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This invited Letter commemorates the life and scientific legacy of Dr. Herbert Tabor (1918–1920), a leading scientist at the National Institutes of Health in Bethesda Maryland and former Chief Editor of the Journal of Biological Chemistry.

The dependence of the conformation of the S-adenosylmethionine (SAM) II riboswitch on the concentration of added Mg(2+) ions and SAM, individually and in mixtures, was monitored by circular dichroism (CD) spectroscopy and by measurement of the diffusion coefficient. The results are analyzed in the context of two complementary quantitative models, both of which are consistent with a single underlying physical model. Magnesium binding sites in the open state have an affinity on average higher than the affinity of those in the compact state, but formation of the compact state is accompanied by an increase in the number of binding sites. Consequently, at low Mg(2+) concentrations, Mg(2+) binds preferentially to the open state, favoring its formation, but at high concentrations, Mg(2+) binds preferentially to the compact state. The affinity of the riboswitch for SAM increases drastically with an increased level of binding of Mg(2+) to the compact pseudoknot conformation. The effect of increasing concentrations of trimethylamine N-oxide (TMAO), a well-studied molecular crowding agent, on the conformation of the riboswitch and its affinity for SAM were also monitored by CD spectroscopy and measurement of diffusion. In the absence of added Mg(2+), high concentrations of TMAO were found to induce a conformational change compatible with the formation of the pseudoknot form but have only a small effect on the affinity of the RNA for SAM.

The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro(216)-Phe(228) loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors.

Saccharomyces cerevisiae antizyme (AZ) resembles mammalian AZ in its mode of synthesis by translational frameshifting and its ability to inhibit and facilitate the degradation of ornithine decarboxylase (ODC). Despite many studies on the interaction of AZ and ODC, the ODC:AZ complex has not been purified from any source and thus clear information about the stoichiometry of the complex is still lacking. In this study we have studied the yeast antizyme protein and the ODC:AZ complex. The far UV CD spectrum of the full-length antizyme shows that the yeast protein consists of 51% β-sheet, 19% α-helix, and 24% coils. Surface plasmon resonance analyses show that the association constant (KA) between yeast AZ and yeast ODC is 6 × 107 (M−1). Using purified His-tagged AZ as a binding partner, we have purified the ODC:AZ inhibitory complex. The isolated complex has no ODC activity. The molecular weight of the complex is 90 kDa, which indicates a one to one stoichiometric binding of AZ and ODC in vitro. Comparison of the circular dichroism (CD) spectra of the two individual proteins and of the ODC:AZ complex shows a change in the secondary structure in the complex.

A wide range of parameters influence allosteric communications between the α- and β-subunits of the Trp synthase α2β2 multienzyme complex with L-Ser, including monovalent cations, pH, temperature, ligands, organic solvents, and hydrostatic pressure. The conformational change from closed to open can be monitored either by absorbance at 423 nm or fluorescence at 495 nm from the pyridoxal-5′-phosphate-L-Ser complex. Pressure perturbation was used to quantify the effects of monovalent cations, ligands, and mutations on the conformational equilibrium of Trp synthase. P-jump kinetics in the presence of Na+, NH4+, and Na+ together with benzimidazole were also examined. The plots of lnk versus P are nonlinear and require a compressibility (β‡o) term to obtain a good fit. β‡o is positive for the Na+ enzyme but negative for NH4+ and Na+ with benzimidazole. These results suggest that there is a large contribution of solvation to the kinetics of the conformational change of Trp synthase. The relaxation kinetics are also different if the P-jumps are made by increasing or decreasing pressure, suggesting that the enzyme conformations are ensembles of microstates.

The extracellular curli proteins of Enterobacteriaceae form fibrous structures that are involved in biofilm formation and adhesion to host cells. These curli fibrils are considered a functional amyloid because they are not a consequence of misfolding, but they have many of the properties of protein amyloid. We confirm that fibrils formed by CsgA and CsgB, the primary curli proteins of Escherichia coli, possess many of the hallmarks typical of amyloid. Moreover we demonstrate that curli fibrils possess the cross-beta structure that distinguishes protein amyloid. However, solid state NMR experiments indicate that curli structure is not based on an in-register parallel beta-sheet architecture, which is common to many human disease-associated amyloids and the yeast prion amyloids. Solid state NMR and electron microscopy data are consistent with a beta-helix-like structure but are not sufficient to establish such a structure definitively.

The cross-linking of polysaccharides to assemble new cell wall in fungi requires mechanisms by which a preexisting linkage is broken for each new one made, to allow for the absence of free energy sources outside the plasma membrane. Previous work showed that Crh1p and Crh2p, putative transglycosylases, are required for the linkage of chitin to β(1–3)glucose branches of β(1–6)glucan in the cell wall of budding yeast. To explore the linking reaction in vivo and in vitro, we used fluorescent sulforhodamine-linked laminari-oligosaccharides as artificial chitin acceptors. In vivo, fluorescence was detected in bud scars and at a lower level in the cell contour, both being dependent on the CRH genes. The linking reaction was also shown in digitonin-permeabilized cells, with UDP-N-acetylglucosamine as the substrate for nascent chitin production. Both the nucleotide and the Crh proteins were required here. A gas1 mutant that overexpresses Crh1p showed very high fluorescence both in intact and permeabilized cells. In the latter, fluorescence was still incorporated in patches in the absence of UDP-GlcNAc. Isolated cell walls of this strain, when incubated with sulforhodamine-oligosaccharide, also showed Crhp-dependent fluorescence in patches, which were identified as bud scars. In all three systems, binding of the fluorescent material to chitin was verified by chitinase digestion. Moreover, the cell wall reaction was inhibited by chitooligosaccharides. These results demonstrate that the Crh proteins act by transferring chitin chains to β(1–6)glucan, with a newly observed high activity in the bud scar. The importance of transglycosylation for cell wall assembly is thus firmly established.

Tryptophan synthase is an alpha2beta2 multienzyme complex that exhibits coupling of the alpha- and beta-subunit reactions by tightly controlled allosteric interactions. A wide range of parameters can affect the allosteric interactions, including monovalent cations, pH, alpha-site and beta-site ligands, temperature, and pressure. Rapid changes in hydrostatic pressure (P-jump) and temperature (T-jump) were used to examine the effects of pressure and temperature on the rates of the interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex. The intense fluorescence emission of the Tryptophan synthase L-Ser external aldimine complex at 495 nm, with 420 nm excitation, provides a probe of the conformational state of Trp synthase. P-jump measurements allowed the determination of rate constants for the reactions in the presence of Na(+), Na(+) with benzimidazole (BZI), and NH4(+). The data require a compressibility term, beta(o)(double dagger), to obtain good fits, especially for the NH4(+) and BZI/Na(+) data. The compressibility changes are consistent with changes in solvation in the transition state. The transition state for the relaxation is more similar in volume to the closed aminoacrylate complex in the presence of Na(+), while it is more similar to the open external aldimine in the presence of NH4(+). Differences between the relaxations for positive and negative P-jumps may arise from changing relative populations of microstates with pressure. T-jump experiments of the Na(+) form of the tryptophan synthase-L-Ser complex show large changes in rate and amplitude over the temperature range from 7 to 45 degrees C. The Arrhenius plots show strong curvature, and hence require a heat capacity term, DeltaC(p)(double dagger), to obtain good fits. The values of DeltaC(p)(double dagger) are very large and negative (-3.6 to -4.4 kJ mol(-1) K(-1)). These changes are also consistent with large changes in solvation in the transition state for interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex.

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of hypusine (Nϵ-(4-amino-2-hydroxybutyl)lysine) in eIF5A. DOHH is a HEAT-repeat protein with eight tandem helical hairpins in a symmetrical dyad. It contains two potential iron coordination sites (one on each dyad) composed of two strictly conserved His-Glu motifs. The purified human recombinant DOHH was a mixture of active holoenzyme containing 2 mol of iron/mol of DOHH and inactive metal-free apoenzyme. The two species could be distinguished by their different mobilities upon native gel electrophoresis. The DOHH apoenzyme exhibited markedly reduced levels of iron and activity. DOHH activity could be restored only by the addition of Fe2+ to the apoenzyme but not by other metals including Cd2+,Co2+,Cr2+,Cu2+,Mg2+,Mn2+,Ni2+, and Zn2+. The role of the strictly conserved His-Glu residues was evaluated by site-directed mutagenesis. Substitution of any single amino acid in the four His-Glu motifs with alanine abolished the enzyme activity. Of these eight alanine substitutions, six, including H56A, H89A, E90A, H207A, H240A, and E241A, caused a severe reduction in the iron content. Our results provide strong evidence that Fe(II) is the active-site-bound metal critical for DOHH catalysis and that the strictly conserved His-Glu motifs are essential for iron binding and catalysis. Furthermore, the iron to DOHH stoichiometry and dependence of iron binding on each of the four conserved His-Glu motifs suggest a binuclear iron mediated reaction mechanism, distinct from that of other Fe(II)-dependent protein hydroxylases, such as prolyl 4-hydroxylase or lysyl hydroxylases.

Spinocerebellar ataxia type 10 is an autosomal dominant disorder caused by expansion of a pentameric repeat tract (ATTCT.AGAAT)(n) in intron 9 of the gene that encodes ataxin-10. We have analyzed duplex DNA containing the repeat, the individual DNA strands, and the RNA that would be generated by transcription of the repeat. Circular dichroism and UV absorbance measurements suggest that the previously reported tendency of the repeat to unpair when supercoiled is probably related simply to GC content rather than reflecting any unusual property of the duplex. DNA containing d(ATTCT)9 forms a folded structure at relatively low temperatures, whereas the antisense strand, d(AGAAT)9, does not form a structure even at 0 degrees C. In contrast r(AUUCU)9 forms a folded structure under physiologically reasonable conditions. S1 nuclease analysis reveals a single region of hypersensitivity in the middle of the repeat tract, whereas V1 digestion is consistent with a hydrogen bonded or well stacked structure. CD spectroscopy shows that the structure is unimolecular and hydrogen bonded and has a significant amount of A-form helix. NMR spectroscopy demonstrates that these hydrogen bonds comprise an equal number of A.U and U.U base pairs. Our data thus suggest that the repeat forms an unusual RNA hairpin. Thus the ability to form an RNA hairpin seems to be a common property of those Repeat Expansion Diseases that are not recessively inherited and are caused by repeats that are transcribed but not translated.

All 20 cysteine residues are accessible to disulphide reagents in the tubulin dimer, whereas only four are accessible in taxol-stabilized microtubules. Reaction rates with disulphide reagents are a function of the reagent, are decreased by G nucleotides, and increased with increase in pH and urea. With transient (stop-flow) kinetics, DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)] and 2,2′-dithiodipyridine progress curves cannot be fitted by the sum of exponential terms based only on classes of cysteines. The mixed disulphide products react further to form both intra- and intermonomer disulphide bonds that can be reversed by reducing agents. With MMTS (methyl methanethiosulphonate) or ODNB (n-octyl-dithio-2-nitrobenzoate), virtually no protein–protein disulphide bonds are formed and the ODNB reaction can be given as the sum of three exponential terms with pseudo-first-order rate constants of 0.206, 0.069 and 0.010 s−1 at pH 6.5, suggesting three classes of thiol reactivities. Limited cysteine substitution leads to only small changes in tryptophan or CD spectra, whereas complete substitution leads to loss of the helix content. MMTS-induced loss of SH groups leads to progressive increases in the critical concentration and loss of polymerization competence that can be reversed by assembly promoters such as higher protein concentration, taxol or high ionic strength. Under such conditions, the substituted tubulin forms protofilament-based structures such as microtubules, open tubules, sheets and/or bundles.

Plakin family members envoplakin and periplakin have been shown to be part of the cornified cell envelope in terminally differentiating stratified squamous epithelia. In the present study, purified recombinant human envoplakin and periplakin were used to investigate their properties and interactions. We found that envoplakin was insoluble at physiological conditions in vitro, and co-assembly with periplakin was required for its solubility. Envoplakin and periplakin formed soluble complexes with equimolar stoichiometry. Chemical cross-linking revealed that the major soluble form of all periplakin constructs and of envoplakin/periplakin rod domains was a dimer, although co-assembly of the full-length proteins resulted in formation of higher order oligomers. Electron microscopy of rotary-shadowed periplakin demonstrated thin flexible molecules with an average contour length of 88 nm for the rod-plus-tail fragment, and immunolabeling EM confirmed the molecule as a parallel, in-register, dimer. Both periplakin and envoplakin/periplakin oligomers were able to bind synthetic lipid vesicles whose composition mimicked the cytoplasmic side of the plasma membrane of eukaryotic cells. This binding was dependent on anionic phospholipids and Ca2+. These findings raise the possibility that envoplakin and periplakin bind to the plasma membrane upon elevation of intracellular [Ca2+] in differentiating keratinocytes, where they serve as a scaffold for cornified cell envelope assembly.

Irrespective of the constituent protein, all amyloid fibrils show similar morphology in the electron microscope and x-ray diffraction patterns characteristic of a “cross-β” structure, with extended β-strands perpendicular to the fibril's long axis. Little is known about the amount or type of this structure. I have measured CD spectra of films formed from a number of amyloid proteins and polypeptides, and estimated their contents of extended secondary structure, by analysis of their g-factor spectra, the ratio of the CD and absorbance signals (P. McPhie, Analytical Biochemistry, 2001, Vol. 293, pp. 109–119). Amyloid films of Aβ-(1–40) peptide, β-2-microglobulin, insulin, and three homopolypeptides show very intense CD spectra, compatible with the presence of a β-helix-like structure, arranged in a common framework in the fibrils. The extent of this structure was estimated as 45–80% in the protein fibrils and 30–80% in the polypeptide fibrils. © 2004 Wiley Periodicals, Inc. Biopolymers, 2004

The zinc-endopeptidase light chain of botulinum A neurotoxin undergoes autocatalytic fragmentation that is accelerated by the presence of the metal cofactor, zinc [Ahmed, S. A. et al. (2001) J. Protein Chem. 20, 221-231]. We show in this paper that >95% fragmented light chain obtained in the absence of added zinc retained 100% of its original catalytic activity against a SNAP-25-derived synthetic peptide substrate. In the presence of zinc chloride, when >95% of the light chain had undergone autocatalytic fragmentation, the preparation retained 35% of its original catalytic activity. On the other hand, in the presence of glycerol, the light chain did not display autocatalysis and retained 100% of the original activity. These results suggest that the activity loss by incubation with zinc was not a direct consequence of autocatalysis and that the environment of the active site was not affected significantly by the fragmentation. The optimum pH 4.2-4.6 for autocatalysis was different than that (pH 7.3) for intrinsic catalytic activity. Inhibition of autocatalysis at low pH by a competitive inhibitor of catalytic activity rules out the presence of a contaminating protease but suggests a rate-limiting step of low pH-induced conformational change suitable for autocatalysis. Our results of LC concentration dependence of the fragmentation reaction indicate that the autocatalysis occurs by both intramolecular and intermolecular mechanisms.

Thermally induced transition curves of hen egg-white lysozyme were measured in the presence of several concentrations of dextran at pH 2.0 by near-UV and far-UV CD. The transition curves were fitted to a two-state model by a non-linear, least-squares method to obtain the transition temperature (T(m)), enthalpy change (deltaH(u)(T(m))), and free energy change (deltaG(u)(T)) of the unfolding transition. An increase in T(m) and almost constant deltaH(u)(T(m)) values were observed in the presence of added dextran at concentrations exceeding ca 100 g l(-1). In addition, dextran-induced conformational changes of fully unfolded protein were investigated by CD spectroscopy. Addition of high concentrations of dextran to solutions of acid-unfolded cytochrome c at pH 2.0 results in a shift of the CD spectrum from that characteristic of the fully unfolded polypeptide to that characteristic of the more compact, salt-induced molten globule state, a result suggesting that the molten globule-like state is stabilized relative to the fully unfolded form in crowded environments. Both observations are in qualitative accord with predictions of a previously proposed model for the effect of intermolecular excluded volume (macromolecular crowding) on protein stability and conformation.

Mutations in the thyroid hormone receptor beta gene (TRbeta) cause resistance to thyroid hormone (RTH). Genetic analyses indicate that phenotypic manifestation of RTH is due to the dominant negative action of mutant TRbeta. However, the molecular mechanisms underlying the dominant negative action of mutants and how the same mutation results in marked variability of resistance in different tissues in vivo are not clear. Here we used a knock-in mouse (TRbetaPV mouse) that faithfully reproduces human RTH to address these questions. We demonstrated directly that TRbeta1 protein was approximately 3-fold higher than TRalpha1 in the liver of TRbeta(+/+) mice but was not detectable in the heart of wild-type and TRbetaPV mice. The abundance of PV in the liver of TRbeta(PV/PV) was more than TRbeta(PV/+) mice but not detectable in the heart. TRalpha1 in the liver was approximately 6-fold higher than that in the heart of wild-type and TRbetaPV mice. Using TR isoforms and PV-specific antibodies in gel shift assays, we found that in vivo, PV competed not only with TR isoforms for binding to thyroid hormone response elements (TRE) but also competed with TR for the retinoid X receptors in binding to TRE. These competitions led to the inhibition of the thyroid hormone (T(3))-positive regulated genes in the liver. In the heart, however, PV was significantly lower and thus could not effectively compete with TRalpha1 for binding to TRE, resulting in activation of the T(3)-target genes by higher levels of circulating thyroid hormones. These results indicate that in vivo, differential expression of TR isoforms in tissues dictates the dominant negative activity of mutant beta receptor, thereby resulting in variable phenotypic expression in RTH.

Development of simple processes to fractionate synthetic mixtures of single-wall carbon nanotubes (SWCNTs) into individual species is crucial to many applications. Existing methods for single-chirality SWCNT purification are cumbersome, often requiring multiple steps and different conditions for different species. Here, we report a method to achieve total fractionation of a synthetic SWCNT mixture by countercurrent chromatography, resulting in purification of many single-chirality SWCNT species in a single run. This method is based on a tunable partition of sodium deoxycholate dispersed SWCNTs in a polyethylene glycol/dextran aqueous two-phase system. By running the mobile phase with 0.02% of sodium deoxycholate and a gradient of sodium dodecyl sulfate from 0.1% to 0.7% (w/w), we observe clear diameter-dependent elution, with ∼ 90% total recovery. Among all the fractions collected, a number of them are enriched in single-chirality (9,4), (7,5), (7,6), (8,3), (6,5) species, while most of the remaining ones contain no more than 2-3 major species. We also observe strong (n,m)-dependent elution peak width due to the enantiomer-resolved partition. These results demonstrate countercurrent chromatography (CCC) as an effective way to obtain high purity (n, m) species, and suggest the potential of CCC as an analytical tool for chirality distribution mapping of synthetic SWCNT mixtures.

Our studies of the reaction mechanism of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme that forms a series of intermediates in the reaction of L-serine and L-homocysteine to form L-cystathionine. To characterize these reaction intermediates, we have carried out rapid-scanning stopped-flow and single-wavelength stopped-flow kinetic measurements under pre-steady-state conditions, as well as circular dichroism and fluorescence spectroscopy under steady-state conditions. We find that the gem-diamine and external aldimine of aminoacrylate are the primary intermediates in the forward half-reaction with L-serine and that the external aldimine of aminoacrylate or its complex with L-homocysteine is the primary intermediate in the reverse half-reaction with L-cystathionine. The second forward half-reaction of aminoacrylate with L-homocysteine is rapid. No primary kinetic isotope effect was obtained in the forward half-reaction with L-serine. The results provide evidence (1) that the formation of the external aldimine of L-serine is faster than the formation of the aminoacrylate intermediate, (2) that aminoacrylate is formed by the concerted removal of the alpha-proton and the hydroxyl group of L-serine, and (3) that the rate of the overall reaction is rate-limited by the conversion of aminoacrylate to L-cystathionine. We compare our results with cystathionine beta-synthase with those of related investigations of tryptophan synthase and O-acetylserine sulfhydrylase.

Human IFN-α is a family of structurally related proteins that exhibit a wide range of antiproliferative activities. To understand the structural basis for these different antiproliferative activities, eight recombinant human IFN-α hybrids (HY) of α21a/α2c (HY-4, HY-5) and mutants (site-directed mutagenesis (SDM)-1, 2 and cassette mutagenesis (CM)-1, 2, 3, and 4) have been expressed, purified, and characterized. The data showed that the amino acid region 81–95 is important for antiproliferative activity. Site-directed mutagenesis and cassette mutagenesis studies showed that if serine (S) 86 and asparagine (N) 90 were replaced by tyrosine (Y), the antiproliferative activity was increased. We have also observed that if Y86 was replaced by isoleucine (I), the antiproliferative activity was comparable. However, if Y86 was replaced by aspartic acid (D), lysine (K), or alanine (A), the antiproliferative activity was substantially decreased. Our results indicate that Y and/or I at position 86 and Y at position 90 are very important in antiproliferative activity of human IFN-α. Circular dichroism spectra showed that the amino acid replacements at position 86 did not change the secondary structure. Thus the biological activity changes among those mutants do not appear to be due to conformational changes. The results also suggest that hydrophobic residue(s) at position 86 may be important for the interaction of the molecule with its receptor. The competitive binding data correlated with the antiproliferative activity. The N-terminal region of the molecule and the hydrophobic residues (including Y and I) on the C-helix region at positions 86 and/or 90 are important for binding and antiproliferative activities of human IFN-αs.

Estimates of the secondary structure of a protein in solution are made by mathematical analyses of its circular dichroism (CD) spectrum below 240 nm. All current procedures require accurate determination of the concentration of the protein sample. Insoluble proteins, such as prions or amyloid, are examined as thin films or gels, but concentrations cannot be precisely defined. The ratio of a sample's CD and absorbance signals is the g-factor, an intensive property. The g-factor spectra of 19 soluble, unconjugated proteins of known structures were measured and used to derive basis spectra, characteristic of the four major structural elements, helix, sheet, turn, and remainder. Using these, the g-factor spectra of other unconjugated proteins, measured in solution or as films, can be analyzed by linear regression to give good estimates of their secondary structures when protein concentration cannot be determined.

The sulfotransferases that are active in the metabolism of xenobiotics represent a large family of enzymes that catalyze the transfer of the sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate to phenols, to primary and secondary alcohols, to several additional oxygen-containing functional groups, and to amines. Restriction of this review to the catalytic processes of phenol or aryl sulfotransferases does not really narrow the field, because these enzymes have overlapping specificity, not only for specific compounds, but also for multiple functional groups. The presentation aims to provide an overview of the wealth of phenol sulfotransferases that are available for study but concentrates on the enzymology of rat and human enzymes, particularly on the predominant phenol sulfotransferase from rat liver. The kinetics and catalytic mechanism of the rat enzyme is extensively reviewed and is compared with observations from other sulfotransferases.

Aryl sulfotransferase IV from rat liver has the very broad substrate range that is characteristic of the enzymes of detoxication. With the conventional assay substrates, 4-nitrophenol and PAPS, sulfation was considered optimal at pH 5.5 whereas the enzyme in the physiological pH range was curiously ineffective. These properties would seem to preclude a physiological function for this cytosolic enzyme. Partial oxidation of the enzyme, however, results not only in a substantial increase in the rate of sulfation of 4-nitrophenol at physiological pH but also in a shift of the pH optimum to this range and radically altered overall substrate specificity. The mechanism for this dependence on redox environment involves oxidation at Cys66, a process previously shown to occur by formation of a mixed disulfide with glutathione or by the formation of an internal disulfide with Cys232. Oxidation at Cys66 acts only as a molecular redox switch and is not directly part of the catalytic mechanism. Underlying the activation process is a change in the nature of the ternary complex formed between enzyme, phenol, and the reaction product, adenosine 3′,5′-bisphosphate. The reduced enzyme gives rise to an inhibitory, dead-end ternary complex, the stability of which is dictated by the ionization of the specific phenol substrate. Ternary complex formation impedes the binding of PAPS that is necessary to initiate a further round of the reaction and is manifest as profound, substrate-dependent inhibition. In contrast, the ternary complex formed when the enzyme is in the partially oxidized state allows binding of PAPS and the unhindered completion of the reaction cycle.

Cystathionine beta-synthase from yeast (Saccharomyces cerevisiae) provides a model system for understanding some of the effects of disease-causing mutations in the human enzyme. The mutations, which lead to accumulation of L-homocysteine, are linked to homocystinuria and cardiovascular diseases. Here we characterize the domain architecture of the heme-independent yeast cystathionine beta-synthase. Our finding that the homogeneous recombinant truncated enzyme (residues 1-353) is catalytically active and binds pyridoxal phosphate stoichiometrically establishes that the N-terminal residues 1-353 compose a catalytic domain. Removal of the C-terminal residues 354-507 increases the specific activity and alters the steady-state kinetic parameters including the K(d) for pyridoxal phosphate, suggesting that the C-terminal residues 354-507 compose a regulatory domain. The yeast enzyme, unlike the human enzyme, is not activated by S-adenosyl-L-methionine. The truncated yeast enzyme is a dimer, whereas the full-length enzyme is a mixture of tetramer and octamer, suggesting that the C-terminal domain plays a role in the interaction of the subunits to form higher oligomeric structures. The N-terminal catalytic domain is more stable and less prone to aggregate than full-length enzyme and is thus potentially more suitable for structure determination by X-ray crystallography. Comparisons of the yeast and human enzymes reveal significant differences in catalytic and regulatory properties.

Monocarboxylate transporters (MCTs) comprise a group of highly homologous proteins that reside in the plasma membrane of almost all cells and which mediate the 1:1 electroneutral transport of a proton and a lactate ion. The isoform MCT3 is restricted to the basal membrane of the retinal pigment epithelium where it regulates lactate levels in the neural retina. Kinetic analysis of this transporter poses formidable difficulties due to the presence of multiple lactate transporters and their complex interaction with MCTs in adjacent cells. To circumvent these problems, we expressed both the MCT3 gene and a green fluorescent protein-tagged MCT3 construct in Saccharomyces cerevisiae. Since L-lactate metabolism in yeast depends on the CYB2 gene, we disrupted CYB2 to study the MCT3 transporter activity free from the complications of metabolism. Under these conditions L-lactate uptake varied inversely with pH, greater uptake being associated with lower pH. Whereas the V(max) was invariant, the K(m) increased severalfold as the pH rose from 6 to 8. In addition, MCT3 was highly resistant to a number of "classical" inhibitors of lactate transport. Last, studies with diethyl pyrocarbonate and p-chloromercuribenzenesulfonate set limitations on the locus of potential residues involved in the critical site of lactate translocation.

This work is aimed at understanding how protein structure and conformation regulate activity and allosteric communication in the tryptophan synthase α2β2 complex from Salmonella typhimurium. Previous crystallographic and kinetic results suggest that both monovalent cations and a salt bridge between α subunit Asp56 and β subunit Lys167 play allosteric roles. Here we show that mutation of either of these salt bridging residues produced deleterious effects that could be repaired by increased temperature in combination with CsCl or with NaCl plus an α subunit ligand, α-glycerol 3-phosphate. Arrhenius plots of the activity data under these conditions were nonlinear. The same conditions yielded temperature-dependent changes in the equilibrium distribution of enzyme-substrate intermediates and in primary kinetic isotope effects. We correlate the results with a model in which the mutant enzymes are converted by increased temperature from a low activity, “open” conformation to a high activity, “closed” conformation under certain conditions. The allosteric ligand and different monovalent cations affected the equilibrium between the open and closed forms. The results suggest that α subunit Asp56 and β subunit Lys167 are not essential for catalysis and for allosteric communication between the α and β subunits but that their mutual interaction is important in stabilization of the active, closed form of the α2β2 complex.

Thyroid hormone (T3) nuclear receptors (TR) are ligand-dependent transcription factors which regulate growth, differentiation, and development. One emerging hypothesis suggests that TR mediate these diverse effects via a large network of coregulators. Recently, we found that TR-mediated transcriptional responses varied in six cell lines derived from different tissues. We therefore used human TR subtype beta1 (TRbeta1) as bait to search for coregulators in human colon carcinoma RKO cells with a yeast two-hybrid system. RKO cells exhibited T3-dependent and -independent transcriptional activation. One of the three positive clones was identified as Ear-2, which is a distant member of the chick ovalbumin upstream promoter-transcription factors of the orphan nuclear receptor family. The physical interaction between Ear-2 and TRbeta1 was further confirmed by specific binding of Ear-2 to glutathione S-transferase-TRbeta1. In addition, Ear-2 was found to associate with TRbeta1 in cells. As a result of this physical interaction, binding of TRbeta1 to the T3 response elements was inhibited. Using reporter systems, we found that both the basal activation and the T3-dependent activation mediated by TRbeta1 were repressed by Ear-2 in CV1 cells. In RKO cells, however, the T3-independent transcriptional activity was more sensitive to the repression effect of Ear-2 than the T3-dependent transcriptional activity. The repression effect of Ear-2 was reversed by steroid hormone receptor coactivator 1. These results suggest that TR-mediated responses reflect a balance of corepressors and coactivators in cells. These findings further strengthen the hypothesis that the diverse activities of TR are achieved via a large network of coregulators that includes Ear-2.

Strategy, Management and Health Policy Venture Capital Enabling Technology Preclinical Research Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics Clinical Development Phases I–III Regulatory, Quality, Manufacturing Postmarketing Phase IV View it in a separate window The hexahistidine-tagged mouse P2X1 receptor (H-mP2X1R), an ATP-gated ion channel receptor, was expressed in a baculovirus system using the pAcHLT-B transfer vector containing a hexahistidine tag. Both widely used denaturing (8M urea) and nondenaturing (such as 1% Triton X-100) solubilization conditions were compared, resulting in about 30% of the P2X1 receptors being solubilized (S1). However, at pH 13 most of the H-mP2X1R from the initially insoluble pellet fraction was solubilized (S2) and remained in the soluble fraction (S3) after dialyzing against a nondenaturing buffer. H-mP2X1Rs were purified sequentially through cobalt and ATP affinity columns. Receptors purified from S3 had higher purity than those from S1 (i.e., ~90% vs. ~75%). Circular dichroism spectra indicated identical protein secondary structures of the receptors from both sources. Autoradiographic data showed that the purified receptors from S3 had higher affinity for 8-azido-ATP-γ-32P than the receptors from S1. The binding of 8-azido-ATP-γ-32P to H-mP2X1R was inhibited by ATP-γ-S, α,β-me-ATP, and PPADS, but not by a nucleoside analog (N6-methyl-2′-deoxy-adenosine). In the presence of 2 mM Ca2+ or Mg2+ the binding was increased, but not when using a partially purified receptor fraction, in which unidentified proteins bound 8-azido-ATP-γ-32P or were phosphorylated at 4°C in the presence of 2 mM Mg2+. These data suggest that the decrease in potency of ATP in the presence of Ca2+ and Mg2+, as observed in functional studies, is not due to a direct effect of the cations on the binding of ATP to the receptor. Both cyanogen bromide and hydroxylamine cleavage further confirmed the peptide structure of the purified H-mP2X1R. Autoradiographic analysis of the cleavage products showed that 8-azido-ATP-γ-32P was crosslinked to the carboxyl side of the extracellular domain of the receptor.

To characterize the conformational transitions that regulate the activity and specificity of the tryptophan synthase alpha 2 beta 2 complex, we have determined some effects of low concentrations of guanidine hydrochloride (GuHCl) and of urea on functional properties. We report the novel finding that GuHCl at low concentrations (0. 02-0.08 M) is a cation activator of the tryptophan synthase alpha 2 beta 2 complex. Molecular modeling studies show that GuH+ could bind at a previously identified cation binding site in the tryptophan synthase beta subunit. Addition of increasing concentrations of GuHCl has strikingly different effects on the rates of different reactions with L-serine or beta-chloro-L-alanine in the presence or absence of indole. Spectroscopic studies demonstrate that GuHCl alters the equilibrium distribution of pyridoxal 5'-phosphate intermediates formed in reactions at the active site of the beta subunit. Data analysis shows that GuHCl binds preferentially with the conformer of the enzyme that predominates when the aldimine of L-serine is formed and shifts the equilibrium in favor of this conformer. These results provide evidence that GuHCl exerts dual effects on tryptophan synthase as a cation, stimulating activity, and as a chaotropic agent, altering the distribution of conformational states that exhibit different reaction specificities. Our finding that the nonionic urea stabilizes the aldimine of L-serine in the presence, but not in the absence, of NaCl shows that cation binding plays an important role in the conformational transitions that regulate activity and the transmission of allosteric signals between the alpha and beta sites.

We purified the extracellular domain (ECD) of the human calcium receptor (hCaR) from the medium of HEK-293 cells stably transfected with a hCaR cDNA containing an isoleucine 599 nonsense mutation. A combination of lectin, anion exchange, and gel permeation chromatography yielded milligram quantities of >95% pure protein from 15 liters of starting culture medium. The purified ECD ran as an approximately 78-kDa protein on SDS-polyacrylamide gel electrophoresis and was found to be a disulfide-linked dimer. Its NH2-terminal sequence, carbohydrate content, and CD spectrum were defined. Tryptic proteolysis studies showed two major sites accessible to cleavage. These studies provide new insights into the structure of the hCaR ECD. Availability of purified ECD protein should permit further structural studies to help define the mechanism of Ca2+ activation of this G protein-coupled receptor.

Estimation of a protein's secondary structure from its circular dichroism spectrum usually requires accurate knowledge of the concentration and pathlength of the sample. Two recently described methods avoid this problem by analysis of g-factor spectra (McPhie, Anal. Biochem. 293, 109-119) or scaling of relative intensities (Raussens et al., Anal. Biochem. 319, 114-121). Application of the two methods to the same samples shows that they can have similar efficacies. Calculation with the latter method is more rapid, but the performance of the former is maintained over reduced wavelength ranges.

Mutations in the pyridoxal phosphate binding site of the tryptophan synthase beta subunit (S377D and S377E) alter cofactor chemistry [Jhee, K.-H., et al. (1998) J. Biol. Chem. 273, 11417-11422]. We now report that the S377D, S377E, and S377A beta2 subunits form alpha2 beta2 complexes with the alpha subunit and activate the alpha subunit-catalyzed cleavage of indole 3-glycerol phosphate. The apparent Kd for dissociation of the alpha and beta subunits is unaffected by the S377A mutation but is increased up to 500-fold by the S377D and S377E mutations. Although the three mutant alpha2 beta2 complexes exhibit very low activities in beta elimination and beta replacement reactions catalyzed at the beta site in the presence of Na+, the activities and spectroscopic properties of the S377A alpha2 beta2 complex are partially repaired by addition of Cs+. The S377D and S377E alpha2 beta2 complexes, unlike the wild-type and S377A alpha2 beta2 complexes and the mutant beta2 subunits, undergo irreversible substrate-induced inactivation by L-serine or by beta-chloro-L-alanine. The rates of inactivation (kinact) are similar to the rates of catalysis (kcat). The partition ratios are very low (kcat/kinact = 0.25-3) and are affected by alpha subunit ligands and monovalent cations. The inactivation product released by alkali was shown by HPLC and by fluorescence, absorption, and mass spectroscopy to be identical to a compound previously synthesized from pyridoxal phosphate and pyruvate. We suggest that alterations in the cofactor chemistry that result from the engineered Asp377 in the active site of the beta subunit may promote the mechanism-based inactivation.

Aryl sulfotransferase IV from rat liver has the broad substrate range that is characteristic of the enzymes of detoxication. With the standard assay substrates, 4-nitrophenol and 3'-phosphoadenosine 5'-phosphosulfate (PAPS), sulfation is optimum at pH 5.4 whereas the reaction is minimal in the physiological pH range. These properties preclude a physiological function for this cytosolic enzyme. Partial oxidation of the enzyme, however, results not only in an increase in the rate of sulfation but also in a shift of the pH optimum to the physiological pH range. The mechanism for this dependence on the redox environment involves oxidation at Cys66, the cysteine residue that is conserved throughout the phenol sulfotransferase family. As documented by mass spectroscopic methods, oxidation by GSSG leads to the formation of an internal disulfide between Cys66 and Cys232; for mutants at Cys232, the oxidation product is a mixed disulfide of Cys66 and glutathione. Both of these disulfide species activate the enzyme and allow it to function at a pH optimum in the physiological range. The activated enzyme differs from the reduced form by a more circumscribed substrate spectrum. All five mutants, in which each of the cysteines of the sulfotransferase subunit have been changed to serine, are catalytically active. Only Cys66 is required for the redox response.

Complementary DNAs for two mutant thyroid hormone alpha1 receptors (TR alpha1) were isolated from hepatocellular carcinomas of two patients. Sequence analyses of the complementary DNAs showed a single Val390Ala and double Pro398Ser/Glu350Lys mutations in mutants H and L, respectively. We characterized their hormone-binding, DNA-binding, and dominant negative activities. Mutants H and L did not bind the hormone T3. Their DNA-binding activities were analyzed using three types of thyroid hormone response elements (TREs) in which the half-site binding motifs are arranged in an everted repeat (Lys), an inverted repeat (Pal), or a direct repeat separated by four nucleotides (DR4). Compared with wild-type TR alpha1 (w-TR alpha1), which bound these TREs with different homodimer/monomer ratios, binding of mutant L to the three TREs as homodimers was reduced by approximately 90%. However, binding of mutant H to these TREs was more complex. Although it bound normally to DR4 as homodimers, its binding to Lys as homodimers was reduced by approximately 80%. Surprisingly, its binding to Pal was markedly enhanced compared with w-TR alpha1. The binding of these two mutants to the three TREs as heterodimers with retinoid X receptors (RXR alpha and -beta) was not significantly affected. Consistent with the lack of T3-binding activity, both mutants had lost their trans-activation capacity. Mutants H and L exhibited dominant negative activity, but differed in their TRE dependency. The dominant negative potency of mutant H was in the rank order of PalDR4Lys, whereas no TRE dependency was observed for mutant L. The present study indicates that mutations of the TR alpha gene do occur in patients and that these novel TR alpha1 mutants provide a valuable tool to further understand the molecular basis of the dominant negative action of mutant TRs.

To understand whether the transcriptional activity of thyroid hormone nuclear receptors (TRs) is modulated by cell-type specific factors, full length TR subtype alpha1 (TRalpha1) and beta1 (TRbeta1) cDNAs were cloned from human hepatoma cell lines: HA22T, SK-Hep-1 and HepG2. The cloned receptor bound to the thyroid hormone 3,3',5-triiodo-L-thyronine (T3) and the thyroid hormone response elements (TREs) similarly to those cloned from other tissues. They exhibited T3- and TRE-dependent transactivation activities, indicating these TRs were transcriptionally active. The lipogenic malic enzyme (ME), a T3-target gene in liver, was stimulated approximately 3- and 1.5-fold by T3 in HA22T and SK-Hep-1, respectively. The T3-stimulated ME gene expression was inhibited in HA22T, but stimulated in SK-Hep-1 cells by insulin. These results suggest that the gene regulating activity of TRs was modulated by cell-type specific factors. Furthermore, these cell-type specific factors could modulate the cross talk between TR- and insulin receptor-mediated pathways.

A phenol sulfotransferase from rat liver (EC 2.8.2.9), expressed in Escherichia coli from a single cDNA, was purified as two separable but catalytically active proteins. The proteins appeared to be identical to each other and to the natural liver sulfotransferase by comparison of their amino acid constitution, amino-terminal end group, and interaction with a polyclonal antibody raised against the liver enzyme. Each of the recombinant forms, alpha and beta, catalyzed the sulfuryl group transfer from 4-nitrophenylsulfate to an acceptor phenol, a reaction in which 3'-phospho-adenosine 5'-phosphate (PAP) is a necessary intermediate. Only form beta, however, catalyzes the physiological transfer of a sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the free phenol. Evidence is presented that sulfotransferase alpha, but not beta, has 1 mol of PAP tightly bound per enzyme dimer. The ability to utilize PAPS as a sulfate donor could be altered: form alpha could be treated and purified as form beta to acquire the ability to use PAPS, whereas form beta was treated by extended incubation with PAP, lost its ability to use PAPS, and was purified as form alpha.

To characterize the conformational transitions that lead to activation of catalysis by the tryptophan synthase α2β2 complex, we have determined the solvent effects of a co-substrate, β-mercaptoethanol, and of a model nonsubstrate, ethanol, on the catalytic and spectroscopic properties of the enzyme. Our results show that ethanol and β-mercaptoethanol both alter the equilibrium distribution of pyridoxal 5′-phosphate intermediates formed in the reactions of L-serine at the β site in the α2β2 complex. Addition of increasing concentrations of ethanol increases the proportion of the external aldimine of L-serine and decreases the proportion of the external aldimine of aminoacrylate. Low concentrations of the co-substrate β-mercaptoethanol (Kd = ∼13 mM) decrease the proportion of the external aldimine of aminoacrylate and induce formation of the quinonoid of S-hydroxyethyl-L-cysteine. Higher concentrations of β-mercaptoethanol decrease the concentration of the quinonoid intermediate and increase the proportion of the external aldimine of L-serine. Data analysis shows that β-mercaptoethanol and ethanol both interact or bind preferentially with the conformer of the enzyme that predominates when the aldimine of L-serine is formed and shift the equilibrium in favor of this conformer. We propose that a nonpolar region of the β subunit, possibly the hydrophobic indole tunnel, becomes less exposed to solvent in the conformational transition that activates the α2β2 complex.

Synexin (annexin VII) is a calcium-dependent, phospholipid-binding and membrane fusion protein in the annexin gene family, which forms calcium channels and may play a role in exocytotic secretion. We report here the cloning and characterization of five novel isoforms of cDNAs encoding Xenopus synexin from brain, oocyte and stage 24 cDNA libraries. The most prevalent Xenopus synexin has 1976 bp of cDNA sequence, which contains a 1539 bp open reading frame of 512 amino acids encoding a 54 kDa protein. This Xenopus protein is 6 kDa larger than the previously reported human and mouse synexins with which it shares approx. 73% identity in the C-terminal region and approx. 44% identity in the N-terminal region. Further studies with PCR revealed the molecular basis of the substantial divergence in the Xenopus synexin's N-terminal domain. The domain equivalent to the mammalian tissue-specific cassette exon occurs at a different position and is variable in size and sequence. The most interesting observation relates to the occurrence of different forms of synexin due to the varying numbers of tandem PGQM repeats that are expressed differently in different adult tissues and embryonic stages. For these reasons we have labelled this set of unique isoforms annexin VIIb, referring to mammalian forms, which lack the PGQM tandem repeats, as annexin VIIa. In spite of these differences from annexin VIIa, the form of recombinant annexin VIIb with three PGQM repeats was found to be catalytically active. We interpret these results to indicate that the actual calcium and phospholipid binding sites are conserved in Xenopus, and that the variations observed between members of the synexin gene family in the regulatory domain clearly point towards the tissue- and stage-specific roles of individual members, possibly involving the exocytotic process.

A reversible thermally induced conformational transition of the beta2 subunit of tryptophan synthase from Salmonella typhimurium has been detected by use of the pyridoxal 5'-phosphate coenzyme as a spectroscopic probe. Increasing the temperature converts the major form of pyridoxal 5'-phosphate bound to the beta2 subunit from a ketoenamine species with lambdamax at 410 nm to a enolimine species with lambdamax at 336 nm (Tm = approximately 43 degrees C) and results in loss of the circular dichroism signal at 410 nm and of fluorescence emission at 510 nm. The results indicate that increasing the temperature favors a conformer of the enzyme that binds pyridoxal 5'-phosphate in a more nonpolar environment and leads to loss of asymmetric pyridoxal 5'-phosphate binding. The internal aldimine between pyridoxal 5'-phosphate and the epsilon-amino group of lysine 87 is not disrupted by increased temperature because sodium borohydride treatment of the enzyme at either 15 or 60 degrees C results in covalent attachment of [4'-3H]pyridoxal 5'-phosphate. The thermal transition of the beta2 subunit below 60 degrees C produces reversible thermal inactivation (Ti = approximately 52 degrees C) and occurs at a much lower temperature than the major reversible unfolding at approximately 80 degrees C (Remeta, D. P., Miles, E. W., and Ginsburg, A. (1995) Pure Appl. Chem. 67, 1859-1866). Our new results indicate that the 410 nm absorbing species of pyridoxal 5'-phosphate is the catalytically active form of the cofactor in the beta2 subunit and that the low temperature reversible conformational transition disturbs the active site and causes loss of catalytic activity.

The clinical manifestations of patients with resistance to thyroid hormone result from inhibition of the functions of wild-type thyroid hormone receptors (wTRs) by the dominant negative effect of mutant TR beta 1 receptors (mTR beta 1). One of the proposed mechanisms by which mTR beta 1 exerts its dominant negative action is via formation of the putative inactive wTR beta 1/mTR beta 1 heterodimer. However, the nature of the wTR beta 1/mTR beta 1 heterodimer is poorly understood. The present study characterizes the wTR beta 1/mTR beta 1 heterodimer by electrophoretic mobility shift assay. The mutant TR beta 1 used was PV, which contains a frame shift mutation in the C-terminal part of TR beta 1 and has less than 1% of the T3 binding affinity of the wTR beta 1. Because of the difficulty in resolving wTR beta 1 and mutant PV dimers, we used a truncated wTR beta 1 in which the A/B domain was deleted (delta TR beta 1) to demonstrate the formation of the heterodimer on thyroid hormone response elements (TREs) in which the half-site binding motifs are oriented in an inverted repeat (F2), a direct repeat separated by four nucleotides (DR4), or an inverted repeat (Pal). Deletion of the A/B domain had no effect on the binding of T3 and TREs to wTR beta 1. In the presence of equal amounts of delta TR beta 1 and PV, three types of molecular complexes. delta TR beta 1 homodimer, delta TR beta 1/PV heterodimer, and PV homodimer bound to each TRE in a ratio of approximately 1:2:1. The identities of these complexes were confirmed by their ability to be supershifted by anti-TR beta 1 and/or anti-PV antibodies. delta TR beta 1/PV heterodimer formation varied with different TREs. The ratio of apparent affinity constant (Ka) in the binding of delta TR beta 1/PV to TREs was F2:DR4:Pal = approximately 6:2:1. The effect of T3 on delta TR beta 1/PV heterodimer formation was TRE dependent. No T3-induced dissociation was observed for the delta TR beta 1/PV heterodimer when bound to F2 and Pal. In contrast, the delta TR beta 1/PV heterodimer bound to DR4 was dissociated by T3 with an ED50 of 3.9 +/- 0.9 nM. The T3-induced dissociation of delta TR beta 1 homodimer bound to F2, DR4, and Pal had ED50 values of 4.1 +/- 1.2, 1.3 +/- 0.3, and more than 100 nM, respectively. By transfection assays, the dominant negative action of PV was found to be TRE dependent with the rank order of F2 >> Pal > ME (a DR4-like TRE in the rat malic enzyme gene). Taken together, these results indicate a strong correlation between wTR beta 1/mTR beta 1 heterodimer formation and the dominant negative potency of PV. These results suggest that the wTR beta 1/mTR beta 1 heterodimer could play an important role in the dominant negative action of mTR beta 1.

The purpose of this study was to bacterially express, purify, and refold combinations of the extracellular immunoglobulin (Ig)-like domains (2-3, 1-3, and 1-5) of the human alpha-platelet-derived growth factor receptor (alpha PDGFR) to characterize molecular interactions with its ligand, platelet-derived growth factor (PDGF). The far UV circular dichroism spectroscopy of the alpha-PDGFR extracellular domains (ECDs) revealed a predominantly beta-sheet protein, with a structure consistent with folded Ig-like domains. The addition of PDGF-BB to these ECD types changed the conformation of all three types with a decrease in mean residue ellipticity in the following rank order: 1-5 = 1-32-3. In striking contrast, addition of PDGF-AA to these ECD types markedly changed the conformation of ECD 2-3, by an increased mean residue ellipticity but no changes were observed for ECDs 1-3 and 1-5. PDGF-AA bound to the immobilized ECD types 2-3, 1-3, and 1-5 at concentrations of 20, 11, and 7.5 nM, respectively. In contrast, PDGF-BB bound the ECD types 2-3, 1-3, and 1-5 at concentrations of 3, 3, and 2.2 nM, respectively. Scatchard analysis of binding studies using labeled ECDs indicated that PDGF-BB bound ECD 1-3 and ECD 2-3 with KD values of 74 and 72 nM, respectively. While, PDGF-AA bound ECD 1-3 and ECD 2-3 with KD values of 33 and 87 nM, respectively. Therefore, our results indicated that the loss of ECD 1 impaired the binding affinity of alpha PDGFR ECD 1-3 toward PDGF-AA without having a similar effect on PDGF-BB binding. Together all of our data suggest that ECD 1 is differentially required for proper orientation of PDGF-AA but not PDGF-BB binding determinant within ECDs 2 and 3.

Pleckstrin homology (PH) domains are approximately 110 amino acid residues in length and are structurally conserved in a number of intracellular signaling proteins. A role for these domains has been postulated for beta ARK, which binds to G beta gamma subunits. We have quantified the binding of individual (His)6-tag PH domains of human Db1, human Sos1, rat IRS-1, human beta ARK, and human beta ARK with an extra 33-residue C-terminal extension (beta ARK + C) to G beta gamma subunits. Our in vitro binding studies show that all of the PH domains (apart from Sos1), bind G beta gamma subunits in a dose-dependent manner, but beta ARK + C binds 4 times as much G beta gamma at saturation as the others. The IRS-1 PH domain has a similar half-maximal concentration of G beta gamma binding (18 nM) to beta ARK + C (30 nM), suggesting that the IRS-1 PH domain has sufficient determinants for G beta gamma binding. The beta ARK PH domain alone has a half-maximal value of 45 nM but a drastically reduced extent of G beta gamma binding, suggesting that both the PH domain and the C-terminal 33 residues are necessary for maximal binding. Db1 has a half-maximum concentration of G beta gamma binding of 45 nM and a maximal extent of binding similar to that of beta ARK, but it is difficult to demonstrate saturable binding of G beta gamma to Sos1. Since it was previously predicted that the C-terminal PH domain of Pleckstrin [Tyers, M., et al. (1988) Nature 333, 470-473] contains a potential calcium binding site, we have tested the different PH domains for calcium binding. Only the PH domain of Db1 bound 45Ca2+ with a Kd of 10 microM. CD spectroscopy of the purified recombinant PH domains indicated that they are predominantly beta-sheet structures.(ABSTRACT TRUNCATED AT 250 WORDS)

To understand the structural basis in the hormone-dependent transcriptional regulation of human beta 1 thyroid hormone receptor (h-TR beta 1), we characterized the region which interacted with the thyroid hormone, 3,3',5-triiodo-L-thyronine (T3). Using the hormone binding domain of h-TR beta 1 (K206-D461) as an immunogen, we screened for monoclonal antibodies which inhibited the binding of T3 to h-TR beta 1. mAb C3, which recognized native h-TR beta 1, was obtained. Analyses of the binding data indicate that binding of T3 to h-TR beta 1 was competitively inhibited by mAb C3. Using a series of truncated mutants of h-TR beta 1 and synthetic peptides, we mapped the binding site of mAb C3 to the region of E248-V256. Thus, part of T3 binding site in h-TR beta 1 is in this nine-amino acid segment, which was shown by circular dichroism spectroscopy to be a random coil. Based on the proposed model of the hormone binding domain as an alpha/beta barrel, E248-V256 contains part of Loop 1 which is on the same side of the DNA binding domain. These results raise the possibility that Loop 1 could be in direct contact with the nearby DNA binding domain to affect the interaction of DNA binding domain with the T3 target genes.

This work is aimed at understanding subunit assembly in the tryptophan synthase α2β2 complex and the importance of the internal aldimine between pyridoxal phosphate and lysine 87 of the β2 subunit of tryptophan synthase for subunit association. We utilize a mutant form of the β2 subunit that is unable to form the internal aldimine because lysine 87 is replaced by threonine (K87T). The K87T α2β2 complex is inactive in reactions catalyzed by the β2 subunit but retains activity in the reaction catalyzed by the α subunit. We find that dialysis removes pyridoxal phosphate much more rapidly from the K87T β2 subunit and α2β2 complex than from the wild type counterparts. Activity measurements, gel filtration, and subunit interchange experiments show that the α subunit dissociates more readily from the K87T β2 subunit than from the wild type β2 subunit. The reaction of L-serine to form an external aldimine with pyridoxal phosphate at the active site of the K87T β2 subunit markedly increases the affinity for the α subunit and slows removal of pyridoxal phosphate by dialysis. We propose that the external aldimine between L-serine and pyridoxal phosphate bridges the N-domain and the C-domain in the K87T β2 subunit. This interdomain bridge may mimic the internal aldimine bond in the wild type β2 subunit and stabilize pyridoxal phosphate binding. The interdomain bridges formed by the internal aldimine with the wild type β2 subunit and by the external aldimine with L-serine in the K87T β2 subunit may further stabilize interaction with the α subunit because the α/β interaction site contains residues from both N- and C-domains of the β2 subunit.

The consensus DNA sequence for binding of the Escherichia coli cyclic AMP receptor protein (CRP) has two symmetrically related inverted recognition elements TGTGA:TCACA, separated by a variable spacer, normally 6 bp long. We have shown that the CRP-cAMP complex, when bound to synthetic binding sites with an extended 8 bp spacer segment, induces an increase in the DNA circular dichroism (CD). The CD change at lambda275 nm agrees with the shift of approximately one helical turn of DNA into A-like form. The B-conformation is preserved for CRP binding sites similar to that in the lac and uxaCA promoters with 6 bp spacers. Another effect accompanying DNA binding is a dramatic increase of the negative CD magnitude in the spectral region of the ligand cAMP, at lambda272 nm. This effect is observed when CRP binds to specific sites with 6 or 8 bp spacers as well as to non-specific DNA. We reason that the A-like form arises by compressing and unwinding the DNA in CRP-DNA complexes having 8 bp central spacers. This serves to maintain a fixed length and twisting angle and is controlled by the protein's relatively rigid frame. This model is consistent with the observation that some binding sites with 6 bp spacers may also show the CD increase inherent to the sites with the extended 8 bp spacers. These 6 bp spacers are characterized by an increased twisting angle that requires their unwinding to bind to CRP. We propose that a mutual adaptation between CRP and binding sites by local untwisting and a B--A-like transition in the DNA is of general importance and may occur in other protein-DNA complexes, such as the complex of RNA polymerase with promoter DNA.

In myositis, disease-specific autoantibodies may be directed against an aminoacyl-tRNA synthetase, usually histidyl-tRNA synthetase. To explore the basis for this phenomenon, we have made recombinant histidyl-tRNA synthetase in the baculovirus system. It was enzymatically active and recognized by human autoantibodies. A truncated protein lacking the first 60 amino acids was inactive as an antigen and as an enzyme. This region is within the first two exons, is predicted to have a coiled-coil configuration, and is found in some other synthetases but not in Escherichia coli or yeast histidyl-tRNA synthetase. Circular dichroism showed that the peptides from this region (amino acids 1-60 and 1-47) have the predicted high alpha-helical content, but smaller fragments (1-30, 14-45, and 31-60) do not. The peptides with a high alpha-helical content could inhibit autoantibodies almost completely, whereas the smaller peptides were unable to do so. The amino acid sequence of this coiled-coil domain in human histidyl-tRNA synthetase resembles the sequence of the extended this coiled-coil arm near the NH2 terminus of bacterial seryl-tRNA synthetase as well as similar regions in some eukaryotic aminoacyl-tRNA synthetases, raising the possibility that this domain serves a similar tRNA-stabilizing role and has been preserved from a common ancestor.

The over-expression of mammalian enzymes in bacterial systems by means of recombinant DNA technology has provided the enzymologist with a supply of catalyst sufficiently abundant to identify suboptimal substrates. Such large quantities are particularly useful when working with the enzymes of detoxication, a family of proteins that are distinguished by their broad substrate specificity for generally lipophilic compounds, i.e., by their very low specificity for features other than the functional group [1]. We have achieved bountiful expression of a sulfotransferase active with phenols [2], an enzyme originally purified and characterized from rat liver [3], and classified as tyrosine-ester sulfotransferase, EC 2.8.2.9 [4,5], but usually referred to as rat liver phenol or aryl sulfotransferase IV. Having improved the sensitivity and versatility of some of the assays for sulfotransferases, we examined the substrate spectrum of this enzyme. As presented here, the results of this examination point to the limitations of enzyme nomenclature and to the danger of equating enzymes isolated from their normal habitat with those formed by recombinant technology in a foreign cell. Our experiments also establish a greater catalytic scope for the natural rat liver enzyme than that previously described.

Src-homology region 2 (SH2) domains are stretches of about 100 amino acids which are found to be structurally conserved in a number of signaling molecules. These regions have been shown to bind with high affinity to phosphotyrosine residues within activated receptor tyrosine kinases. Here we report the bacterial expression and purification of individual N-terminal SH2 (NSH2) domains of phosphatidylinositol 3-kinase (PI-3K) binding subunit (p85) and Ras GTPase activating protein (GAP) in amounts suitable for structure-function studies. The p85NSH2 domain stains dark purple and absorbs around 620-640 nm with Stains-all, a dye known to bind to calcium binding proteins. This effect was not observed for the GAPNSH2 domain. Circular dichroism analysis of the N-terminal SH2 domain of these proteins shows that p85NSH2, but not GAPNSH2, undergoes a significant dose-dependent change in conformation in the presence of increasing calcium concentrations. Moreover, the conformational change of p85NSH2 induced by calcium could be replicated by addition of a phosphorylated hexapeptide (DYpMDMK) representing the alpha-PDGFR binding site for p85. Limited proteolysis studies showed a significant calcium-dependent increase in protection of p85NSH2 but not GAPNSH2 from degradation by subtilisin. Our results further indicate that holmium, a trivalent lanthanide ion, which has been previously shown to substitute for calcium, could also protect the p85NSH2 domain from proteolysis even at 10-fold lower concentrations. In vitro binding studies using purified preparations of activated alpha-PDGFR show that calcium did not affect the binding of GAPNSH2 domains to activated alpha-PDGFR.(ABSTRACT TRUNCATED AT 250 WORDS)