Joseph E. Coleman

Crystal structure of the RAG1 dimerization domain reveals multiple zinc-binding motifs including a novel zinc binuclear cluster.

The crystal structure of the dimerization domain of the V(D)J recombination-activating protein, RAG1, was solved using zinc anomalous scattering. The structure reveals an unusual combination of multi-class zinc-binding motifs, including a zinc RING finger and a C2H2 zinc finger, that together form a single structural domain. The domain also contains a unique zinc binuclear cluster in place of a normally mononuclear zinc site in the RING finger. Together, four zinc ions help organize the entire domain, including the two helices that form the dimer interface.

The RING finger motif of photomorphogenic repressor COP1 specifically interacts with the RING-H2 motif of a novel Arabidopsis protein.

The constitutive photomorphogenic 1 (COP1) protein of Arabidopsis functions as a molecular switch for the seedling developmental fates: photomorphogenesis under light conditions and skotomorphogenesis in darkness. The COP1 protein contains a cysteine-rich zinc-binding RING finger motif found in diverse groups of regulatory proteins. To understand the role of the COP1 RING finger in mediating protein-protein interaction, we have performed a yeast two-hybrid screen and isolated a novel protein with a RING-H2 motif, a variant type of the RING finger. This protein, designated COP1 Interacting Protein 8 (CIP8), is encoded by a single copy gene and localized to cytosol in a transient assay. In addition to the RING-H2 motif, the predicted protein has a C4 zinc finger, an acidic region, a glycine-rich cluster, and a serine-rich cluster. The COP1 RING finger and the CIP8 RING-H2 domains are sufficient for their interaction with each other bothin vitro and in yeast, whereas neither motif displayed significant self-association. Moreover, site-directed mutagenesis studies demonstrated that the expected zinc-binding ligands of the RING finger and RING-H2 fingers are essential for their interaction. Our findings indicate that the RING finger motif, in this case, serves as autonomous protein-protein interaction domain. The allele specific effect of cop1 mutations on the CIP8 protein accumulation in seedlings indicates that its stability in vivo is dependent on the COP1 protein.

113Cd NMR as a probe of the active sites of metalloenzymes

113Cd NMR has been used to study the active site metal ion(s) of the 113Cd(II) derivatives of four Zn(II) metalloenzymes, carboxypeptidase A, carbonic anhydrases, alkaline phosphatase, and superoxide dismutase. The resonances of the enzyme-bound 113Cd(II) ions are extremely sensitive to ligand exchange, including solvent and inhibitor, and to changes in the metal ion coordination sphere. The nature of this behavior can be shown to parallel the known structural properties and proposed roles of the metal ion in the catalytic mechanisms. Models accounting for the exchange mechanisms which may be modulating the chemical shift, linewidth, and coupling are discussed.

The role of Zn(II) in transcription by T7 RNA polymerase

Abstract Homogeneous T7 RNA polymerase contains from 2–4 gm atoms of zinc per mole of M.W. 107,000. Inactivated molecules which can be separated from the active molecules by repeated chromatography contain less zinc, from 0.4 to 1 gm at per mole. Instability of the enzyme makes it difficult to relate maximal activity to a specific stoichiometry of Zn. The enzyme is inhibited by 1,10-phenanthroline, EDTA, CN − , SH − , N 3 − and by incubation with Chelex resin. Zinc is retained on gel filtration, but can be removed by dialysis for 96 hr against 5 mM 1,10-phenanthroline which totally inactivates the enzyme. Catalytic activity requires the presence of thiol reagents. Preparations with low activity can be activated by exogenous Zn ions.

Autoxidation of fatty materials in emulsions. I. Pro-oxidant effect of histidine and trace metals on the oxidation of linoleate esters

Aqueous emulsions of methyl or ethyl linoleate (sodium dodecylsulfate as emulsifier) together with such added components as 1-histidine, metal chlorides, buffers, and acid or alkali, were oxidized in the dark with shaking in an oxygen atmosphere. Under optimum conditions (pH 6.5), the linoleate peroxide content, after 2 hr autoxidation at 20C, was increased more than 3-fold by the addition of 1 ppm of ferrous or ferric ions, approximately 20-fold by a 0.01 M concentration of histidine and more than 60-fold by the addition of both histidine and ionic iron. The pro-oxidative effect of other transition metal ions (Cu++, Co++, Cr+++, Mn++, and Ni++) also was investigated. None of these ions had a significant effect alone. Combined with 0.01 M histidine, only Mn++ increased peroxidation over that when when histidine alone was added.The pro-oxidative action of histidine was retarded approximately 60% by 0.1 N acetate buffer and completely repressed by 0.05 M phosphate, nonionic emulsifiers, and low and high pH. The threshold concentration of histidine necessary for pro-oxidative action was greater than 0.0001 M.

Zinc metalloproteins involved in replication and transcription

RNA polymerase (RPase) from E. coli contains two tightly incorporated Zn(II) ions, while the monomeric RPase from bacteriophage T7 does not contain zinc and does not require Zn(II) in the assay. One of the two Zn(II) ions can be differentially removed from E. coli RPase with p-hydroxymercuriphenylsulfonate (PMPS) combined with EDTA and thiol. The resultant Znl or ZnA RPase shows no alteration in transcription initiation and elongation rate from sigma-specific promoters. Biosynthesis of a Co2 RPase and formation of CoA RPase by similar treatment shows the tetrahedral-type Co(II) d-d absorption bands to be associated only with the Co(II) at the A site with maxima at 760 (epsilon = 800), 710 (epsilon = 900), 602 (epsilon = 1500), and 484 (epsilon = 4000) nm. Sulfur to Co(II) charge transfer bands are present at 350 (epsilon = 9600) and 370 (epsilon = 9500) nm. The absorption characteristics strongly suggest that the A site is a tetrathiolate site. While DNA polymerases do not in general appear to contain zinc, gene 32 protein (g32P) from bacteriophage T4, an accessory protein essential for DNA replication and recombination and translational control in the T4 life cycle, is a Zn(II) metalloprotein and contains 1 gram atom of tightly incorporated Zn(II). PMPS displaces the zinc by reacting with three SH groups. Apo-g32P shows markedly altered DNA binding properties. Co(II) substitution gives a protein with intense d-d transitions typical of a tetrahedral Co(II) complex with absorption maxima at 680 (epsilon = 480), 645 (epsilon = 660), 605 (epsilon = 430), 355 (epsilon = 2250), and 320 (epsilon = 3175) nm. The data support a 3 Cys, 1 His coordination site located in the middle of the DNA binding domain of g32P. Data thus far suggest that the Zn(II) binding sites in multisubunit RNA polymerases and in accessory proteins involved in polynucleotide biosynthesis are more likely to play structural or allosteric (regulatory) roles rather than directly participating in catalysis.

Reactions of fatty materials with oxygen. XVII. Some observations on the secondary products of autoxidation of methyl oleate

SummaryMethyl oleate, autoxidized for short and long periods of time, has been fractionated with urea. Up to a peroxide content of about 15% the autoxidation mixture can be cleanly separated into a peroxide concentrate containing 90% peroxide and unoxidized methyl oleate. From about 15% peroxide to the maximum peroxide content (35–40%) concentration to only about 70% peroxide can be obtained, and the remaining material is largely a mixture of oxygenated compounds and residual methyl oleate.If the autoxidation is conducted beyond the peak value in peroxide content, little, if any, concentration of peroxide can be obtained. Also, beyond the peak in peroxide value and in the range of 30–20% peroxide, methyl oleate is substantially absent and the autoxidation mixture consists almost entirely of oxygenated compounds containing only one functional group in the chain.Evidence is presented which shows that in the autoxidation of methyl oleate substantially all of it undergoes single attack by oxygen (or peroxides) before any significant quantity of multiple attack occurs.α,β-Unsaturated carbonyl compounds are among the important secondary products of autoxidation.

Reactions of fatty materials with oxygen. XX. Recent developments in the autoxidation of methyl oleate and other monounsaturated fatty materials

SummarySome significant developments since 1947 in the autoxidation of methyl oleate and other monounsaturated fatty materials have been reviewed and critically evaluated. Subjects discussed are preparation and characterization of hydroperoxides, and mechanism, kinetics, and secondary products of autoxidation. Major developments in the field have resulted largely from the use of newer instruments (polarograph, infrared spectrophotometer) and separation techniques (urea complexes, molecular distillation, countercurrent distribution).Direct experimental evidence is now available which demonstrates that a) hydroperoxides are the predominating, but not the exclusive, primary products of autoxidation; b) the hydroperoxides obtained from methyl oleate are mostly, if not entirely,trans; c) substantially all the methyl oleate undergoes single attack in the chain before any significant amount of multiple attack occurs, and d) α,β-unsaturated carbonyl compounds are among the most important secondary products of autoxidation.

Metalloalkaline phosphatases from Bacillus subtilis: Physicochemical and enzymatic properties

The physicochemical, enzymatic, and metal-binding properties of the alkaline phosphatase from the gram positive organism, B. subtilis, are described. The native protein is a dimer of MW ∼100,000 containing 2.48±0.5 g atoms of zinc. The most active preparations of the homogeneous enzyme catalyze the hydrolysis of p-nitrophenylphosphate at a maximum velocity of ∼8100 μmoles/hr/mg enzyme dimer, over twice the specific activity of the crystalline enzyme from the gram negative organism, E. coli. The metal-free apoenzyme prepared by removal of the metal atom with Chelex resin is inactive. Of the first transition and IIB metals only Zn(II) and Co(II) restore activity to the metal-free apoenzyme. Addition of Co(II) results in an enzyme with twice the activity of the reconstituted. Zn(II) enzyme. The enzyme activity is stimulated threefold by increasing the ionic strength from 0.1 to 1.0. The enzyme also catalyzes phosphoryl transfer from p-nitrophenylphosphate to Tris. The visible absorption spectrum of the Co(II) enzyme shows the metal coordination site to be almost identical to that present in the enzyme from E. coli. Visible absorption maxima of the Co(II) enzyme occur at 620 (ɛ=305), 596 (ɛ=335), 567 (ɛ=500), and 517 nm (ɛ=382).

Autoxidation of fatty materials in emulsion. II. Factors affecting the histidine-catalyzed autoxidation of emulsified methyl linoleate

Several factors which affect autoxidation of methyl linoleate in emulsion have been examined. Data are presented which indicate: 1) In the presence of histidine, the ionic (anionic) emulsifiers examined promote autoxidation of emulsified methyl linoleate, but nonionic emulsifiers do not. 2) The concentration of an emulsifier affects the rate of oxygen absorption. 3) Inorganic salts (0.1 M or less) such as sodium chloride, sodium acetate and sodium sulfate affect oxygen absorption of emulsified methyl linoleate prepared with either ionic or nonionic emulsifiers. In histidine-catalyzed autoxidation there is a suppressing effect in the case of the ionic and a promotional effect in the case of the nonionic. In uncatalyzed autoxidation, these salts have a promotional effect in ionic emulsions and none in nonionic emulsions. 4) Sodium phosphate buffers completely suppress autoxidation due to histidine catalysis, but do not suppress the normal uncatalyzed autoxidation of emulsified methyl linoleate. 5) The pro-oxidative effects of histidine and histidine-metal ion complexes on emulsified unsaturated materials is not limited to polyolefins but also includes mono-olefinic compounds.