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The Position of Arginine 124 Controls the Rate of Iron Release from the N-lobe of Human Serum Transferrin

2003; Elsevier BV; Volume: 278; Issue: 8 Linguagem: Inglês

10.1074/jbc.m210349200

1083-351X

Ty E. Adams, Anne B. Mason, Qing‐Yu He, Peter J. Halbrooks, Sara K. Briggs, Valerie C. Smith, Ross T. A. MacGillivray, Stephen J. Everse,

Porphyrin Metabolism and Disorders

Human serum transferrin (hTF) is a bilobal iron-binding and transport protein that carries iron in the blood stream for delivery to cells by a pH-dependent mechanism. Two iron atoms are held tightly in two deep clefts by coordination to four amino acid residues in each cleft (two tyrosines, a histidine, and an aspartic acid) and two oxygen atoms from the "synergistic" carbonate anion. Other residues in the binding pocket, not directly coordinated to iron, also play critical roles in iron uptake and release through hydrogen bonding to the liganding residues. The original crystal structures of the iron-loaded N-lobe of hTF (pH 5.75 and 6.2) revealed that the synergistic carbonate is stabilized by interaction with Arg-124 and that both the arginine and the carbonate adopt two conformations (MacGillivray, R. T. A., Moore, S. A., Chen, J., Anderson, B. F., Baker, H., Luo, Y. G., Bewley, M., Smith, C. A., Murphy, M. E., Wang, Y., Mason, A. B., Woodworth, R. C., Brayer, G. D., and Baker, E. N. (1998) Biochemistry 37, 7919–7928). In the present study, we show that the two conformations are also found for a structure at pH 7.7, indicating that this finding was not strictly a function of pH. We also provide structures for two single point mutants (Y45E and L66W) designed to force Arg-124 to adopt each of the previously observed conformations. The structures of each mutant show that this goal was accomplished, and functional studies confirm the hypothesis that access to the synergistic anion dictates the rate of iron release. These studies highlight the importance of the arginine/carbonate movement in the mechanism of iron release in the N-lobe of hTF. Access to the carbonate via a water channel allows entry of protons and anions, enabling the attack on the iron. Human serum transferrin (hTF) is a bilobal iron-binding and transport protein that carries iron in the blood stream for delivery to cells by a pH-dependent mechanism. Two iron atoms are held tightly in two deep clefts by coordination to four amino acid residues in each cleft (two tyrosines, a histidine, and an aspartic acid) and two oxygen atoms from the "synergistic" carbonate anion. Other residues in the binding pocket, not directly coordinated to iron, also play critical roles in iron uptake and release through hydrogen bonding to the liganding residues. The original crystal structures of the iron-loaded N-lobe of hTF (pH 5.75 and 6.2) revealed that the synergistic carbonate is stabilized by interaction with Arg-124 and that both the arginine and the carbonate adopt two conformations (MacGillivray, R. T. A., Moore, S. A., Chen, J., Anderson, B. F., Baker, H., Luo, Y. G., Bewley, M., Smith, C. A., Murphy, M. E., Wang, Y., Mason, A. B., Woodworth, R. C., Brayer, G. D., and Baker, E. N. (1998) Biochemistry 37, 7919–7928). In the present study, we show that the two conformations are also found for a structure at pH 7.7, indicating that this finding was not strictly a function of pH. We also provide structures for two single point mutants (Y45E and L66W) designed to force Arg-124 to adopt each of the previously observed conformations. The structures of each mutant show that this goal was accomplished, and functional studies confirm the hypothesis that access to the synergistic anion dictates the rate of iron release. These studies highlight the importance of the arginine/carbonate movement in the mechanism of iron release in the N-lobe of hTF. Access to the carbonate via a water channel allows entry of protons and anions, enabling the attack on the iron. Human serum transferrin (hTF) 1The abbreviations used are: TF, transferrin; hTF, human serum transferrin; hTF/2N, the N-lobe of hTF; oTF, ovotransferrin; LTF, lactoferrin; MES, morpholinoethanesulfonic acid; Tiron, 4,5-dihydroxy-m-benzenedisulfonic acid sodium salt; WT, wild type 1The abbreviations used are: TF, transferrin; hTF, human serum transferrin; hTF/2N, the N-lobe of hTF; oTF, ovotransferrin; LTF, lactoferrin; MES, morpholinoethanesulfonic acid; Tiron, 4,5-dihydroxy-m-benzenedisulfonic acid sodium salt; WT, wild type is a member of the transferrin family of iron-binding proteins, which includes ovotransferrin (oTF), found in avian egg white, lactoferrin (LTF), found in milk, tears, and other bodily secretions, and melanotransferrin, found on the surface of melanocytes (1Evans R.W. Crawley J.B. Joannou C.L. Sharma N.D. Bullen J.J. Griffiths E. Iron and Infection: Molecular, Physiological and Clinical Aspects. John Wiley and Sons, Chichester, UK1999: 27-86Google Scholar, 2He Q.-Y. Mason A.B. Templeton D.M. Molecular and Cellular Iron Transport. Marcel Dekker, Inc., New York2002: 95-123Google Scholar). The hTF binds iron reversibly in blood plasma and transports it to cells requiring iron. Full-length transferrin molecules (∼80 kDa) consist of a single polypeptide chain folded into two similar lobes, the N-lobe and C-lobe. The two lobes display significant sequence similarity and appear to have evolved from the duplication of an ancestral gene coding for a protein with a single metal-binding site (3Williams J. Trends Biochem. Sci. 1982; 7: 394-397Google Scholar). Each homologous lobe contains an iron-binding site deep within a cleft that subdivides it into two dissimilar domains (designated NI and NII domains for the N-lobe and CI and CII for the C-lobe). Iron is bound in a distorted octahedral coordination involving four amino acid ligands and two oxygen atoms from a synergistically bound carbonate ion. The synergistic relationship of metal and anion refers to the fact that neither binds tightly in the absence of the other (4Faber H.R. Baker C.J. Day C.L. Tweedie J.W. Baker E.N. Biochemistry. 1996; 35: 14473-14479Google Scholar). This synergy is a unique characteristic of the transferrin family and is not observed in other metal-binding proteins. Although hTF, oTF, and LTF share identical iron-binding ligands and display high sequence homology, substantial differences in the binding affinity for iron both within and between the TFs are found and are not well understood (5Bali P.K. Harris W.R. J. Am. Chem. Soc. 1989; 111: 4457-4461Google Scholar, 6El Hage Chahine J.-M. Fain D. Eur. J. Biochem. 1994; 223: 581-587Google Scholar, 7Marques H.M. Walton T. Egan T.J. J. Inorg. Biochem. 1995; 57: 11-21Google Scholar, 8Zak O. Tam B. MacGillivray R.T.A. Aisen P. Biochemistry. 1997; 36: 11036-11043Google Scholar, 9Muralidhara B.K. Hirose M. J. Biol. Chem. 2000; 275: 12463-12469Google Scholar). An obvious experimental approach to explore these differences involves the mutation of specific residues followed by assessment of the changes in function.A critical function of hTF in serum is to deliver iron to actively dividing cells. Diferric hTF binds with high affinity to specific receptors on the surface of cells (10Aisen P. Enns C. Wessling-Resnick M. Int. J. Biochem. Cell Biol. 2001; 33: 940-959Google Scholar). The apo- or iron-depleted hTF is poorly recognized by the receptor at physiological pH. The hTF-receptor complex is endocytosed; within the endosome, a proton pump results in a drop in the pH to ∼5.6, triggering iron release. This step is not well defined despite intensive research efforts. The entire cycle occurs in 2–3 min (11Dautry-Varsat A. Ciechanover A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2258-2262Google Scholar).Iron binding and release is characterized by a large conformational change. When iron is released, the two domains move away from each other by means of a hinge located in two antiparallel β-strands that lie behind the iron-binding site in each lobe. In the N-lobe of transferrin, one domain rotates 63° relative to the other about this hinge (12MacGillivray R.T.A. Moore S.A. Chen J. Anderson B.F. Baker H. Luo Y.G. Bewley M. Smith C.A. Murphy M.E. Wang Y. Mason A.B. Woodworth R.C. Brayer G.D. Baker E.N. Biochemistry. 1998; 37: 7919-7928Google Scholar, 13Jeffrey P.D. Bewley M.C. MacGillivray R.T.A. Mason A.B. Woodworth R.C. Baker E.N. Biochemistry. 1998; 37: 13978-13986Google Scholar). The precise mechanism for this movement remains unclear, but protonation of several key amino acid residues appears to be critical to the release of iron (see below).Many studies have clearly demonstrated that an arginine located near the iron-binding site (Arg-124 in hTF/2N) stabilizes the synergistic carbonate anion. This arginine is highly conserved in each lobe of all mammalian TFs. As detailed previously (4Faber H.R. Baker C.J. Day C.L. Tweedie J.W. Baker E.N. Biochemistry. 1996; 35: 14473-14479Google Scholar), those lobes with amino acids other than arginine are unable to bind iron with high affinity and specificity. Mutational studies in both hTF and LTF further confirm the important role of the arginine in iron release (14Zak O. Aisen P. Crawley J.B. Joannou C.L. Patel K.J. Rafiq M. Evans R.W. Biochemistry. 1995; 34: 14428-14434Google Scholar, 15Peterson N.A. Anderson B.F. Jameson G.B. Tweedie J.W. Baker E.N. Biochemistry. 2000; 39: 6625-6633Google Scholar, 16He Q.Y. Mason A.B. Nguyen V. MacGillivray R.T.A. Woodworth R.C. Biochem. J. 2000; 350: 909-915Google Scholar, 17Li Y.J. Harris W.R. Maxwell A. MacGillivray R.T.A. Brown T. Biochemistry. 1998; 37: 14157-14166Google Scholar); all mutants feature accelerated rates of iron release.The two high resolution structures (1.6 and 1.8 Å) of the Fe(III) form of hTF/2N revealed the presence of electron density indicative of positional disorder near the metal-binding sites of both crystal forms. The density was fitted by placement of the synergistic carbonate and the side chain of Arg-124 into two positions. In the A or "near" conformer, the carbonate is fully engaged in binding to the iron and is stabilized by bonds to the NE and NH2 atoms of Arg-124, the OG1 atom of Thr-120, and the main chain amide nitrogen atoms of Ala-126 and Gly-127, which reside on helix 5 (Fig.1 A). This form is found in both lobes of oTF and LTF. In contrast, in the B or "far" conformer, the carbonate has rotated 30°, Arg-124 has moved away from the site, and the carbonate is detached from helix 5 (Fig.1 B). We hypothesized that protonation of the carbonate (resulting in bicarbonate) might be responsible for these changes in the position of the arginine and that this protonation could well be the first step in iron release, an idea originally suggested by Aisen and Leibman in 1973 (18Aisen P. Leibman A. Biochim. Biophys. Acta. 1973; 304: 797-804Google Scholar). Obviously, changes in access to carbonate could have a profound effect on the release rate. In the wild-type N-lobe of hTF, the carbonate is surrounded by a network of ordered water molecules that may function in the transport of protons from the outside of the protein into the iron-binding site. Of interest is that no heterogeneity has been observed in the position of the arginine and carbonate in either lobe of any of the other TF structures reported to date. These include many different LTF structures (19Baker E.N. Anderson B.F. Baker H.M. MacGillivray R.T.A. Moore S.A. Peterson N.A. Shewry S.C. Tweedie J.W. Adv. Exp. Med. Biol. 1998; 443: 1-14Google Scholar, 20Baker E.N. Baker H.M. Kidd R.D. Biochem. Cell Biol. 2002; 80: 27-34Google Scholar), oTF (21Kurokawa H. Mikami B. Hirose M. J. Mol. Biol. 1995; 254: 196-207Google Scholar), rabbit and pig serum transferrin (22Hall D.R. Hadden J.M. Leonard G.A. Bailey S. Neu M. Winn M. Lindley P.F. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 70-80Google Scholar), and the iron-containing C-lobe of hTF (23Zuccola H.J. The Crystal Structure of Monoferric Human Serum TransferrinPh.D. thesis. Georgia Institute of Technology, Atlanta, GA1993Google Scholar).The current study was undertaken to determine the significance of the alternate positions of Arg-124 in the function of the N-lobe of hTF. It is important to mention that the present study includes the structure of wild-type hTF/2N crystallized at pH 7.7 as a control and that it also features alternate positions for both the carbonate and Arg-124. This implies that the original observation was not directly due to the lower pH used in these earlier studies as postulated previously (12MacGillivray R.T.A. Moore S.A. Chen J. Anderson B.F. Baker H. Luo Y.G. Bewley M. Smith C.A. Murphy M.E. Wang Y. Mason A.B. Woodworth R.C. Brayer G.D. Baker E.N. Biochemistry. 1998; 37: 7919-7928Google Scholar). Tyrosine 45 resides at the lip of the iron-binding cleft and is hydrogen-bonded to a water molecule; it was mutated to a glutamic acid residue (Y45E). We hypothesized that the presence of the negatively charged glutamic acid might force Arg-124 into a single position, namely the B or far conformer, allowing easier access to the site. By replacing the tyrosine with glutamic acid in the wild-type structure, the carboxyl head group is the proper distance from Arg-124 to produce a salt bridge (Fig.2 A).Figure 2Position of Y45E and L66W mutations. The structure of the hTF N-lobe showing the carbonate and Arg-124 in their far (green) (inset A) and near (purple) (inset B) conformations is shown. The Y45E and L66W mutations are shown in red in theinsets. The NI domain is depicted in dark blue, and the NII domain is depicted in blue. Note that the two mutants (Y45E and L66W) reside in the NI domain, whereas Arg-124 is in the NII domain.View Large Image Figure ViewerDownload (PPT)Alternatively Leu-66, which in the closed iron-containing N-lobe resides near Arg-124 on the edge of the solvent cavity adjacent to the iron-binding site, was converted to a tryptophan (L66W) with the premise that substitution of the bulky, hydrophobic Trp residue would force Arg-124 into the A or near conformer and thus restrict access to the synergistic anion (Fig. 2 B). Functional and structural data appear to support these hypotheses and confirm the importance of the alternate conformations of carbonate and Arg-124 in the mechanism of iron release from the N-lobe of hTF.EXPERIMENTAL PROCEDURESMaterialsAll chemicals used were of reagent quality. Stock solutions of HEPES, MES, and other buffers were prepared by dissolving the anhydrous salts in Milli-Q (Millipore) purified water, and adjusting the pH to the desired values with 1 n NaOH or HCl. EDTA was purchased from Mann Research Laboratories, Inc.; nitrilotriacetate was from Sigma, and Tiron was from Fisher. Tiron stock solutions were prepared by dissolving the Tiron in the appropriate buffers and adjusting the pH to the desired values with 1 n NaOH. Centricon 10 microconcentrators were from Amicon. Polyethylene glycol 3350 was from Hampton Research, Inc.Molecular BiologyThe mutant Y45E was introduced into the N-lobe of transferrin using a polymerase chain reaction-based mutagenesis procedure (24Nelson R.M. Long G.L. Anal. Biochem. 1989; 180: 147-151Google Scholar). The following synthetic oligonucleotide was used to introduce the mutations (the mutagenic nucleotides are in bold type and underlined): Y45E: 5′-AAA GCC TCC GAA CTT GAT TGC-3′.The conditions for the PCR reactions were as follows: denaturation at 94° for 15 s, annealing at 50° for 30 s, and extension at 72° for 30 s. Step I of the PCR mutagenesis procedure consisted of 30 cycles, step II consisted of 1 cycle, and step III consisted of 30 cycles (24Nelson R.M. Long G.L. Anal. Biochem. 1989; 180: 147-151Google Scholar). The nucleotide sequence of the insert confirmed the introduction of the specific mutation. The mutated hTF/2N cDNA was excised from the Bluescript vector, the ends were made blunt, and the fragment was ligated into the SmaI site of the pNUT expression vector. Restriction endonuclease mapping was used to confirm the correct orientation.The L66W mutation was introduced into the transferrin N-lobe using the QuikChange mutagenesis kit (Stratagene). The mutation was made directly in the transferrin N-lobe cDNA sequence cloned in the pNUT vector. Two complimentary mutagenic oligonucleotide primers containing the L66W mutation were used: primer 1, 5′-ACA CTG GAT GCA GGT TGG GTG TAT GAT GCT TAC TTG GC-3′, and primer 2, 5′-GCC AAG TAA GCA TCA TAC ACC CAA CCT GCA TCC AGT GT-3′. The mutagenic nucleotides are shown in bold type and underlined.The conditions for the PCR reaction were as follows: 95° for 30 s followed by 18 cycles with denaturation at 95° for 30 s, annealing at 55° for 1 min, and extension at 68° for 13 min. To determine the presence of the correct mutation and absence of other mutations, the complete sequence of the transferrin cDNA and flanking pNUT sequence was determined prior to the introduction of the plasmid into baby hamster kidney cells.Recombinant Protein Production and PurificationThe production and purification of hTF/2N and mutants of hTF/2N in baby hamster kidney cells using the pNUT expression vector system has been described previously in detail (25Mason A.B. Funk W.D. MacGillivray R.T.A. Woodworth R.C. Protein Expression Purif. 1991; 2: 214-220Google Scholar, 26He Q.Y. Mason A.B. Lyons B.A. Tam B.M. Nguyen V. MacGillivray R.T.A. Woodworth R.C. Biochem. J. 2001; 354: 423-429Google Scholar). Briefly, the recombinant hTF/2N that is secreted into the tissue culture medium is saturated with iron and exchanged into 5 mm Tris-HCl buffer, pH 8.0, using a spiral cartridge concentrator. A Poros 50 HQ anion-exchange column is used to eliminate most of the serum albumin and all of the phenol red from the sample. Pooled fractions are concentrated and applied to a Sephacryl S-200 HR column (5 × 80 cm) equilibrated and run in 0.1 m ammonium bicarbonate. Following passage through a 0.2-μm syringe filter and concentration, the samples are stored at −20 °C in 0.1 m ammonium bicarbonate. Purity of the recombinant protein was determined using SDS-polyacrylamide gel electrophoresis.Kinetics of Iron RemovalIron removal from the wild-type N-lobe and the mutants (∼40 μm) was measured using the chelator Tiron (12 mm) in 50 mm HEPES at pH 7.4 and 25 °C. The reaction was monitored by following an increase in absorbance at 480 nm for the iron-Tiron complex formation. For experiments at pH 5.6, the chelator EDTA was used to remove iron at a concentration of 4 mm in 50 mm MES. In this case, the reaction was monitored by the decrease in absorbance at 470 or 293 nm, which follows the release of iron from the protein. For slower release rates, a Cary 100 spectrophotometer (Varian) was used. For faster rates, iron release kinetics were measured using an OLIS RSM-1000 stop-flow spectrophotometer (On-Line Instrumentation Systems, Inc). One syringe contained the protein sample in water, and the other contained 8 mm EDTA in 100 mm MES buffer, pH 5.6. Absorbance spectra were collected 5 ms after mixing and continued for at least four half-lives.pH Dependence on Iron ReleaseThe retention of iron as a function of pH was measured for each mutant. Aliquots of iron-saturated protein (∼50 μm) in 100 mm ammonium bicarbonate were incubated in a buffer containing 33.3 mm HEPES, MES, and sodium acetate adjusted to the appropriate pH (between 3 and 8) with either 1n NaOH or 9 lacial acetic acid and maintained at 4 °C for a period of 1 week to allow each sample to reach equilibrium. The percentage of iron remaining bound to the transferrin samples was determined by measuring the absorbance at the visible absorption maxima and comparing this absorbance to the fully iron-loaded protein. The pH was measured on identical aliquots at the end of 1 week. The data were plotted and analyzed using Origin software (Microcal).Crystallization of hTF/2N and the Two MutantsRecombinant hTF/2N and both mutants were crystallized using the sitting drop method at 20 °C. Protein (35 mg/ml) in 0.1m ammonium bicarbonate was mixed with an equal amount of the reservoir solution composed of 0.2 m potassium acetate (pH 7.7) and 20% polyethylene glycol 3350. In all cases, large red crystals (2.0 mm × 0.9 mm × 0.4 mm) formed in 5–14 days. All proteins were essentially isomorphous with wild-type hTF/2N (Protein Data Bank accession number 1A8E), showing similar cell dimensions and crystallizing in the orthorhombic space group P212121 with one molecule in each asymmetric unit (Table I).Table IData collection and model refinement statisticshTF/2NY45EL66WSpace groupP212121P212121P212121Unit cell (Å)a = 45.02a = 43.92a = 45.18b = 57.88b = 56.57b = 57.92c = 135.66c = 135.84c = 135.51Resolution limits (Å)30–2.0530–2.130–2.1R sym(%)aData in parentheses are for the outermost shell (2.12–2.05 Å for hTF/2N, 2.18–2.10 Å for Y45E, 2.18–2.10 Å for L66W).7.7 (17.8)6.2 (13.9)9.3 (30.2)I/ςaData in parentheses are for the outermost shell (2.12–2.05 Å for hTF/2N, 2.18–2.10 Å for Y45E, 2.18–2.10 Å for L66W).19.5 (9.4)17.7 (11.0)17.5 (6.1)Completeness (%)aData in parentheses are for the outermost shell (2.12–2.05 Å for hTF/2N, 2.18–2.10 Å for Y45E, 2.18–2.10 Å for L66W).88.1 (91.6)92.6 (90.2)98.4 (96.0)Molecules in asymmetric unit111No. of unique reflectionsaData in parentheses are for the outermost shell (2.12–2.05 Å for hTF/2N, 2.18–2.10 Å for Y45E, 2.18–2.10 Å for L66W).20284 (2046)19129 (1813)21203 (2018)R-factor (R free) (%)19.8 (22.9)19.3 (24.2)19.6 (23.8)Model detailsNo. of protein atoms254825482548No. of solvent atoms14416733No. of ionsFe3+, 2CO32−Fe3+, 2CO32−Fe3+, CO32−Average B-factor (Å2)protein main chain atoms23.721.626.2protein side chain atoms26.824.532.3solvent molecules43.350.838.5r.m.s.br.m.s., root mean squared. deviation from ideal geometrybond distances (Å)0.0080.0060.008bond angles (deg)1.31.31.3Residues in favored Ramachandran regions (%)87.187.187.1a Data in parentheses are for the outermost shell (2.12–2.05 Å for hTF/2N, 2.18–2.10 Å for Y45E, 2.18–2.10 Å for L66W).b r.m.s., root mean squared. Open table in a new tab Data Collection, Structure Determination, and RefinementData for hTF/2N and the two mutants were collected at room temperature using a Mar345 image plate detector on a Rigaku RU-300 generator with a copper rotating anode. All data sets were indexed using DENZO (27Otwinowski Z. Minor W. Carter C.W. Sweet R.M. Methods in Enzymology, Vol. 270A. Academic Press, Inc., San Diego1997: 307-326Google Scholar) and scaled and merged using SCALEPACK (27Otwinowski Z. Minor W. Carter C.W. Sweet R.M. Methods in Enzymology, Vol. 270A. Academic Press, Inc., San Diego1997: 307-326Google Scholar). Statistics are given in Table I. For all structures, the wild-type pH 5.75 structure (Protein Data Bank accession number 1A8E) was used with molecular replacement. In all cases Arg-124, carbonate, iron, and waters were removed from the search model to eliminate any bias from these atomsWild-type hTF/2N, pH 7.7Since the unit cell was identical to the previously solved wild-type hTF/2N, the rigid-body alignment routine of CNS (in which the NI and NII domains were also allowed to move independently) was used to solve the wild-type hTF/2N, pH 7.7, structure (28Brunger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Google Scholar). This reduced the R-factor to 0.30 for data from 30 to 2.05 Å. The model was refined using successive rounds of simulated annealing, occupancy, and B-factor refinement. This was followed by revision of the model based upon map interpretation using an SGI work station with the graphics program O (29Jones T.A. Zou J.Y. Cowan S.W. Acta Crystallogr. Sect. A. 1991; 47: 110-119Google Scholar, 30Kleywegt G.J. Jones T.A. Carter C.W. Sweet R.M. Methods in Enzymology, Vol. 270A. Academic Press, Inc., San Diego1997: 208-230Google Scholar). In the later stages of refinement, Arg-124, carbonate, and solvent molecules were added and allowed to move freely. Conformations of Arg-124 and carbonate were determined by fitting observedF o − F c electron density maps. Only residues 3–331 from the polypeptide chain were observed in the electron density and deposited in the final model (Protein Data Bank accession number 1N84).Y45EThe structure of the Y45E mutant was solved by using the molecular replacement routines in CNS. The cross-rotation search yielded a single peak 8.8 ς above the mean, and the translational search found a peak 3.8 ς above the mean. Rigid-body refinement reduced the R-factor to 0.31 for data from 30 to 2.1 Å. Refinement was carried out as described above; again, only residues 3–331 were deposited in the final model (Protein Data Bank accession number 1N7X).L66WAs described for the wild-type pH 7.7 structure above, the rigid-body alignment routine of CNS was used to solve the L66W mutant; this reduced the R-factor to 0.29 for data from 30 to 2.1 Å. Refinement was carried out as described above, again with only residues 3–331 deposited in the final model (Protein Data Bank accession number 1N7W). Human serum transferrin (hTF) 1The abbreviations used are: TF, transferrin; hTF, human serum transferrin; hTF/2N, the N-lobe of hTF; oTF, ovotransferrin; LTF, lactoferrin; MES, morpholinoethanesulfonic acid; Tiron, 4,5-dihydroxy-m-benzenedisulfonic acid sodium salt; WT, wild type 1The abbreviations used are: TF, transferrin; hTF, human serum transferrin; hTF/2N, the N-lobe of hTF; oTF, ovotransferrin; LTF, lactoferrin; MES, morpholinoethanesulfonic acid; Tiron, 4,5-dihydroxy-m-benzenedisulfonic acid sodium salt; WT, wild type is a member of the transferrin family of iron-binding proteins, which includes ovotransferrin (oTF), found in avian egg white, lactoferrin (LTF), found in milk, tears, and other bodily secretions, and melanotransferrin, found on the surface of melanocytes (1Evans R.W. Crawley J.B. Joannou C.L. Sharma N.D. Bullen J.J. Griffiths E. Iron and Infection: Molecular, Physiological and Clinical Aspects. John Wiley and Sons, Chichester, UK1999: 27-86Google Scholar, 2He Q.-Y. Mason A.B. Templeton D.M. Molecular and Cellular Iron Transport. Marcel Dekker, Inc., New York2002: 95-123Google Scholar). The hTF binds iron reversibly in blood plasma and transports it to cells requiring iron. Full-length transferrin molecules (∼80 kDa) consist of a single polypeptide chain folded into two similar lobes, the N-lobe and C-lobe. The two lobes display significant sequence similarity and appear to have evolved from the duplication of an ancestral gene coding for a protein with a single metal-binding site (3Williams J. Trends Biochem. Sci. 1982; 7: 394-397Google Scholar). Each homologous lobe contains an iron-binding site deep within a cleft that subdivides it into two dissimilar domains (designated NI and NII domains for the N-lobe and CI and CII for the C-lobe). Iron is bound in a distorted octahedral coordination involving four amino acid ligands and two oxygen atoms from a synergistically bound carbonate ion. The synergistic relationship of metal and anion refers to the fact that neither binds tightly in the absence of the other (4Faber H.R. Baker C.J. Day C.L. Tweedie J.W. Baker E.N. Biochemistry. 1996; 35: 14473-14479Google Scholar). This synergy is a unique characteristic of the transferrin family and is not observed in other metal-binding proteins. Although hTF, oTF, and LTF share identical iron-binding ligands and display high sequence homology, substantial differences in the binding affinity for iron both within and between the TFs are found and are not well understood (5Bali P.K. Harris W.R. J. Am. Chem. Soc. 1989; 111: 4457-4461Google Scholar, 6El Hage Chahine J.-M. Fain D. Eur. J. Biochem. 1994; 223: 581-587Google Scholar, 7Marques H.M. Walton T. Egan T.J. J. Inorg. Biochem. 1995; 57: 11-21Google Scholar, 8Zak O. Tam B. MacGillivray R.T.A. Aisen P. Biochemistry. 1997; 36: 11036-11043Google Scholar, 9Muralidhara B.K. Hirose M. J. Biol. Chem. 2000; 275: 12463-12469Google Scholar). An obvious experimental approach to explore these differences involves the mutation of specific residues followed by assessment of the changes in function. A critical function of hTF in serum is to deliver iron to actively dividing cells. Diferric hTF binds with high affinity to specific receptors on the surface of cells (10Aisen P. Enns C. Wessling-Resnick M. Int. J. Biochem. Cell Biol. 2001; 33: 940-959Google Scholar). The apo- or iron-depleted hTF is poorly recognized by the receptor at physiological pH. The hTF-receptor complex is endocytosed; within the endosome, a proton pump results in a drop in the pH to ∼5.6, triggering iron release. This step is not well defined despite intensive research efforts. The entire cycle occurs in 2–3 min (11Dautry-Varsat A. Ciechanover A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2258-2262Google Scholar). Iron binding and release is characterized by a large conformational change. When iron is released, the two domains move away from each other by means of a hinge located in two antiparallel β-strands that lie behind the iron-binding site in each lobe. In the N-lobe of transferrin, one domain rotates 63° relative to the other about this hinge (12MacGillivray R.T.A. Moore S.A. Chen J. Anderson B.F. Baker H. Luo Y.G. Bewley M. Smith C.A. Murphy M.E. Wang Y. Mason A.B. Woodworth R.C. Brayer G.D. Baker E.N. Biochemistry. 1998; 37: 7919-7928Google Scholar, 13Jeffrey P.D. Bewley M.C. MacGillivray R.T.A. Mason A.B. Woodworth R.C. Baker E.N. Biochemistry. 1998; 37: 13978-13986Google Scholar). The precise mechanism for this movement remains unclear, but protonation of several key amino acid residues appears to be critical to the release of iron (see below). Many studies have clearly demonstrated that an arginine located near the iron-binding site (Arg-124 in hTF/2N) stabilizes the synergistic carbonate anion. This arginine is highly conserved in each lobe of all mammalian TFs. As detailed previously (4Faber H.