Biomedical subjects

A B Mason

Publications and source records attributed to A B Mason.

At least 37 records · Page 2Linked to original sources

PubMed1998

Mutations at nonliganding residues Tyr-85 and Glu-83 in the N-lobe of human serum transferrin. Functional second shell effects.

The x-ray crystal structure of the N-lobe of human serum transferrin has shown that there is a hydrogen bond network, the so-called "second shell," around the transferrin iron binding site. Tyrosine at position 85 and glutamic acid at position 83 are two nonliganding residues in this network in the human serum transferrin N-lobe (hTF/2N). Mutation of each of these two amino acids has a profound effect on the metal binding properties of hTF/2N. When Tyr-85 is mutated to phenylalanine, iron release from the resulting mutant Y85F is much more facile than from the parent protein. Elimination of the hydrogen bond between Tyr-85 and Lys-296 appears to interfere with the "di-lysine (Lys-206-Lys-296) trigger," which affects the iron binding stability of the protein. Surprisingly, mutation of Glu-83 to alanine leads to the absence of one of the normal iron binding ligands; introduction of a monovalent anion is able to restore the normal first coordination sphere. The missing ligand appears to be His-249, as revealed by comparison of the metal binding behaviors of mutants H249Q and E83A and structural analysis. Glu-83 has a strong H bond linkage with His-249 in apo-hTF/2N, which helps to hold the His-249 in the proper position for iron binding. Disabling Glu-83 by mutation to an alanine seriously disturbs the H bond network, allowing His-249 to move away. A monovalent anion can help reestablish the normal network by providing a negative charge near the position of Glu-83 to reach charge balance, so that ligand His-249 is available again for iron binding.

Base Sequence↗

PubMed1998

Two high-resolution crystal structures of the recombinant N-lobe of human transferrin reveal a structural change implicated in iron release.

The N-lobe of human serum transferrin (hTF/2N) has been expressed in baby hamster kidney cells and crystallized in both orthorhombic (P212121) and tetragonal (P41212) space groups. Both crystal forms diffract to high resolution (1.6 and 1.8 A, respectively) and have been solved by molecular replacement. Subsequent refinement resulted in final models for the structure of hTF/2N that had crystallographic R-factors of 18.1 and 19.7% for the two crystal forms, respectively; these models represent the highest-resolution transferrin structures determined to date. The hTF/2N polypeptide has a folding pattern similar to those of other transferrins, including the presence of a deep cleft that contains the metal-binding site. In contrast to other transferrins, both crystal forms of hTF/2N display disorder at the iron-binding site; model building suggests that this disorder consists of alternative conformations of the synergistically bound carbonate anion, the side chain for Arg-124, and several solvent molecules. Subsequent refinement revealed that conformation A has an occupancy of 0.63-0. 65 and corresponds to the structure of the iron-binding site found in other transferrins. The alternative conformation B has an occupancy of 0.35-0.37; in this structure, the carbonate has rotated 30 degrees relative to the iron and the side chain for Arg-124 has moved to accommodate the new carbonate position. Several water molecules appear to stabilize the carbonate anion in the two conformations. These structures are consistent with the protonation of the carbonate and resulting partial removal of the anion from the metal; these events would occur prior to cleft opening and metal release.

Animals↗

PubMed1998

[1H,13C] NMR determination of the order of lobe loading of human transferrin with iron: comparison with other metal ions.

Human serum transferrin (hTF) is a single-chain bilobal glycoprotein (80 kDa) which transports Fe3+ and a variety of other metal ions in blood. Only diferric transferrin, not the apo-protein, binds strongly to transferrin receptors and is taken up by cells via receptor-mediated endocytosis. We show here that 2D [1H,13C] NMR studies of recombinant epsilon-[13C]Met-hTF allow the order of lobe loading with various metal ions, including Fe3+, to be determined. In particular, the resonance for Met-464, a residue in the hydrophobic patch of helix 5, is very sensitive to iron binding in the C-lobe. The selectivity of lobe loading with Fe3+ is compared to loading with Fe2+ (which binds as Fe3+), Al3+, Ga3+ and Bi3+. Similar changes in shifts of the Met residues are observed for these metal ions, suggesting that they induce similar conformational changes in the protein.

Aluminum↗

PubMed1997

Inequivalence of the two tyrosine ligands in the N-lobe of human serum transferrin.

Human serum transferrin N-lobe (hTF/2N) has four iron-binding ligands, including one histidine, one aspartate, and two tyrosines. The present report elucidates the inequivalence of the two tyrosine ligands (Tyr 95 and Tyr 188) on the metal-binding properties of hTF/2N by means of site-directed mutagenesis, metal release kinetics, and absorption and electron paramagnetic resonance (EPR) spectroscopies. When the liganding tyrosines were mutated individually to phenylalanine, the resulting mutant Y95F showed a weak binding affinity for iron and no affinity for copper, whereas, mutant Y188F completely lost the ability to bind iron but formed a stable complex with copper. Since other studies have demonstrated that mutations of the other two ligands, histidine and aspartate, did not completely abolish iron binding, the present findings suggest that the tyrosine ligand at position 188 is essential for binding of iron to occur. Replacement of Tyr 188 with phenylalanine created a favorable chemical environment for copper coordination but a fatal situation for iron binding. The positions of the two liganding tyrosines in the metal-binding cleft suggest a reason for the inequivalence.

Aspartic Acid↗

PubMed1997

Iron release from recombinant N-lobe and single point Asp63 mutants of human transferrin by EDTA.

Transferrins bind ferric ion and deliver the iron to cells. The mechanism of the iron release has been studied kinetically, in vitro, with the aid of single point mutants in which the iron-binding ligand, Asp63 (aspartic acid-63, D63), has been changed to Ser, Asn, Glu and Ala. Iron release from the unmutated N-lobe of human serum transferrin (hTF/2N) by EDTA is influenced by a variety of factors. The rate-determining conformational-change mechanism may be a major pathway for iron release from hTF/2N's having a 'closed' conformation, which leads to a saturation kinetic mode with respect to ligand concentration. The effect of chloride depends on the protein conformation, showing a negative action in the case of tight binding and a positive action when the protein has an 'open' or 'loose' conformation. The negative effect of chloride could originate from the binding competition between chloride and the chelate to the active site for iron release, and the positive effect could derive from the synergistic participation of chloride in iron removal. The 'open' conformation may be induced by decreasing pH: the transitional point appears to be at about pH 6.3 for the wild-type hTF/2N; the 'loose' conformation may be facilitated by mutations at D63, which result in the loss of a key linking component in interdomain interactions of the protein. In the latter case, structural factors dominate over other potential negative effects because the weak interdomain contacts derived from the mutation of D63 cause the binding site to open easily, even at pH 7.4. Therefore chloride exhibits an accelerating action on iron release by EDTA from all the D63 mutants.

Aspartic Acid↗

PubMed1997

Receptor recognition sites reside in both lobes of human serum transferrin.

The binding of iron by transferrin leads to a significant conformational change in each lobe of the protein. Numerous studies have shown that the transferrin receptor discriminates between iron-saturated and iron-free transferrin and that it modulates the release of iron. Given these observations, it seems likely that there is contact between each lobe of transferrin and the receptor. This is the case with chicken transferrin, in which it has been demonstrated unambiguously that both lobes are required for binding and iron donation to occur [Brown-Mason and Woodworth (1984) J. Biol. Chem. 259, 1866-1873]. Further support to this contention is added by the ability of both N- and C-domain-specific monoclonal antibodies to block the binding of a solution containing both lobes [Mason, Brown and Church (1987) J. Biol. Chem. 262, 9011-9015]. In the present study a similar conclusion is reached for the binding of human serum transferrin to the transferrin receptor. With the use of recombinant N- and C-lobes of human transferrin produced in a mammalian expression system, we show that both lobes are required to achieve full binding. (Production of recombinant C-lobe in the baby hamster kidney cell system is reported here for the first time.) Each lobe is able to donate iron to transferrin receptors on HeLa S3 cells in the presence of the contralateral lobe. The results are not identical with the chicken system, because the C-lobe alone shows a limited ability to bind to receptors and to donate iron. Further complications arise from the relatively weak re-association between the two lobes of human transferrin compared with the re-association of the ovotransferrin lobes. However, domain-specific monoclonal antibodies to either lobe block the binding of N- and C-lobe mixtures in the human system, thus substantiating the need for both.

Animals↗

PubMed1997

Exploring an antifungal target in the plasma membrane H(+)-ATPase of fungi.

The plasma membrane H(+)-ATPase is a promising new antifungal target that is readily probed with the sulfhydryl-reactive reagent omeprazole. Inhibition of the H(+)-ATPase by omeprazole is closely linked to cell killing, and it has been suggested that enzyme inhibition may result from a covalent interaction within the first two transmembrane segments (M1 and M2) (Monk et al. (1995) Biochim. Biophys. Acta 1239, 81-90). In this study, the molecular nature of this interaction was examined by screening a series of 26 well-characterized pma1 mutations residing in the first two transmembrane segments of the H(+)-ATPase from Saccharomyces cerevisiae. Only two pma1 mutants, A135G and G158D,G156C, were found to significantly decrease the sensitivity of cells for omeprazole. In contrast, enhanced sensitivity was observed at a number of positions, with D140C(A) and M128C producing the most significant increases in sensitivity. The introduction of cysteine at various locations within this region only marginally affected omeprazole sensitivity, suggesting that this region was not a direct site of covalent modification. Rather, its conformation influences omeprazole binding at some other locus. In order to determine the sidedness of the omeprazole interaction, a novel in vitro assay system was exploited that utilized liposomes co-reconstituted with the H(+)-ATPase and the light-driven proton pump bacteriorhodopsin. Omeprazole was found to completely inhibit proton transport by the H(+)-ATPase at 50 microM in this system. An asymmetrically-distributed chemical trap system involving glutathione was used to demonstrate that this inhibition appears localized to the extracellular portion of the enzyme. This work indicates that omeprazole can inhibit the H(+)-ATPase from its extracellular face, and this inhibition is influenced by changes in the M1, M2 region of the protein.

Acridine Orange↗

PubMed1997

Effects of mutations of aspartic acid 63 on the metal-binding properties of the recombinant N-lobe of human serum transferrin.

Mutations of the aspartic acid residue at position 63 of the N-lobe of human serum transferrin substantially alter the metal ion- and anion-binding properties of the protein. Substitution of serine, asparagine, glutamic acid, or alanine results in the loss of a key component of the interface in the interdomain cleft and the metal-binding ligand, aspartic acid, leading in all cases to an increased preference for NTA rather than carbonate as the "synergistic" anion relative to the wild-type protein. Excess bicarbonate is required to eliminate the NTA and obtain the "correct" visible spectrum. Carbonate replaces NTA via an intermediate. Blue shifts for the characteristic absorption band of each mutant show a range of effects on the Fe-O (Tyr) interaction. Titration with Co(III) yielded the molecular absorption coefficient for each mutant except D63A, where Co(III) appeared to oxidize the tyrosine residues and damage the ability of the mutant to bind metal. The chelator, Tiron, removes iron from hTF/2N with a simple saturation kinetic mode with respect to the ligand concentration. Chloride inhibits the release in an interesting manner: the effect is initially sharp and then levels off with a minimum k(obs) at [KCl] = 0.5 M. However, the reaction of the D63 mutants with Tiron results in the formation of the ternary complexes Fe-hTF/2N-Tiron. Significant red shifts for the characteristic absorption bands of these complexes suggest a different ligation of Tiron in the mutants from that in wild-type hTF/2N.

Aspartic Acid↗

PubMed1997

Trp128Tyr mutation in the N-lobe of recombinant human serum transferrin: 1H- and 15N-NMR and metal binding studies.

The conserved Trp residue within helix 5 of the N-lobe of human serum transferrin (hTF/2N, 40 kDa) has been mutated to Tyr. NMR and CD spectra and energy calculations show that the mutation causes little perturbation of the overall structure of hTF/2N although the chelating agent Tiron removed Fe3+ from the mutant protein about three times faster than from wild-type hTF/2N. 1H-NMR resonances of residues in the Leu122-Trp128-Ile132 hydrophobic patch are assigned both by ring current calculations and with the aid of the mutation. [1H, 15N]-NMR resonances for 11 of the 14 Tyr residues were observed in the spectra of 15N-Tyr-hTF/2N and a resonance for Tyr128 was assignable in spectra of the mutant. The 15N resonance of Y128 was sensitive to oxalate and Ga3+ binding, and Ga3+ binding perturbed 15N resonances for most of the Tyr residues. Since these are well distributed over the N-lobe, it can be concluded that metal-induced structural changes are not merely local to the binding site.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗

PubMed1996

Functional complementation between transmembrane loops of Saccharomyces cerevisiae and Candida albicans plasma membrane H(+)-ATPases.

Saccharomyces cerevisiae PMA1 sequences encoding a putative antifungal target site comprising transmembrane loops 1 + 2 and/or 3 + 4 were replaced with the homologous sequences from Candida albicans PMA1 by using PCR-mediated domain transfer. The chimeric pma1 mutants and an isogenic wild type S. cerevisiae strain had similar growth rates, growth yields, glucose-dependent proton pumping rates, acid-activated omeprazole sensitivities, salt tolerances and antifungal sensitivities. The yields and kinetic properties of H(+)-ATPases in plasma membranes of mutant and wild type strains were comparable. Single heterologous transmembrane loops caused deleterious phenotypes at low pH and elevated temperature. Inclusion of both heterologous transmembrane loops fully suppressed the temperature sensitivity caused by heterologous transmembrane loop 1 + 2, partially suppressed the pH sensitivity and gave Candida-like in vitro sensitivity to vanadate, suggesting that the loops operate as a domain. The fully functional chimeric H(+)-ATPase containing C. albicans transmembrane loops 1 + 2 and 3 + 4 demonstrates this domain's complementarity to the equivalent region of the S. cerevisiae enzyme and validates the wild type S. cerevisiae H(+)-ATPase as an antifungal screening target.

Amino Acid Sequence↗

PubMed1996

Association of the two lobes of ovotransferrin is a prerequisite for receptor recognition. Studies with recombinant ovotransferrins.

Different recombinant N-lobes of chicken ovotransferrin (oTF/2N) have been isolated from the tissue-culture medium of baby hamster kidney cells transfected with the plasmid pNUT containing the relevant DNA coding sequence. Levels of up to 40, 55 and 30 mg/1 oTF/2N were obtained for constructs defining residues 1-319, 1-332 and 1-337-(Ala)3 respectively. In addition, a full-length non-glycosylated oTF was expressed at a maximum of 80 mg/1 and a foreshortened oTF consisting of residues 1-682 was expressed at a level of 95 mg/l. These preparations were then used to produce, proteolytically, two different C-lobes (oTF/2C) comprising residues 342-686 and 342-682. The purified recombinant N-lobes (oTF/2N) are similar to the proteolytically derived half-molecule with regard to immunoreactivity and spectral properties; they show some interesting differences in thermal stability. A sequence analysis of the cDNA revealed six changes at the nucleotide level that led to six differences in the amino acid sequence compared with that reported by Jeltsch and Chambon [(1982) Eur. J. Biochem. 122, 291-295]. Electrospray mass spectrometry gives results consistent with these six changes. Interaction between the various N- and C-lobes was measured by titration calorimetry. Studies show that only those lobes that associate in solution are able to bind to the receptors on chick embryo red blood cells. These findings do not support a previous report by Oratore et al.

Animals↗

PubMed1996

Spectrophotometric titration with cobalt(III) for the determination of accurate absorption coefficients of transferrins.

A rapid and sensitive technique, involving difference spectral titration with cobalt(III), to measure the epsilon values of chicken ovotransferrin, human serum transferrin, the N-lobe of human transferrin and several single point mutants is reported. The resulting epsilon values were compared with the values calculated from the equation proposed by Pace, Vajdos, Fee, Grimsley and Gray [(1995) Protein Sci. 4, 2411-2423]. The titrations with cobalt feature sharp break-points and do not destroy the protein samples. The choice of buffer was found to be important, depending on the metal-binding avidity of the proteins. Cobalt titration should prove useful for studying the comparative metal-binding properties of transferrin and mutants of transferrin being generated by recombinant technology.

Animals↗

PubMed1996

Interlobe communication in 13C-methionine-labeled human transferrin.

[1H, 13C] NMR investigations of metal-induced conformational changes in the blood serum protein transferrin (80 kDa) are reported. These are thought to play an important role in the recognition of this protein by its cellular receptors. [1H, 13C] NMR resonance assignments are presented for all nine methionine 13CH3 groups of recombinant deglycosylated human transferrin on the basis of studies of recombinant N-lobe (40 kDa, five Met residues), NOESY-relayed [1H, 13C] HMQC spectra, and structural considerations. The first specific assignments for C-lobe resonances of transferrin are presented. Using methionine 13CH3 resonances as probes, it is shown that, with oxalate as the synergistic anion, Ga3+ binds preferentially to the C-lobe and subsequently to the N-lobe. The NMR shifts of Met464, which is in the Trp460-centered hydrophobic patch of helix 5 in the C-lobe in contact with the anion and metal binding site, show that Ga3+ binding causes movement of side chains within this helix, as is also the case in the N-lobe. The C-lobe residue Met382, which contacts the N-lobe hinge region, is perturbed when Ga3+ binds to the N-lobe, indicative of interlobe communication, a feature which may control the recognition of fully-metallated transferrin by its receptor. These results demonstrate that selective 13C labeling is a powerful method for probing the structure and dynamics of high-molecular-mass proteins.

Amino Acid Sequence↗

PubMed1996

Production and isolation of the recombinant N-lobe of human serum transferrin from the methylotrophic yeast Pichia pastoris.

The N-lobe of human serum transferrin has been expressed in the methylotrophic yeast Pichia pastoris by placing the hTF/2N cDNA under the control of the methanol-inducible alcohol oxidase promoter. Following induction with methanol, the N-lobe was efficiently secreted into a basal salt medium in shake flasks at a level of 150-240 mg/liter. As judged by mobility on SDS-PAGE, immunoreactivity with two domain-specific monoclonal antibodies, and both thermal stability and spectral properties (indictative of correct folding and ability to bind iron), the recombinant N-lobe produced by the yeast cells appears to be identical to that produced in a mammalian expression system. Electrospray-mass spectrometry and a third domain specific antibody, however, show that approximately 80% of the protein from the yeast cells contains one or two hexose residues.

Alcohol Oxidoreductases↗

PubMed1995

pH dependence of specific divalent anion binding to the N-lobe of recombinant human transferrin.

The binding of the two synergistic anion mimics, phosphate and sulfate, and of the synergistic anions, malonate and oxalate, to the N-lobe of recombinant human serum transferrin (hTF/2N) wild-type and H207E mutant protein was assessed by difference ultraviolet (UV) spectroscopy at 246 nm as a function of pH. The absolute values of both the maximum delta epsilon 246 and the Kd decreased with decreasing pH. A plot of -log Kd vs pH gave a straight line with a slope of -1.0. Furthermore, the sum of -log Kd and pH is a constant for each anion binding to each protein. We interpret these data to mean that each anion binds in divalent form along with an H+. The binding equilibrium then appears to be H+ + hTF/2N + X2- reversible K' H-hTF/2N(X) and log K' = -log Kd + pH. A plot of delta epsilon 246 vs pH was sigmoidal with a pKa = 7.4 for both proteins with phosphate and sulfate. When synergistic anions were used with hTF/2N, malonate and oxalate gave pKas of ca. 6.9 and 7.1 for dependence of delta epsilon 246 on pH, but values of 7.3 and 7.6 for the H207E mutant protein. In an attempt to locate the anion binding site in hTF/2N, the binding of sulfate to the single point mutants of the N-lobe of human transferrin, K296E, K296Q, and K206Q, was carried out by difference UV spectroscopy at pH 7.4. In the case of K296E, sulfate binding gave delta epsilon 246 = 0, while for K296Q, it gave a slightly positive delta epsilon 246.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

The yeast plasma membrane proton pumping ATPase is a viable antifungal target. I. Effects of the cysteine-modifying reagent omeprazole.

The yeast plasma membrane proton pumping ATPase (H(+)-ATPase) was investigated as a potential molecular target for antifungal drug therapy by examining the inhibitory effects of the sulfhydryl-reactive reagent omeprazole on cell growth, glucose-induced medium acidification and H(+)-ATPase activity. Omeprazole inhibits the growth of Saccharomyces cerevisiae and the human pathogenic yeast Candida albicans in a pH dependent manner. Omeprazole action is closely correlated with inhibition of the H(+)-ATPase and is fungicidal. Glucose-dependent medium acidification is correspondingly blocked by omeprazole and appears to require the H(+)-ATPase to proceed through its reaction cycle. A strong correlation is observed between inhibition of medium acidification and H(+)-ATPase activity in plasma membranes isolated from treated cells. The inhibitory properties of omeprazole are blocked by pre-treatment of activated drug with beta-mercaptoethanol, which is consistent with the expected formation of a sulfhydryl-reactive sulfenamide derivative. Mutagenesis of the three putative membrane sector cysteine residues (C148S, C312S, C867A) in the S. cerevisiae H(+)-ATPase suggests that covalent modification of the conserved C148 residue may be important for inhibition of ATPase activity and cell growth. Other mutations (M128C and G158D/G156C) mapping near C148 support the importance of this region by modulating omeprazole inhibition of the H(+)-ATPase. These findings suggest that the plasma membrane H(+)-ATPase may serve as an important molecular target for antifungal intervention.

Candida albicans↗

PubMed1995

The effect of salt and site-directed mutations on the iron(III)-binding site of human serum transferrin as probed by EPR spectroscopy.

The effects of site-directed mutation and salt on the iron(III)-binding site of the recombinant half-molecule of the N-terminal lobe (hTf/2N) of human transferrin was studied by EPR spectroscopy. Changes were observed in the EPR spectra of all variants investigated (D63S, D63C, G65R, K206Q, H207E, H249E, H249Q, K296E and K296Q) compared with that of the wild-type protein. The most pronounced changes in the metal site were caused by replacement of the coordinating residues, Asp-63 and His-249, and the non-coordinating residue Lys-296, which is located in the hinge region of the iron-binding cleft. The EPR spectral changes from replacement of other non-coordinating residues were more subtle, indicating small changes in Fe3+ coordination to the protein. The EPR spectrum of variant G65R suggests that it adopts two distinct conformations in solution, one in which the two domains forming the iron-binding cleft are closed and one in which they are open; in the latter instance Asp-63 is no longer coordinated to the Fe3+. Chloride-binding studies on hTf/2N, K206Q, H207E, K296Q and K296E showed similar binding isotherms, indicating that none of the hinge region residues replaced, i.e. Lys-206, His-207 or Lys-296, are the sites of chloride binding. The results show that the coordination environment of the Fe3+ is sensitive to structural changes from site-directed mutation of both remote and coordinated residues and also to chloride-binding and ionic strength effects.

Binding Sites↗

PubMed1995

Oral Candida: clearance, colonization, or candidiasis?

Candida albicans is frequently isolated from the human mouth, yet few carriers develop clinical signs of candidiasis. Oral candidiasis presents clinically in many forms. This reflects the ability of the yeast to colonize different oral surfaces and the variety of factors which predispose the host to Candida colonization and subsequent infection. Colonization of the oral cavity appears to be facilitated by several specific adherence interactions between C. albicans and oral surfaces which enable the yeast to resist host clearance mechanisms. Thus, Candida has been shown to adhere to complement receptors, various extracellular matrix proteins, and specific sugar residues displayed on host or bacterial surfaces in the oral cavity. Oral candidiasis results from yeast overgrowth and penetration of the oral tissues when the host's physical and immunological defenses have been undermined. Tissue invasion may be assisted by secreted hydrolytic enzymes, hyphal formation, and contact sensing. While these and other phenotypic characteristics may endow certain Candida species or strains with a competitive advantage in the oral cavity, it is the host's immune competence that ultimately determines whether clearance, colonization, or candidiasis occurs.

Antifungal Agents↗