modification displays the highest nuclease resistance among all phosphodiester-based analogues and its RNA binding affinity surpasses that of phosphorothioate DNA by 1°C per modified residue. We found that oligodeoxynucleotides containing AP-RNA residues at their 3′ ends competitively inhibit the degradation of single-stranded DNA by the Escherichia coli Klenow fragment (KF) 3′-5′ exonuclease and snake venom phosphodiesterase. To shed light on the origins of nuclease resistance brought about by the AP modification, we determined the crystal structure of an A-form DNA duplex with AP-RNA modifications at 1.6-Å resolution. In addition, the crystal structures of complexes between short DNA fragments carrying AP-RNA modifications and wild-type KF were determined at resolutions between 2.2 and 3.0 Å and compared with the structure of the complex between oligo(dT) and the D355A/E357A KF mutant. The structural models suggest that interference of the positively charged 2′-O-substituent with the metal ion binding site B of the exonuclease allows AP-RNA to effectively slow down degradation.

Classification of mononuclear zinc metal sites in protein structures

Our study of the extended metal environment, particularly of the second shell, focuses in this paper on zinc sites. Key findings include: (i) The second shell of mononuclear zinc centers is generally more polar than hydrophobic and prominently features charged residues engaged in an abundance of hydrogen bonding with histidine ligands. Histidine–acidic or histidine–tyrosine clusters commonly overlap the environment of zinc ions. (ii) Histidine tautomeric metal bonding patterns in ligating zinc ions are mixed. For example, carboxypeptidase A, thermolysin, and sonic hedgehog possess the same ligand group (two histidines, one unibidentate acidic ligand, and a bound water), but their histidine tautomeric geometries markedly differ such that the carboxypeptidase A makes only Nδ1 contacts, thermolysin makes only Nɛ2 contacts, and sonic hedgehog uses one of each. Thus the presence of a similar ligand cohort does not necessarily imply the same topology or function at the active site. (iii) Two close histidine ligands HXmH, m ≤ 5, rarely both coordinate a single metal ion in the Nδ1 tautomeric conformation, presumably to avoid steric conflicts. Mononuclear zinc sites can be classified into six types depending on the ligand composition and geometry. Implications of the results are discussed in terms of divergent and convergent evolution.

Detection of alkali metal ions in DNA crystals using state-of-the-art X-ray diffraction experiments

The observation of light metal ions in nucleic acids crystals is generally a fortuitous event. Sodium ions in particular are notoriously difficult to detect because their X-ray scattering contributions are virtually identical to those of water and Na+…O distances are only slightly shorter than strong hydrogen bonds between well-ordered water molecules. We demonstrate here that replacement of Na+ by K+, Rb+ or Cs+ and precise measurements of anomalous differences in intensities provide a particularly sensitive method for detecting alkali metal ion-binding sites in nucleic acid crystals. Not only can alkali metal ions be readily located in such structures, but the presence of Rb+ or Cs+ also allows structure determination by the single wavelength anomalous diffraction technique. Besides allowing identification of high occupancy binding sites, the combination of high resolution and anomalous diffraction data established here can also pinpoint binding sites that feature only partial occupancy. Conversely, high resolution of the data alone does not necessarily allow differentiation between water and partially ordered metal ions, as demonstrated with the crystal structure of a DNA duplex determined to a resolution of 0.6 Å.