Chemical and biochemical issues related to X-ray crystallography of the ligand-binding domain of estrogen receptor alpha.

Careful attention to technical issues preceded successful crystallography of the ligand-binding domain of estrogen receptor alpha (ERalpha) complexed with CP-336156, a nonsteroidal estrogen agonist/antagonist. An affinity column based on immobilized estradiol was prepared according to the scheme of Greene et al. (Greene, G. L., Nolan, C., Engler, J. P., and Jensen, E. V. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5115-5119). It was shown by X-ray crystallography that the major and less polar isomer of the affinity column precursor was 17alpha-((S)-2',3'-epoxyprop-1'-yl)estra-1,3,5(10)-triene-3,17beta-diol. This diastereomer was coupled to Thiopropyl Sepharose, with coupling monitored by observing loss of the phenolic absorption band of estradiol from the reaction supernatant, and gave an affinity matrix containing about 9 micromol of estradiol per milliliter of wet gel. Recombinant ERalpha ligand binding domain was selectively removed from E. coli cell lysate by binding to the column and was partly S-carboxymethylated by treatment with iodoacetic acid while bound to the column as described by previous workers. After being eluted from the column as a complex with drug, the receptor fragment was shown by mass spectrometry to be a mixture of differently modified forms. It was further S-carboxymethylated in solution, after which anion-exchange chromatography was used to isolate protein in which two of the four cysteine residues were S-carboxymethylated. This material, which afforded diffraction-quality crystals, was subjected to digestion with trypsin and peptide mapping analysis by HPLC coupled with mass spectrometry. For this experiment, the two previously unmodified cysteines were alkylated with 4-vinylpyridine to allow definitive identification. It was shown that Cys-417 and Cys-530 were S-carboxymethylated in the crystallized protein, while Cys-381 and Cys-447 remained unmodified. Close attention to such technical issues may be important in structural studies of other nuclear receptors, a very important class of potential drug targets.

Identification, characterization, and crystal structure of the Omega class glutathione transferases.

A new class of glutathione transferases has been discovered by analysis of the expressed sequence tag data base and sequence alignment. Glutathione S-transferases (GSTs) of the new class, named Omega, exist in several mammalian species and Caenorhabditis elegans. In humans, GSTO 1-1 is expressed in most tissues and exhibits glutathione-dependent thiol transferase and dehydroascorbate reductase activities characteristic of the glutaredoxins. The structure of GSTO 1-1 has been determined at 2.0-A resolution and has a characteristic GST fold (Protein Data Bank entry code ). The Omega class GSTs exhibit an unusual N-terminal extension that abuts the C terminus to form a novel structural unit. Unlike other mammalian GSTs, GSTO 1-1 appears to have an active site cysteine that can form a disulfide bond with glutathione.

Dye-pair reporter systems for protein-peptide molecular interactions.

Modifying a linear peptide near each terminus with a fluorescent dye can make it able to signal its own binding to a protein. As originally described, the dye pair is composed of fluorescein and tetramethylrhodamine [Wei, A.-P., Blumenthal, D. K., and Herron, J. N. (1994) Anal. Chem. 66, 1500-1506]. This paper shows that it may also be two molecules of tetramethylrhodamine. In aqueous solution, mutual affinity of the dyes causes fluorescence-quenching contact between them. When the peptide is bound by an antibody or cleaved by a proteinase, or when acetonitrile is added, dye-to-dye contact decreases and fluorescence increases 3-15-fold. When five peptides of 4-20 amino acid residues were doubly modified with tetramethylrhodamine, each product had the absorption spectrum of a tetramethylrhodamine dimer. As the peptides were not known to have special conformational features, self-affinity of the dye appeared to be the main cause of dimerization. Disruption of the dye dimers by acetonitrile suggested that dimerization of the dye(s) in aqueous solution was largely an effect of hydrophobicity. Dye-tagged peptides were used in fluorometric assays for two peptide-protein interactions. First, a peptide from type II collagen recognized by a monoclonal antibody was derivatized with two different dye pairs. The monoclonal bound each modified peptide, disrupting dye-to-dye contact and increasing fluorescence up to 4-fold. Second, a phosphopeptide recognized by an SH2 domain was tagged with fluorescein and tetramethylrhodamine, and its binding to the SH2 domain was detected through fluorescence. Doubly dye-tagged peptides offer a direct, solution-phase assay for protein-peptide binding.

Spontaneous alpha-N-6-phosphogluconoylation of a "His tag" in Escherichia coli: the cause of extra mass of 258 or 178 Da in fusion proteins.

Several proteins expressed in Escherichia coli with the N-terminus Gly-Ser-Ser-[His]6- consisted partly (up to 20%) of material with 178 Da of excess mass, sometimes accompanied by a smaller fraction with an excess 258 Da. The preponderance of unmodified material excluded mutation, and the extra masses were attributed to posttranslational modifications. As both types of modified protein were N-terminally blocked, the alpha-amino group was modified in each case. Phosphatase treatment converted +258-Da protein into +178-Da protein. The modified His tags were isolated, and the mass of the +178-Da modification estimated as 178.06 +/- 0.02 Da by tandem mass spectrometry. As the main modification remained at +178 Da in 15N-substituted protein, it was deemed nitrogen-free and possibly carbohydrate-like. Limited periodate oxidations suggested that the +258-Da modification was acylation with a 6-phosphohexonic acid, and that the +178-Da modification resulted from its dephosphorylation. NMR spectra of cell-derived +178-Da His tag and synthetic alpha-N-d-gluconoyl-His tag were identical. Together, these results suggested that the +258-Da modification was addition of a 6-phosphogluconoyl group. A plausible mechanism was acylation by 6-phosphoglucono-1,5-lactone, produced from glucose 6-phosphate by glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Supporting this, treating a His-tagged protein with excess d-glucono-1,5-lactone gave only N-terminal gluconoylation.

Heme accessibility in the ferricytochrome c-cytochrome c peroxidase complex.

Complex formation between ferricytochrome c and cytochrome c peroxidase inhibits the rate of cyanide binding by ferricytochrome c nearly 90%. The reactions between cytochrome c peroxidase and fluoride or hydrogen peroxide are not significantly affected by complex formation with cytochrome c.