NMR identification of heavy metal-binding sites in a synthetic zinc finger peptide: toxicological implications for the interactions of xenobiotic metals with zinc finger proteins.

Lead (Pb), mercury (Hg), and cadmium (Cd) are toxic and interfere with protein metal-binding sites. The Cys(2)/His(2) zinc finger is a structural motif required for sequence-specific DNA binding and is present in zinc finger transcription factors (ZFP): Sp1, Egr-1, and TFIIIA. Neurotoxic studies have shown that heavy metals directly inhibit the DNA binding of ZFP and result in adverse cellular effects. Recently, we demonstrated the ability of heavy metals to alter the DNA binding of a synthetic Cys(2)/His(2) finger peptide (Razmiafshari and Zawia, Toxicol. Appl. Pharmacol. 166, 1-12, 2000). To determine the precise site of interactions between heavy metals and this protein domain, Pb, Hg, Cd, and Ca were reconstituted with the synthetic apopeptide and studied by one- and two-dimensional NMR spectroscopy. In the presence of Zn, Cd, Hg, and Pb, but not Ca, distinct peptide NMR signal changes in the aliphatic region were observed and attributed to metal-cystiene interactions. However, chemical shifts indicative of metal-histidine binding were elicited by all the metals in the peptide's aromatic region. Chemical shift assignments and sequential connectivity were established in the presence and absence of Zn, Pb, and Ca through TOCSY and NOESY spectra. Cysteine and histidine residues showed a distinct change in their amide and beta resonances in the presence of Zn and Pb, suggesting the metal-ligand binding sites were near these residues. However, Ca led to no significant spectral changes in these regions, suggesting that it is not actively involved in the binding site. These studies reveal this structure as a mediator of metal-induced alterations in protein function.

3-Bromotyrosine and 3,5-dibromotyrosine are major products of protein oxidation by eosinophil peroxidase: potential markers for eosinophil-dependent tissue injury in vivo.

Detection of specific reaction products is a powerful approach for dissecting out pathways that mediate oxidative damage in vivo. Eosinophil peroxidase (EPO), an abundant protein secreted from activated eosinophils, has been implicated in promoting oxidative tissue injury in conditions such as asthma, allergic inflammatory disorders, cancer, and helminthic infections. This unique heme protein amplifies the oxidizing potential of H2O2 by utilizing plasma levels of Br- as a cosubstrate to form potent brominating agents. Brominated products might thus serve as powerful tools for identifying sites of eosinophil-mediated tissue injury in vivo; however, structural identification and characterization of specific brominated products formed during EPO-catalyzed oxidation have not yet been reported. Here we explore the role of EPO and myeloperoxidase (MPO), a related leukocyte protein, in promoting protein oxidative damage through bromination and demonstrate that protein tyrosine residues serve as endogenous traps of reactive brominating species forming stable ring-brominated adducts. Exposure of the amino acid L-tyrosine to EPO, H2O2, and physiological concentrations of halides (100 mM Cl-,

Synthesis, isolation, and characterization of the adduct formed in the reaction of p-hydroxyphenylacetaldehyde with the amino headgroup of phosphatidylethanolamine and phosphatidylserine.

The adducts that form when aldehydes modify proteins have been implicated in the pathogenesis of vascular disease and aging. Our previous studies indicated that p-hydroxyphenylacetaldehyde (pHA), the major product of L-tyrosine oxidation by the myeloperoxidase/hydrogen peroxide/chloride system of phagocytes, covalently modifies the epsilon-amino group of lysine residues at sites of inflammation. Here, we report that pHA also reacts with the amino group of synthetic phospholipids and red blood cell model systems. Using fast atom bombardment mass spectrometric analysis of ethanolamine glycerophospholipid or serine glycerophospholipid incubated with pHA and NaBH3CN, we detected products that were consistent with reduced phospholipid Schiff base adducts. We confirmed the reaction of the aldehyde with the amino group through 1H NMR and mass spectrometric analysis of polar headgroups recovered from the modified and reduced parent lipid. When phospholipid model systems and cell membranes were exposed to physiological levels of L-tyrosine and the myeloperoxidase/hydrogen peroxide/chloride system followed by treatment with NaBH3CN, reduced Schiff base adducts of pHA with ethanolamine glycerophospholipid and serine glycerophospholipid (pHA-PE and pHA-PS, respectively) were produced. The reaction required myeloperoxidase, hydrogen peroxide, L-tyrosine, and chloride ion; it was inhibited by catalase or heme poisons, implicating hydrogen peroxide and peroxidase in the pathway. Collectively, these results demonstrate that an aldehyde generated by the myeloperoxidase system of phagocytes can covalently modify the amino groups of phosphatidylethanolamine and phosphatidylserine. Because amino glycerophospholipids are critical components of cell membranes and circulating lipoproteins such as LDL, similar reactions may play important roles in the initiation or progression of disease at sites of inflammation.

Human neutrophils employ myeloperoxidase to convert alpha-amino acids to a battery of reactive aldehydes: a pathway for aldehyde generation at sites of inflammation.

We have recently demonstrated that activated phagocytes employ the heme protein myeloperoxidase, H2O2, and Cl- to oxidize the aromatic amino acid l-tyrosine to the reactive aldehyde p-hydroxyphenylacetaldehyde. We now present evidence for the generality of this reaction by demonstrating that neutrophils employ the myeloperoxidase-H2O2-Cl- system to oxidize nearly all of the common alpha-amino acids to yield a family of reactive aldehydes. Chemical characterization suggested that reactive carbonyl moieties were generated during amino acid oxidation by myeloperoxidase. The structures of amino-acid-derived aldehydes were confirmed using a variety of mass spectrometric methods. Aldehyde production required myeloperoxidase, H2O2, Cl-, and an amino acid; it was inhibited by heme poisons and catalase. Hypochlorous acid was the apparent oxidizing intermediate because its addition to alpha-amino acids resulted in the formation of the anticipated aldehyde. Stimulated human neutrophils likewise generated aldehydes from all classes of alpha-amino acids by a pathway inhibited by heme poisons and catalase, implicating myeloperoxidase and H2O2 in the cell-mediated reaction. Aldehyde production accounted for a significant fraction of the H2O2 generated by stimulated neutrophils at physiological concentrations of amino acids. Collectively, these results suggest that amino-acid-derived aldehydes represent a product of reactive oxidant species generated by activated phagocytes.

Human neutrophils employ the myeloperoxidase-hydrogen peroxide-chloride system to oxidize alpha-amino acids to a family of reactive aldehydes. Mechanistic studies identifying labile intermediates along the reaction pathway.

We have recently demonstrated that neutrophils oxidize nearly all of the amino acids commonly found in plasma to a corresponding family of aldehydes in high yield. The reaction is mediated by hypochlorous acid (HOCl), the major oxidant generated by the myeloperoxidase-H2O2-Cl- system of phagocytes. We now present evidence for the underlying mechanism of this reaction, including the structural requirements and reaction intermediates formed. Utilizing mass spectrometry and isotopically labeled amino acids, we rule out hydrogen atom abstraction from the alpha-carbon as the initial event in aldehyde formation during amino acid oxidation, a pathway known to occur with ionizing radiation. Aldehyde generation from amino acids required the presence of an alpha-amino moiety; beta- and epsilon-amino acids did not form aldehydes upon oxidation by either the myeloperoxidase system or HOCl, generating stable monochloramines instead. UV difference spectroscopy, high pressure liquid chromatography, and multinuclear (1H,15N) NMR spectroscopy established that the conversion of alpha-amino acids into aldehydes begins with generation of an unstable alpha-monochloramine, which subsequently decomposes to yield an aldehyde. Precursor product relationships between alpha-amino acid and alpha-monochloramine, and alpha-monochloramine and aldehyde were confirmed by high pressure liquid chromatography purification of the reaction intermediate and subsequent 1H and 15N NMR spectroscopy. Collectively, these results detail the chemical mechanism and reaction intermediates generated during conversion of amino acids into aldehydes by myeloperoxidase-generated HOCl.

p-Hydroxyphenylacetaldehyde, the major product of L-tyrosine oxidation by the myeloperoxidase-H2O2-chloride system of phagocytes, covalently modifies epsilon-amino groups of protein lysine residues.

Activated human phagocytes employ the myeloperoxidase-H2O2-Cl- system to convert L-tyrosine to p-hydroxyphenylacetaldehyde (pHA). We have explored the possibility that pHA covalently reacts with proteins to form Schiff base adducts, which may play a role in modifying targets at sites of inflammation. Because Schiff bases are labile to acid hydrolysis, prior to analysis the adducts were rendered stable by reduction with NaCNBH3. Purified pHA reacted with Nalpha-acetyllysine, an analog of protein lysine residues. The reduced reaction product was identified as Nalpha-acetyl-Nepsilon-(2-(p-hydroxyphenyl)ethyl)lysine by 1H NMR spectroscopy and mass spectrometry. The compound Nepsilon-(2-(p-hydroxyphenyl)ethyl)lysine (pHA-lysine) was likewise identified in acid hydrolysates of bovine serum albumin (BSA) that were first exposed to myeloperoxidase, H2O2, L-tyrosine, and Cl- and then reduced with NaCNBH3. Other halides (F-, Br-, I-) and the pseudohalide SCN- could not replace Cl- as a substrate in the myeloperoxidase-H2O2-L-tyrosine system. In the absence of the enzymatic system, pHA-lysine was detected in reduced reaction mixtures of BSA, L-tyrosine, and reagent HOCl. In contrast, pHA-lysine was undetectable when BSA was incubated with L-tyrosine and HOBr, peroxynitrite, hydroxyl radical, or a variety of other peroxidases, indicating that the aldehyde-protein adduct was selectively produced by HOCl. Human neutrophils activated in the presence of tyrosine also modified BSA lysine residues. pHA-lysine formation required L-tyrosine and cell activation; it was inhibited by peroxidase inhibitors and catalase, implicating myeloperoxidase and H2O2 in the reaction pathway. pHA-lysine was detected in inflamed human tissues that were reduced, hydrolyzed, and then analyzed by mass spectrometry, indicating that the reaction of pHA with proteins may be of physiological importance. These observations raise the possibility that the identification of pHA-lysine in tissues will pinpoint targets where phagocytes inflict oxidative damage in vivo.

Differential binding of retinol analogs to two homologous cellular retinol-binding proteins.

A comparative study of the interactions of rat cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) with a number of synthetic phenyl-substituted analogs of all-trans-retinol was performed using fluorescence and nuclear magnetic resonance analysis. These studies indicate that CRBP II is more sensitive to modifications of the ring moiety than CRBP. Removal of the two methyl substituents on the ring which are ortho to the polyene chain abolishes binding to CRBP II. Conformational analysis of the ligands indicates that these two methyl groups influence the planarity of the ligand. The identification of monospecific ligands may prove useful for studying the physiological roles of these two proteins.

Alteration of the binding specificity of cellular retinol-binding protein II by site-directed mutagenesis.

Rat cellular retinol-binding protein II (CRBP II) is an abundant 134-residue intestinal protein that binds all-trans-retinol and all-trans-retinal. It belongs to a family of homologous, 15-kDa cytoplasmic proteins that bind hydrophobic ligands in a noncovalent fashion. These binding proteins include a number of proteins that bind long chain fatty acids. X-ray analyses of the structure of two family members, rat intestinal fatty acid-binding protein and bovine myelin P2 protein, indicate that they have a high degree of conformational similarity and that the carboxylate group of their bound fatty acid interacts with a delta-guanidium group of at least 1 of 2 "buried" arginine residues. These 2 Arg residues are conserved in other family members that bind long chain fatty acids and in cellular retinoic acid-binding protein, but are replaced by Gln109 and Gln129 in CRBP II. We have genetically engineered two amino acid substitutions in CRBP II: 1) Gln109 to Arg and 2) Gln129 to Arg. The purified Escherichia coli-derived CRBP II mutant proteins were analyzed by fluorescence and nuclear magnetic resonance spectroscopy. Both mutants exhibit markedly decreased binding of all-trans-retinol and all-trans-retinaldehyde, but no increased binding of all-trans-retinoic acid. Arg substitution for Gln109 but not for Gln129 produces a dramatic increase in palmitate binding activity. Analysis of the endogenous fatty acids associated with the purified E. coli-derived proteins revealed that E. coli-derived intestinal fatty acid binding protein and the Arg109 CRBP II mutant are complexed with endogenous fatty acids in a qualitatively and quantitatively similar manner. These results provide evidence that this internal Arg may play an important role in the binding of long chain fatty acids by members of this protein family.

Fluorine nuclear magnetic resonance analysis of the ligand binding properties of two homologous rat cellular retinol-binding proteins expressed in Escherichia coli.

Comparative 19F NMR studies were performed on rat cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBPII) to better understand their role in intracellular retinol metabolism within the polarized absorptive epithelial cells (enterocytes) of the intestine. Efficient incorporation of 6-fluorotryptophan (6-FTrp) into these homologous proteins was achieved by growing a tryptophan auxotroph of Escherichia coli, harboring prokaryotic expression vectors with either a full-length rat CRBPII or CRBP cDNA on defined medium supplemented with the analog. It is possible to easily distinguish resonances corresponding to 6-FTrp-apoCRBP, 6-FTrp-CRBP-retinol (or retinal), 6-FTrp-apoCRBPII, and 6-FTrp-CRBPII-retinol (or retinal). We were thus able to use 19F NMR spectroscopy to monitor transfer of all-trans-retinol and all-trans-retinal between CRBPII and CRBP in vitro. Retinol complexed to CRBPII is readily transferred to CRBP, whereas retinol complexed to CRBP is not readily transferred to CRBPII. We estimated that the Kd for CRBP-retinol is approximately 100-fold less than the Kd for CRBPII-retinol. Transfer of all-trans-retinal occurs readily from CRBPII to CRBP and from CRBP to CRBPII. Results from competitive binding studies with retinol and retinal indicated that there is a much larger difference between the affinities of CRBP for retinol and retinal than between the affinities of CRBPII for these two ligands. However, the differences in binding specificities reflect differences in how the two proteins interact with retinol, rather than with retinal. 19F NMR analysis of recombinant isotopically labeled proteins represents a sensitive new and useful method for monitoring retinoid flux between the CRBPs in vitro.

19F nuclear magnetic resonance studies of 6-fluorotryptophan-substituted rat cellular retinol binding protein II produced in Escherichia coli. An analysis of four tryptophan substitution mutants and their interactions with all-trans-retinol.

Rat cellular retinol binding protein (CRBP II) is a 134-amino acid intracellular protein synthesized in the polarized absorptive cells of the intestine. We have previously used 19F nuclear magnetic resonance (NMR) spectroscopy to survey the structural effects of ligand binding on the apoprotein. For these studies, all 4 Trp residues of rat CRBP II were efficiently labeled with 6-fluorotryptophan (6-F-Trp) by inducing its expression in a tryptophan auxotroph of Escherichia coli. Resonances corresponding to 2 of its Trp residues underwent large downfield shifts upon binding of all-trans-retinol and retinal, while resonances corresponding to the other 2 Trp residues underwent only minor perturbations in chemical shifts. To identify which Trp residues undergo changes in their environment upon ligand binding, we have constructed four CRBP II mutants where Trp9, Trp89, Trp107, or Trp110 have been replaced by another hydrophobic amino acid. By comparing the 19F NMR spectrum of each 6-F-Trp-labeled mutant with that of wild type 6-F-Trp CRBP II, we demonstrate that the 19F resonance corresponding to Trp107 undergoes the largest change in chemical shift upon ligand binding (2.0 ppm downfield). This is consistent with the position of this residue predicted from molecular modeling studies. The 19F resonance corresponding to Trp9 also undergoes a downfield change in chemical shift of 0.5 ppm associated with retinol binding even though it is predicted to be removed from the ligand binding site. By contrast, the resonances assigned to Trp89 and Trp110 undergo only minor perturbations in chemical shifts. These results have allowed us to identify residue-specific probes for evaluating the interactions of all-trans-retinol (and other retinoids) with this intracellular binding protein.

Nuclear magnetic resonance studies of 6-fluorotryptophan-substituted rat cellular retinol-binding protein II produced in Escherichia coli. Analysis of the apoprotein and the holoprotein containing bound all-trans-retinol and all-trans-retinal.

Rat cellular retinol-binding protein II (CRBP II) is a 15.6-kDa intestinal protein which binds all-trans-retinol and all-trans-retinal but not all-trans-retinoic acid. We have previously analyzed the interaction of Escherichia coli-derived rat apoCRBP II with several retinoids using fluorescence spectroscopic techniques. Interpretation of these experiments is complicated, because the protein has 4 tryptophan residues. To further investigate ligand-protein interactions, we have utilized 19F nuclear magnetic resonance (NMR) spectroscopy of CRBP II labeled at its 4 tryptophan residues with 6-fluorotryptophan. Efficient incorporation of 6-fluorotryptophan (93%) was achieved by growing a tryptophan auxotroph of E. coli harboring a prokaryotic expression vector with a full-length rat CRBP II cDNA on defined medium supplemented with the analog. Comparison of the 19F NMR spectra of 6-fluorotryptophan-substituted CRBP II with and without bound all-trans-retinol revealed that resonances corresponding to 2 tryptophan residues (designated WA and WB) undergo large downfield changes in chemical shifts (2.0 and 0.5 ppm, respectively) associated with ligand binding. In contrast, 19F resonances corresponding to two other tryptophan residues (WC and WD) undergo only minor perturbations in chemical shifts. The 19F NMR spectra of 6-fluorotryptophan-substituted CRBP II complexed with all-trans-retinal and all-trans-retinol were very similar, suggesting that the interactions of these two ligands with the protein are similar. Molecular model building, based on the crystalline structures of two homologous proteins was used to predict the positions of the 4 tryptophan residues of CRBP II and to make tentative resonance assignments. The fact that ligand binding produced residue-specific changes in the chemical shifts of resonances in CRBP II suggests that NMR analysis of isotopically labeled retinoid-binding proteins expressed in E. coli will provide an alternate, albeit it complementary, approach to fluorescence spectroscopy for examining the structural consequences of their association with ligand.