Extracellular histidine residues identify common structural determinants in the copper/zinc P2X2 receptor modulation

To assess the mechanism of P2X2 receptor modulation by transition metals, the cDNA for the wild-type receptor was injected to Xenopus laevis oocytes and examined 48-72 h later by the two-electrode voltage-clamp technique. Copper was the most potent of the trace metals examined; at 10 microm it evoked a 25-fold potentiation of the 10 microm ATP-gated currents. Zinc, nickel or mercury required 10-fold larger concentrations to cause comparable potentiations, while palladium, cobalt or cadmium averaged only 12- and 3-fold potentiations, respectively. Platinum was inactive. The non-additive effect of copper and zinc at 10-100 microm suggests a common site of action; these metals also shifted to the left the ATP concentration-response curves. To define residues necessary for trace metal modulation, alanines were singly substituted for each of the nine histidines in the extracellular domain of the rat P2X2 receptor. The H120A and H213A mutants were resistant to the modulator action of copper, zinc and other metals with the exception of mercury. Mutant H192A showed a reduction but not an abrogation of the copper or zinc potentiation. H245A showed less affinity for copper while this mutant flattened the zinc-induced potentiation. Mutant H319A reduced the copper but not the zinc-induced potentiation. In contrast, mutants H125A, H146A, H152A and H174A conserved the wild-type receptor sensitivity to trace metal modulation. We propose that His120, His192, His213 and His245 form part of a common allosteric metal-binding site of the P2X2 receptor, which for the specific coordination of copper, but not zinc, additionally involves His319.     

Zinc and copper modulate differentially the P2X4 receptor

The rat ATP P2X4 receptor was expressed in Xenopus laevis oocytes to assess the effect of zinc and copper as possible regulators of purinergic mechanisms. ATP applied for 20 s evoked an inward cationic current with a median effective concentration (EC50) of 21.4+/-2.8 microM and a Hill coefficient (nH) of 1.5+/-0.1. Coapplication of ATP plus 10 microM zinc displaced leftward, in a parallel fashion, the ATP concentration-response curve, reducing the EC50 to 8.4+/-1.8 microM (p < 0.01) without altering the receptor nH. The zinc potentiation was fast in onset, easily reversible, and voltage-independent and did not require metal preexposure. The zinc EC50 was 2-5 microM, with a bell-shaped curve. At concentrations of 100-300 microM, zinc produced less potentiation, and at 1 mM, it inhibited 50% the ATP current. The effect of zinc was mimicked by cadmium. In contrast, copper inhibited the ATP-evoked currents in a time- and concentration-dependent fashion, reducing the maximal current (Imax) without altering the EC50. The copper-induced inhibition was slow in onset, slowly reversible, and voltage-independent. Whereas coapplication of 300 microM copper plus ATP reduced Imax to 36.2+/-5%, the coapplication of, or 60-s preexposure by, 10 microM copper reduced Imax to 79+/-9.2% (p < 0.05) and 39.6+/-8.7% (p < 0.01), respectively. The inhibition was noncompetitive in nature and mimicked by mercury. Cobalt, barium, and manganese did not modify significantly the ATP-evoked current, demonstrating metal specificity. The simultaneous 1-min preapplication of both metals revealed that the 10 microM zinc-induced potentiation was obliterated by 10 microM copper, whereas 30 microM copper not only reduced the potentiation, but inhibited the ATP response. Following coapplication of both metals for 20 s with ATP, at least 100 microM copper was required to counteract the 10 microM zinc-induced potentiation. The simultaneous preincubation with both metals provided evidence for a noncompetitive interaction. We hypothesize the existence of metal binding site(s), which are most likely localized in the extracellular domain of the P2X4 receptor structure. These sites are selective and accessible to extracellular metal applications and bind micromolar concentrations of metals. The present results are compatible with the working hypothesis that trace metals, such as copper and zinc, are physiological modulators of the P2X4 receptor. The modulation of brain purinergic transmission by physiologically and toxicologically relevant trace metal cations is highlighted.     

Trace metals in the brain: allosteric modulators of ligand-gated receptor channels,the case of ATP-gated P2X receptors

Zinc and copper are indispensable trace metals for life with a recognized role as catalysts in enzyme actions. We now review evidence supporting the role of trace metals as novel allosteric modulators of ionotropic receptors: a new and fundamental physiological role for zinc and copper in neuronal and brain excitability. The review is focussed on ionotropic receptor channels including nucleotide receptors, in particular the P2X receptor family. Since zinc and copper are stored within synaptic vesicles in selected brain regions, and released to the synaptic cleft upon electrical nerve ending depolarization, it is plausible that zinc and copper reach concentrations in the synapse that profoundly affect ligand-gated ionic channels, including the ATP-gated currents of P2X receptors. The identification of key P2X receptor amino acids that act as ligands for trace metal coordination, carves the structural determinants underlying the allosteric nature of the trace metal modulation. The recognition that the identified key residues such as histidines, aspartic and glutamic acids or cysteines in the extracellular domain are different for each P2X receptor subtype and may be different for each metal, highlights the notion that each P2X receptor subtype evolved independent strategies for metal coordination, which form upon the proper three-dimensional folding of the receptor channels. The understanding of the molecular mechanism of allosteric modulation of ligand-operated ionic channels by trace metals is a new contribution to metallo-neurobiology.     

Formation of carnosine-Cu(II) complexes prevents and reverts the inhibitory action of copper in P2X4 and P2X7 receptors

To further analyze the action of copper on brain synaptic mechanisms, the brain dipeptide carnosine (beta-alanyl-L-histidine) was tested in Xenopus laevis oocytes expressing the rat P2X4 or P2X7 receptors. Ten micromolar copper halved the currents evoked by ATP in both receptors; co-application of carnosine plus copper prevented the metal induced-inhibition with a median effective concentration of 12.1 +/- 3.9 and 12.0 +/- 5.5 microm for P2X4 and P2X7, respectively. Zinc potentiated only the P2X4 ATP-evoked currents; carnosine had no effect over this metal. The relative potency and selectivity of classical metal chelators to prevent the copper inhibition was compared between carnosine and penicillamine (PA), bathophenanthroline (BPh) or L-histidine (His). Their rank order of potency in P2X4 and P2X7 receptors was carnosine = PA = His > BPh > Glycine (Gly) and carnosine = BPh = His > PA > Gly, respectively. The potency to prevent the zinc-induced potentiation in the P2X4 receptor was BPh > PA > His; carnosine, Gly and beta-alanine were inactive. Whereas 1-100 microm carnosine or His alone did not modify the ATP-evoked currents, 10-100 microm PA augmented and 100 microm BPh decreased the ATP-evoked currents. Carnosine was able to revert the copper-induced inhibition restoring the maximal ATP gated current in a concentration-dependent manner. Electronic spectroscopy confirm the formation of carnosine-Cu(II) complexes, mechanism that can account for the prevention and reversal of the copper inhibition, revealing its potential in copper intoxication treatment.     

Histidine 140 plays a key role in the inhibitory modulation of the P2X4 nucleotide receptor by copper but not zinc

To elucidate the role of extracellular histidines in the modulation of the rat P2X4 receptor by trace metals, we generated single, double, and triple histidine mutants for residues 140, 241, and 286, replacing them with alanines. cDNAs for the wild-type and receptor mutants were expressed in Xenopus laevis oocytes and in human embryonic kidney 293 cells and examined by the two electrode and patch clamp techniques, respectively. Whereas copper inhibited concentration-dependently the ATP-gated currents in the wild-type and in the single or double H241A and H286A receptor mutants, all receptors containing H140A were insensitive to copper in both cell systems. The characteristic bell-shaped concentration-response curve of zinc observed in the wild-type receptor became sigmoid in both oocytes and human embryonic kidney cells expressing the H140A mutant; in these mutants, the zinc potentiation was 2.5-4-fold larger than in the wild-type. Results with the H140T and H140R mutants further support the importance of a histidine residue at this position. We conclude that His-140 is critical for the action of copper, indicating that this histidine residue, but not His-241 or His-286, forms part of the inhibitory allosteric metal-binding site of the P2X4 receptor, which is distinct from the putative zinc facilitator binding site.     

Differential role of extracellular histidines in copper, zinc, magnesium and proton modulation of the P2X7 purinergic receptor

The P2X7 receptor is a non-selective cationic channel activated by extracellular ATP, belonging to the P2X receptor family. To assess the role of extracellular histidines on the allosteric modulation of the rat P2X7 receptor by divalent metals (copper, zinc and magnesium) and protons, these amino acid residues were singly substituted for corresponding alanines. Wild-type and mutated receptors were injected to Xenopus laevis oocytes; metal-related effects were evaluated by the two-electrode voltage-clamp technique. Copper inhibited the ATP-gated currents with a median inhibitory concentration of 4.4 +/- 1.0 micromol/L. The inhibition was non-competitive and time-dependent; copper was 60-fold more potent than zinc. The mutant H267A, resulted in a copper resistant receptor; mutants H201A and H130A were less sensitive to copper inhibition (p < 0.05). The rest of the mutants examined, H62A, H85A, and H219A, conserved the copper-induced inhibition. Only mutants H267A and H219A were less sensitive to the modulator action of zinc. Moreover, the magnesium-induced inhibition was abolished exclusively on the H130A and H201A mutants, suggesting that this metal may act at a novel cationic modulator site. Media acidification inhibited the ATP-gated current 87 +/- 3%; out of the six mutants examined, only H130A was significantly less sensitive to the change in pH, suggesting that His-130 could be involved as a pH sensor. In conclusion, while His-267 is critically involved in the copper or zinc allosteric modulation, the magnesium inhibitory effects is related to His-130 and His-201, His-130 is involved in proton sensing, highlighting the role of defined extracellular histidines in rat P2X7 receptor allosteric modulation.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Role of the electrostatic loop of Cu,Zn superoxide dismutase in the copper uptake process.

Cu,Zn superoxide dismutases are characterized by the presence of four highly conserved charged residues (Lys120, Glu/Asp130, Glu131 and Lys134), which are placed at the edge of the active site channel and have been shown to be individually involved in the electrostatic attraction of the substrate toward the catalytically active copper ion. By genetic engineering we mutated these four residues into neutrally charged ones (Leu120, Gln130, Gln131, Thr134). The effects of these mutations on the rate of superoxide dismutation were not dramatic. In fact, at two different pH and ionic strength values, the mutant enzyme had a catalytic constant even higher with respect to the wild-type protein, showing that electrostatic interaction at these surface sites is not essential for high catalytic efficiency of the enzyme. The mutant and the wild-type enzyme showed the same degree of inhibition by CN(-), and both were not affected by I(-), showing that mutations did not alter the sensitivity of the enzyme to anions. On the other hand, reconstitution of active enzyme from either the wild-type or mutant copper-free enzymes with a copper(I)-glutathione [Cu(I)-GSH] complex showed that metal uptake by the mutant was much slower than by the wild-type enzyme. The demonstration that the 'electrostatic loop' is apparently conserved to assure optimal copper uptake by the enzyme, rather than fast dismutation, may provide further support to the idea that Cu,Zn superoxide dismutase is a bifunctional protein, acting in cellular defense against oxidative stress both as a copper buffer and as a superoxide radical scavenger.     

Inactivation of human fibroblast growth factor-1 (FGF-1) activity by interaction with copper ions involves FGF-1 dimer formation induced by copper-catalyzed oxidation.

Although the angiogenic proteins acidic fibroblast growth factor (FGF-1) and basic fibroblast growth factor (FGF-2) both interact with the transition metal copper, itself a putative modulator of angiogenesis, a role for copper in FGF function has not been established. Using nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we detect the complete conversion of recombinant forms of human FGF-1 monomer protein to FGF-1 homodimers after exposure to copper ions. In contrast, not all forms of bovine FGF-1 isolated from bovine brain or a recombinant preparation of human FGF-2 completely formed homodimers after exposure to copper ions under similar conditions. Since the copper-induced FGF-1 homodimers reverted to the monomer form in the presence of dithiothreitol, specific alkylation of cysteine residues by pyridylethylation prevented FGF-1 homodimer formation, and preformed FGF-1 homodimers could not be dissociated by the metal chelator EDTA, FGF-1 dimer formation appeared to result from the formation of intermolecular disulfide bonds by copper-induced oxidation of sulfhydryl residues. FGF-1 homodimers bound with similar apparent affinity as FGF-1 monomers to immobilized copper ions, both eluting at 60 mM imidazole. Both human FGF-1 monomer and dimer forms had a 6-fold higher apparent affinity for immobilized copper ions, as compared with human FGF-2, which eluted in the monomer form at 10 mM imidazole. Further, in contrast to FGF-1 monomers, which dissociate from immobilized heparin in 1.0 M NaCl, preformed FGF-1 homodimers had reduced apparent affinity for immobilized heparin and eluted at 0.4 M NaCl. In contrast, the apparent affinity of human FGF-2 for immobilized heparin was unaffected after exposure to copper ions. Heparin appeared to modulate the formation of copper-induced intermolecular disulfide bonds for FGF-1 but not FGF-2, since co-incubation of heparin and copper with FGF-1 monomers resulted in dimers and other oligomeric complexes. FGF-1 copper-induced homodimers failed to induce mitogenesis in [3H]thymidine incorporation assays, an effect which could be reversed by treatment with dithiothreitol, whereas FGF-2-induced mitogenic activity was relatively unaffected by pretreatment with copper. The differences between human FGF-1 and FGF-2 in protein-copper interactions may be due to differing free thiol content and arrangement between the two proteins. A recombinant human FGF-1 mutant containing the two cysteines conserved throughout the FGF family of proteins but lacking a cysteine residue (Cys 131) present in wild-type human FGF-1 but not human FGF-2 readily formed copper-induced dimers.(ABSTRACT TRUNCATED AT 400 WORDS)     

The sulfhydryl group of Cys138 of rusticyanin from Acidithiobacillus ferrooxidans is crucial for copper binding

Rusticyanin is a small blue copper protein isolated from Acidithiobacillus ferrooxidans with extreme acid stability and redox potential. The protein is thought to be a principal component in the iron respiratory electron transport chain in this microorganism, but its exact role in electron transfer remains controversial. The gene of rusticyanin was cloned then overexpressed in Escherichia coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. It was reported that Cys138, His85 and His143 were important residues for copper binding, but the significance of Cys138 was not verified so far. We constructed the mutant expression plasmids of these three residues using site-directed mutagenesis. Mutant proteins were expressed in E. coli and purified with a nickel metal affinity column. The EPR and atomic absorption spectroscopy results confirmed that Cys138 was crucial for copper binding. Removal of the sulfhydryl group of Cys138 resulted in copper loss. Mutations of His85 and His143 showed little effect on copper binding.     

Insight to pyrazinamide resistance inMycobacterium tuberculosisby molecular docking

Pyrazinamide (PZA) - an important drug in the anti-tuberculosis therapy, activated by an enzyme Pyrazinamidase (PZase). The basis of PZA resistance in Mycobacterium tuberculosis was owing to mutation in pncA gene coding for PZase. Homology modeling of PZase was performed using software Discovery Studio (DS) 2.0 based on the crystal structure of the PZase from Pyrococcus horikoshii (PDB code 1im5), in this study. The model comprises of one sheet with six parallel strands and seven helices with the amino acids Asp8, Asp49, Trp68, Lys96, Ala134, Thr135 and Cys138 at the active site. Five mutants were generated with Gly at position 8, Thr at position 96, Arg at position 104, Tyr and Ser at position 138. The Wild-type (WT) and five mutant models were docked with PZA. The results indicate that the mutants Lys96Thr, Ser104Arg Asp8Gly and Cys138Tyr may contribute to higher level drug resistance than Cys138Ser. These models provide the first in-silico evidence for the binding interaction of PZA with PZase and form the basis for rationalization of PZA resistance in naturally occurring pncA mutant strains of M. tuberculosis.     

Zinc Potentiation of the Glycine Receptor Chloride Channel Is Mediated by Allosteric Pathways

Abstract: Molecular mechanisms of zinc potentiation were investigated in recombinant human α1 glycine receptors (GlyRs) by whole-cell patch-clamp recording and [³H]strychnine binding assays. In the wild-type (WT) GlyR, 1 µ M zinc enhanced the apparent binding affinity of the agonists glycine and taurine and reduced their concentrations required for half-maximal activation. Thus, in the WT GlyR, zinc potentiation apparently occurs by enhancing agonist binding. However, analysis of GlyRs incorporating mutations in the membrane-spanning domain M1–M2 and M2–M3 loops, which are both components of the agonist gating mechanism, indicates that most mutations uncoupled zinc potentiation from glycine-gated currents but preserved zinc potentiation of taurine-gated currents. One such mutation in the M2–M3 loop, L274A, abolished the ability of zinc to potentiate taurine binding but did not inhibit zinc potentiation of taurine-gated currents. In this same mutant where taurine acts as a partial agonist, zinc potentiated taurine-gated currents but did not potentiate taurine antagonism of glycine-gated currents, suggesting that zinc interacts selectively with the agonist transduction pathway. The intracellular M246A mutation, which is unlikely to bind zinc, also disrupted zinc potentiation of glycine currents. Thus, zinc potentiation of the GlyR is mediated via allosteric mechanisms that are independent of its effects on agonist binding.     

The human prion octarepeat fragment prevents and reverses the inhibitory action of copper in the P2X4 receptor without modifying the zinc action

Human prion protein fragments (PrP60-67 or PrP59-91) prevented and reversed the inhibition elicited by 5 micro m copper on the P2X4 receptor expressed in Xenopus laevis oocytes. A 60-s pre-application of 5 micro m copper caused a 69.2 +/- 2.6% inhibition of the 10 micro m adenosine triphosphate (ATP)-evoked currents, an effect that was prevented by mixing 5 micro m copper with 0.01-10 micro m of the PrP fragments 1-min prior to application. This interaction was selective, as PrP59-91 did not alter the facilitatory action of zinc. The EC50 of PrP60-67 and PrP59-91 for the reduction of the copper inhibition were 4.6 +/- 1 and 1.3 +/- 0.4 micro m, respectively. A synthetic PrP59-91 variant in which all four His were replaced by Ala was inactive. However, the replacement of Trp in each of the four putative copper-binding domains by Ala slightly decreased its potency. Furthermore, the application of 10 micro m PrP59-91 reversed the copper-evoked inhibition, restoring the ATP concentration curve to the same level as the non-inhibited state. Fragment 139-157 of betaA4 amyloid precursor protein also prevented the action of copper; its EC50 was 1.6 +/- 0.1 micro m; the metal chelator penicillamine was equipotent with PrP60-67, but carnosine was significantly less potent. Our findings highlight the role of PrP in copper homeostasis and hint at its possible role as a modulator of synapses regulated by this trace metal.     

Alanine-scanning mutagenesis in the signature disulfide loop of the glycine receptor alpha 1 subunit: critical residues for activation and modulation

The glycine receptor enables the generation of inhibitory postsynaptic currents at synapses via neurotransmitter-dependent activation. These receptors belong to the ligand-gated ion channel gene superfamily, in which all members are comprised of five subunits, each of which possesses a signature 13-residue disulfide loop (Cys loop) in the extracellular domain. In this study, we used alanine-scanning mutagenesis of the residues between C138 and C152 of the Cys loop of the glycine receptor alpha1 subunit to identify residues critical for receptor activation and allosteric modulation. Mutation of L142, F145, or P146 to alanine produced decreases in the potency, maximal amplitude, and Hill coefficient for currents elicited by glycine and impaired receptor activation by the agonist taurine. These residues, along with D148, are positionally conserved in the family of LGIC subunits. Mutation at several other positions had little or no effect. The inhaled anesthetics halothane and isoflurane potentiate submaximal agonist responses at wild-type receptors, via an allosteric site. The mutations L142A, F145A, P146A, and D148A abolished positive modulation by these anesthetics, in some cases revealing a small inhibitory effect. A molecular model of the glycine receptor alpha1 subunit suggests that the Cys loop is positioned in a region of the receptor at the interface between the extracellular and transmembrane domains and that the critical functional residues identified here lie along the face of a predominantly hydrophobic surface. The present data implicate the Cys loop as an important functional moiety in the process of glycine receptor activation and allosteric regulation by anesthetics.     

A new zinc-protein coordination site in intracellular metal trafficking: solution structure of the Apo and Zn(II) forms of ZntA(46-118)

Zinc, a metal ion that functions in a wide variety of catalytic and structural sites in metalloproteins, is shown here to adopt a novel coordination environment in the Escherichia coli transport protein ZntA. The ZntA protein is a P-type ATPase that pumps zinc out of the cytoplasm and into the periplasm. It is physiologically selective for Zn(II) and functions with metalloregulatory proteins in the cell to keep the zinc quota within strict limits. Yet, the N-terminal cytoplasmic domain contains a region that is highly homologous to the yeast Cu(I) metallochaperone Atx1. To investigate how the structure of this region may influence its function, this fragment, containing residues 46-118, has been cloned out of the gene and overexpressed. We report here the solution structure of this fragment as determined by NMR. Both the apo and Zn(II)-ZntA(46-118) structures have been determined. It contains a previously unknown protein coordination site for zinc that includes two cysteine residues, Cys59 and Cys62, and a carboxylate residue, Asp58. The solvent accessibility of this site is also remarkably high, a feature that increasingly appears to be a characteristic of domains of heavy metal ion transport proteins. The participation of Asp58 in this ZntA metal ion binding site may play an important role in modulating the relative affinities and metal exchange rates for Zn(II)/Pb(II)/Cd(II) as compared with other P-type ATPases, which are selective for Cu(I) or Ag(I).     

Molecular basis for the interaction of histamine with the histamine H2 receptor.

We undertook these studies to characterize the molecular basis of the interaction of histamine with the H2 receptor. Key areas of homology in the structures of the histamine H2 and beta 2 adrenergic receptor suggested specific transmembrane amino acids that might be important for binding of histamine. A third transmembrane aspartic acid of the histamine receptor (Asp98), thought to serve as a counter anion that interacts with the cationic amine moiety of histamine, was mutated to Asn98, and the mutated receptor was expressed in Hepa cells. Removal of the negatively charged amino acid abolished both binding of the H2 receptor antagonist [methyl-3H]tiotidine and histamine stimulated increases in cellular cAMP content. Mutation of a fifth transmembrane aspartic acid (Asp186) to Ala186 or Asn186 by itself or in conjunction with mutation of another fifth transmembrane amino acid (Thr190 to Ala190) resulted in a loss of [methyl-3H] tiotidine binding, although the generation of cAMP in response to histamine was maintained. The histamine receptor with only a Thr190 to Ala190 or Cys190 mutation retained the ability to bind [methyl-3H]tiotidine, but both the affinity and efficacy of binding were reduced. These data lead us to propose a model for histamine binding in which Asp98 is essential for histamine binding and action, Asp186 defines H2 selectivity, and Thr190 is important in establishing the kinetics of histamine binding, but is not essential for H2 selectivity.     

Zinc coordination sphere in biochemical zinc sites

Zinc is known to be indispensable to growth and development and transmission of the genetic message. It does this through a remarkable mosaic of zinc binding motifs that orchestrate all aspects of metabolism. There are now nearly 200 three dimensional structures for zinc proteins, representing all six classes of enzymes and covering a wide range of phyla and species. These structures provide standards of reference for the identity and nature of zinc ligands in other proteins for which only the primary structure is known. Three primary types of zinc sites are apparent from examination of these structures: structural, catalytic and cocatalytic. The most common amino acids that supply ligands to these sites are His, Glu, Asp and Cys. In catalytic sites zinc generally forms complexes with water and any three nitrogen, oxygen and sulfur donors with His being the predominant amino acid chosen. Water is always a ligand to such sites. Structural zinc sites have four protein ligands and no bound water molecule. Cys is the preferred ligand in such sites. Cocatalytic sites contain two or three metals in close proximity with two of the metals bridged by a side chain moiety of a single amino acid residue, such as Asp, Glu or His and sometimes a water molecule. Asp and His are the preferred amino acids for these sites. No Cys ligands are found in such sites. The scaffolding of the zinc sites is also important to the function and reactivity of the bound metal. The influence of zinc on quaternary protein structure has led to the identification of a fourth type of zinc binding site, protein inteface. In this case zinc sites are formed from ligands supplied from amino acid residues residing in the binding surface of two proteins. The resulting zinc site usually has the coordination properties of a catalytic or structural zinc binding site.     

Zinc and Copper Concentrations in Human Preterm Milk

Zinc and copper are important trace elements in the nutrition of preterm infants. This study determined and compared the concentrations of zinc and copper in preterm milk of mothers receiving and not receiving zinc supplementation diets. The effects of maternal supplementation on the blood levels and anthropometric parameters were evaluated. Thirty-eight mothers and their preterm infants were enrolled in the study. Eighteen mothers were given a daily supplementation of 50 mg zinc, whereas the other 20 were not. Zinc and copper levels in milk were determined at 15-day intervals, as were blood zinc levels along with anthropometric parameters. Zinc and copper contents were determined by atomic absorption spectrophotometry. No significant differences were found between groups either for zinc values in maternal milk and infant blood or for anthropometric measurements. A pronounced decrease in copper levels was observed in mothers receiving supplemental zinc. Zinc supplementation given to mothers who were breastfeeding preterm infants had no significant effect on zinc secretion in milk.     

Catalytic function and local proton structure at the type 2 copper of nitrite reductase: the correlation of enzymatic pH dependence,conserved residues,and proton hyperfine structure

Electron nuclear double resonance (ENDOR) of protons at Type 2 and Type 1 cupric active sites correlates with the enzymatic pH dependence, the mutation of nearby conserved, nonligating residues, and electron transfer in heterologously expressed Rhodobacter sphaeroides nitrite reductase. Wild-type enzyme showed a pH 6 activity maximum but no kinetic deuterium isotope effect, suggesting protons are not transferred in the rate-limiting step of nitrite reduction. However, protonatable Asp129 and His287, both located near the Type 2 center, modulated enzyme activity. ENDOR of the wild-type Type 2 center at pH 6.0 revealed an exchangeable proton with large hyperfine coupling. Dipolar distance estimates indicated that this proton was 2.50-2.75 or 2.25-2.45 A from Type 2 copper in the presence or absence of nitrite, respectively. This proton may provide a properly oriented hydrogen bond to enhance water formation upon nitrite reduction. This proton was eliminated at pH 5.0 and showed a diminished coupling at pH 7.5. Mutations of Asp129 and His287 reduced enzyme activity and altered the exchangeable proton hyperfine spectra. Mutation of Asp129 prevented a pH-dependent change at the Type 1 Cys167 ligand as observed by Cys C(beta) proton ENDOR, implying there is a Type 2 and pH-dependent alteration of the Type 1 center. Mutation of the Type 1 center ligand Met182 to Thr and mutation of Asp129 increased the activation energy for nitrite reduction. Involvement of both the Type 1 center and Asp129 in modulating activation energy shows that electron transfer from the Type 1 center to a nitrite-ligated Type 2 center is rate-limiting for nitrite reduction. Mutation of Ile289 to Ala and Val caused minor perturbation to enzyme activity, but as detected by ENDOR, allowed formate binding. Thus, bulky Ile289 may exclude non-nitrite ligands from the Type 2 active site.