Ligand variation in the transferrin family: the crystal structure of the H249Q mutant of the human transferrin N-lobe as a model for iron binding in insect transferrins.

Proteins of the transferrin (Tf) family play a central role in iron homeostasis in vertebrates. In vertebrate Tfs, the four iron-binding ligands, 1 Asp, 2 Tyr, and 1 His, are invariant in both lobes of these bilobal proteins. In contrast, there are striking variations in the Tfs that have been characterized from insect species; in three of them, sequence changes in the C-lobe binding site render it nonfunctional, and in all of them the His ligand in the N-lobe site is changed to Gln. Surprisingly, mutagenesis of the histidine ligand, His249, to glutamine in the N-lobe half-molecule of human Tf (hTf/2N) shows that iron binding is destabilized and suggests that Gln249 does not bind to iron. We have determined the crystal structure of the H249Q mutant of hTf/2N and refined it at 1.85 A resolution (R = 0.221, R(free) = 0.246). The structure reveals that Gln249 does coordinate to iron, albeit with a lengthened Fe-Oepsilon1 bond of 2.34 A. In every other respect, the protein structure is unchanged from wild-type. Examination of insect Tf sequences shows that the K206.K296 dilysine pair, which aids iron release from the N-lobes of vertebrate Tfs, is not present in the insect proteins. We conclude that substitution of Gln for His does destabilize iron binding, but in the insect Tfs this is compensated by the loss of the dilysine interaction. The combination of a His ligand with the dilysine pair in vertebrate Tfs may have been a later evolutionary development that gives more sophisticated pH-mediated control of iron release from the N-lobe of transferrins.

Crystal structures and iron release properties of mutants (K206A and K296A) that abolish the dilysine interaction in the N-lobe of human transferrin.

Human transferrin (Tf) is responsible for the binding and transport of iron in the bloodstream of vertebrates. Delivery of this bound iron to cells occurs by a process of receptor-mediated endocytosis during which Tf releases its iron at the reduced endosomal pH of approximately 5.6. Iron release from Tf involves a large conformational change in which the two domains that enclose the binding site in each lobe move apart. We have examined the role of two lysines, Lys206 and Lys296, that form a hydrogen-bonded pair close to the N-lobe binding site of human Tf and have been proposed to form a pH-sensitive trigger for iron release. We report high-resolution crystal structures for the K206A and K296A mutants of the N-lobe half-molecule of Tf, hTf/2N, and quantitative iron release data on these mutants and the double mutant K206A/K296A. The refined crystal structures (for K206A, R = 19.6% and R(free) = 23.7%; for K296A, R= 21.2% and R(free) = 29.5%) reveal a highly conserved hydrogen bonding network in the dilysine pair region that appears to be maintained even when individual hydrogen bonding groups change. The iron release data show that the mutants retain iron to a pH 1 unit lower than the pH limit of wild type hTf/2N, and release iron much more slowly as a result of the loss of the dilysine interaction. Added chloride ions are shown to accelerate iron release close to the pH at which iron is naturally lost and the closed structure becomes destabilized, and to retard it at higher pH.

The chloride effect is related to anion binding in determining the rate of iron release from the human transferrin N-lobe.

The major function of human transferrin is to deliver iron from the bloodstream to actively dividing cells. Upon iron release, the protein changes its conformation from 'closed' to 'open'. Extensive studies in vitro indicate that iron release from transferrin is very complex and involves many factors, including pH, the chelator used, an anion effect, temperature, receptor binding and intra-lobe interactions. Our earlier work [He, Mason and Woodworth (1997) Biochem. J. 328, 439-445] using the isolated transferrin N-lobe (recombinant N-lobe of human transferrin comprising residues 1-337; hTF/2N) has shown that anions and pH modulate iron release from hTF/2N in an interdependent manner: chloride retards iron release at neutral pH, but accelerates the reaction at acidic pH. The present study supports this idea and further details the nature of the dual effect of chloride: the anion effect on iron release is closely related to the strength of anion binding to the apoprotein. The negative effect seems to originate from competition between chloride and the chelator for an anion-binding site(s) near the metal centre. With decreasing pH, the strength of anion binding to hTF/2N increases linearly, decreasing the contribution of competition with the chelator. In the meantime, the 'open' or 'loose' conformation of hTF/2N, induced by the protonation of critical residues such as the Lys-206/Lys-296 pair at low pH, enables chloride to enter the cleft and bind to exposed side chains, thereby promoting cleft opening and synergistically allowing removal of iron by the chelator, leading to a positive anion effect. Disabling one or more of the primary anion-binding residues, namely Arg-124, Lys-206 and Lys-296, substantially decreases the anion-binding ability of the resulting mutant proteins. In these cases, the competition for the remaining binding residue(s) is increased, leading to a negative chloride effect or, at most, a very small positive effect, even at low pH.

Crystal structures of two mutants (K206Q, H207E) of the N-lobe of human transferrin with increased affinity for iron.

The X-ray crystallographic structures of two mutants (K206Q and H207E) of the N-lobe of human transferrin (hTF/2N) have been determined to high resolution (1.8 and 2.0 A, respectively). Both mutant proteins bind iron with greater affinity than native hTF/2N. The structures of the K206Q and H207E mutants show interactions (both H-bonding and electrostatic) that stabilize the interaction of Lys296 in the closed conformation, thereby stabilizing the iron bound forms.

Mutations at the histidine 249 ligand profoundly alter the spectral and iron-binding properties of human serum transferrin N-lobe.

Human serum transferrin is an iron-binding and -transport protein which carries iron from the blood stream into various cells. Iron is held in two deep clefts located in the N- and C-lobes by coordinating to four amino acid ligands, Asp 63, Tyr 95, Tyr 188, and His 249 (N-lobe numbering), and to two oxygens from carbonate. We have previously reported the effect on the iron-binding properties of the N-lobe following mutation of the ligands Asp 63, Tyr 95, and Tyr 188. Here we report the profound functional changes which result from mutating His 249 to Ala, Glu, or Gln. The results are consistent with studies done in lactoferrin which showed that the histidine ligand is critical for the stability of the iron-binding site [H. Nicholson, B. F. Anderson, T. Bland, S. C. Shewry, J. W. Tweedie, and E. N. Baker (1997) Biochemistry 36, 341-346]. In the mutant H249A, the histidine ligand is disabled, resulting in a dramatic reduction in the kinetic stability of the protein toward loss of iron. The H249E mutant releases iron three times faster than wild-type protein but shows significant changes in both EPR spectra and the binding of anion. This appears to be the net effect of the metal ligand substitution from a neutral histidine residue to a negative glutamate residue and the disruption of the "dilysine trigger" [MacGillivray, R. T. A., Bewley, M. C., Smith, C. A., He, Q.-Y., Mason, A. B., Woodworth, R. C., and Baker, E. N. (2000) Biochemistry 39, 1211-1216]. In the H249Q mutant, Gln 249 appears not to directly contact the iron, given the similarity in the spectroscopic properties and the lability of iron release of this mutant to the H249A mutant. Further evidence for this idea is provided by the preference of both the H249A and H249Q mutants for nitrilotriacetate rather than carbonate in binding iron, probably because NTA is able to provide a third ligation partner. An intermediate species has been identified during the kinetic interconversion between the NTA and carbonate complexes of the H249A mutant. Thus, mutation of the His 249 residue does not abolish iron binding to the transferrin N-lobe but leads to the appearance of novel iron-binding sites of varying structure and stability.

Mutation of the iron ligand His 249 to Glu in the N-lobe of human transferrin abolishes the dilysine "trigger" but does not significantly affect iron release.

Serum transferrin is the major iron transport protein in humans. Its function depends on its ability to bind iron with very high affinity, yet to release this bound iron at the lower intracellular pH. Possible explanations for the release of iron from transferrin at low pH include protonation of a histidine ligand and the existence of a pH-sensitive "trigger" involving a hydrogen-bonded pair of lysines in the N-lobe of transferrin. We have determined the crystal structure of the His249Glu mutant of the N-lobe half-molecule of human transferrin and compared its iron-binding properties with those of the wild-type protein and other mutants. The crystal structure, determined at 2.4 A resolution (R-factor 19.8%, R(free) 29.4%), shows that Glu 249 is directly bound to iron, in place of the His ligand, and that a local movement of Lys 296 has broken the dilysine interaction. Despite the loss of this potentially pH-sensitive interaction, the H249E mutant is only slightly more acid-stable than wild-type and releases iron slightly faster. We conclude that the loss of the dilysine interaction does make the protein more acid stable but that this is counterbalanced by the replacement of a neutral ligand (His) by a negatively charged one (Glu), thus disrupting the electroneutrality of the binding site.

Identification of platination sites on human serum transferrin using (13)C and (15)N NMR spectroscopy.

Reactions between various apo and metal-bound forms of human serum transferrin (80 kDa) and the recombinant N-lobe (40 kDa) with [Pt(en)Cl(2)] or cis-[PtCl(2)(NH(3))(2)] have been investigated in solution via observation of [(1)H,(15)N] NMR resonances of the Pt complexes, [(1)H,(13)C] resonances of the eCH(3) groups of the protein methionine residues, and by chromatographic analysis of single-site methionine mutants. For the whole protein, the preferred Pt binding site appears to be Met256. Additional binding occurs at the other surface-exposed methionine (Met499), which is platinated at a slower rate than Met256. In contrast, binding of similar Pt compounds to the N-lobe of the protein occurs at Met313, rather than Met256. Met313 is buried in the interlobe contact region of intact transferrin. After loss of one chloride ligand from Pt and binding to methionine sulfur of the N-lobe, chelate-ring closure appears to occur with binding to a deprotonated backbone amide nitrogen, and the loss of the other chloride ligand. Such chelate-ring closure was not observed during reactions of the whole protein, even after several days.

Dual role of Lys206-Lys296 interaction in human transferrin N-lobe: iron-release trigger and anion-binding site.

The unique structural feature of the dilysine (Lys206-Lys296) pair in the transferrin N-lobe (hTF/2N) has been postulated to serve a special function in the release of iron from the protein. These two lysines, which are located in opposite domains, hydrogen bond to each other in the iron-containing hTF/2N at neutral pH but are far apart in the apo-form of the protein. It has been proposed that charge repulsion resulting from the protonation of the dilysines at lower pH may be the trigger to open the cleft and facilitate iron release. The fact that the dilysine pair is positively charged and resides in a location close to the metal-binding center has also led to the suggestion that the dilysine pair is an anion-binding site for chelators. The present report provides comprehensive evidence to confirm that the dilysine pair plays this dual role in modulating release of iron. When either of the lysines is mutated to glutamate or glutamine or when both are mutated to glutamate, release of iron is much slower compared to the wild-type protein. This is due to the fact that the driving force for cleft opening is absent in the mutants or is converted to a lock-like interaction (in the case of the K206E and K296E mutants). Direct titration of the apo-proteins with anions as well as anion-dependent iron release studies show that the dilysine pair is part of an active anion-binding site which exists with the Lys296-Tyr188 interaction as a core. At this site, Lys296 serves as the primary anion-binding residue and Tyr188 is the main reporter for electronic spectral change, with smaller contributions from Lys206, Tyr85, and Tyr95. In iron-loaded hTF/2N, anion binding becomes invisible as monitored by UV-vis difference spectra since the spectral reporters Tyr188 and Tyr95 are bound to iron. Our data strongly support the hypothesis that the apo-hTF/2N exists in equilibrium between the open and closed conformations, because only in the closed form is Lys296 in direct contact with Tyr188. The current findings bring together observations, ideas, and experimental data from a large number of previous studies and shed further light on the detailed mechanism of iron release from the transferrin N-lobe. In iron-containing hTF/2N, Lys296 may still function as a target to introduce an anion (or a chelator) near to the iron-binding center. When the pH is lowered, the protonation of carbonate (synergistic anion for metal binding) and then the dilysine pair form the driving force to loosen the cleft, exposing iron; the nearby anion (or chelator) then binds to the iron and releases it from the protein.

X-ray crystallography and mass spectroscopy reveal that the N-lobe of human transferrin expressed in Pichia pastoris is folded correctly but is glycosylated on serine-32.

The ferric form of the N-lobe of human serum transferrin (Fe(III)-hTF/2N) has been expressed at high levels in Pichia pastoris. The Fe(III)-hTF/2N was crystallized in the space group P41212, and X-ray crystallography was used to solve the structure of the recombinant protein at 2.5 A resolution. This represents only the second P. pastoris-derived protein structure determined to date, and allows the comparison of the structures of recombinant Fe(III)-hTF/2N expressed in P. pastoris and mammalian cells with serum-derived transferrin. The polypeptide folding pattern is essentially identical in all of the three proteins. Mass spectroscopic analyses of P. pastoris- hTF/2N and proteolytically derived fragments revealed glycosylation of Ser-32 with a single hexose. This represents the first localization of an O-linked glycan in a P. pastoris-derived protein. Because of its distance from the iron-binding site, glycosylation of Ser-32 should not affect the iron-binding properties of hTF/2N expressed in P. pastoris, making this an excellent expression system for the production of hTF/2N.

Regulation and pH-dependent expression of a bilaterally truncated yeast plasma membrane H+-ATPase.

Constitutive, chromosomal expression of yeast pma1 deletion alleles in Saccharomyces cerevisiae yielded functional, truncated forms of the plasma membrane H+-ATPase which were independently capable of supporting wild type yeast growth rates. Deletion of 27 amino-terminal residues affected neither the enzyme's activity nor its responsiveness to changes in glucose metabolism. By contrast, removal of 18 carboxy-terminal amino acids produced an enzyme with a Vmax that was relatively insensitive to glucose-dependent metabolic status and with a Km that was significantly lower than that of the wild type enzyme. These effects were exaggerated when the amino- and carboxy-terminal deletions were combined in a bilaterally truncated H+-ATPase, suggesting that the amino terminus may have a subtle role in modulating ATPase activity. In pma1DeltaDelta cells cultured at pH 6, plasma membrane H+-ATPase levels were much lower than those in cells expressing a wild type ATPase. Increased expression levels could be achieved by growing the pma1DeltaDelta mutant at pH 3, a result that was at least partially due to a sustained, elevated transcription of pma1DeltaDelta mRNA. Our observations suggest that intracellular proton balance can be maintained by regulation of the activity and/or quantity of H+-ATPase in the plasma membrane.

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.

[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.

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.

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.

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.

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.

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.

Targeting the fungal plasma membrane proton pump.

The need for new mechanistic classes of broad spectrum antifungal agents has prompted development of the membrane sector and ectodomain of the plasma membrane proton pumping ATPase as an antifungal target. The fungal proton pump is a highly abundant, essential enzyme in Saccharomyces cerevisiae. It belongs to the family of P-type ATPases, a class of enzymes that includes the Na+,K(+)-ATPase and the gastric H+,K(+)-ATPase. These enzymes are cell surface therapeutic targets for the cardiac glycosides and several anti-ulcer drugs, respectively. The effects of acid-activated omeprazole show that extensive inhibition of the S. cerevisiae ATPase is fungicidal. Fungal proton pumps possess elements within their transmembrane loops that distinguish them from other P-type ATPases. These loops, such as the conformationally sensitive transmembrane loop 1+2, can attenuate the activity of the enzyme. Expression in S. cerevisiae of fully functional chimeric ATPases that contain a foreign target comprising transmembrane loops 1+2 and/or 3+4 from the fungal pathogen Candida albicans suggests that these loops operate as a domain. The chimera containing C. albicans transmembrane loops 1+2 and 3+4 provides a prototype for mutational analysis of the target region and the screening of inhibitors directed against opportunistic fungal pathogens. Panels of mutants with modified ATPase regulation or with altered cell surface cysteine residues are also described. Information about the ATPase membrane sector and ectodomain has been integrated into a model of this region.

Expression and initial characterization of five site-directed mutants of the N-terminal half-molecule of human transferrin.

Five site-directed mutants of the N-terminal half-molecule of human serum transferrin have been expressed in baby hamster kidney cells and purified to homogeneity. Expression levels and overall yields varied considerably from the wild-type protein, depending on the mutant in question. The mutants are D63S, D63C, G65R, K206Q, and H207E and are based on mutations observed in a variety of transferrins of known sequence. Their molecular masses, determined by electrospray mass spectrometry, agree with theory, except for the D63C mutant, which appears to be cysteinylated. All mutants bind iron but with varying affinities; qualitatively, in increasing order D63S approximately D63C approximately G65R much less than wild type less than or equal to H207E much less than K206Q. In general, reduction of formal negative charge within the binding cleft shifts the visible spectral maximum of the iron complex toward the blue and reduces the affinity for iron, and increasing the formal negative charge shifts the visible maximum toward the red and increases the affinity for iron. The K206Q mutant is exceptional inasmuch as its visible maximum shows a blue shift, but its affinity for iron is the greatest of all of the mutants studied. All mutants reported, in addition to the wild-type protein, exhibit very similar visible molar extinction coefficients for the iron complex and very similar changes in extinction coefficients at 240 nm on binding Fe(III) or Ga(III). These results suggest that in all cases the bound metal ion is coordinated by two tyrosyl side chains.