FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2090296940> ?p ?o ?g. }

• W2090296940 endingPage "24944" @default.
• W2090296940 startingPage "24934" @default.
• W2090296940 abstract "Serum transferrin reversibly binds iron in each of two lobes and delivers it to cells by a receptor-mediated, pH-dependent process. The binding and release of iron result in a large conformational change in which two subdomains in each lobe close or open with a rigid twisting motion around a hinge. We report the structure of human serum transferrin (hTF) lacking iron (apo-hTF), which was independently determined by two methods: 1) the crystal structure of recombinant non-glycosylated apo-hTF was solved at 2.7-Å resolution using a multiple wavelength anomalous dispersion phasing strategy, by substituting the nine methionines in hTF with selenomethionine and 2) the structure of glycosylated apo-hTF (isolated from serum) was determined to a resolution of 2.7Å by molecular replacement using the human apo-N-lobe and the rabbit holo-C1-subdomain as search models. These two crystal structures are essentially identical. They represent the first published model for full-length human transferrin and reveal that, in contrast to family members (human lactoferrin and hen ovotransferrin), both lobes are almost equally open: 59.4° and 49.5° rotations are required to open the N- and C-lobes, respectively (compared with closed pig TF). Availability of this structure is critical to a complete understanding of the metal binding properties of each lobe of hTF; the apo-hTF structure suggests that differences in the hinge regions of the N- and C-lobes may influence the rates of iron binding and release. In addition, we evaluate potential interactions between apo-hTF and the human transferrin receptor. Serum transferrin reversibly binds iron in each of two lobes and delivers it to cells by a receptor-mediated, pH-dependent process. The binding and release of iron result in a large conformational change in which two subdomains in each lobe close or open with a rigid twisting motion around a hinge. We report the structure of human serum transferrin (hTF) lacking iron (apo-hTF), which was independently determined by two methods: 1) the crystal structure of recombinant non-glycosylated apo-hTF was solved at 2.7-Å resolution using a multiple wavelength anomalous dispersion phasing strategy, by substituting the nine methionines in hTF with selenomethionine and 2) the structure of glycosylated apo-hTF (isolated from serum) was determined to a resolution of 2.7Å by molecular replacement using the human apo-N-lobe and the rabbit holo-C1-subdomain as search models. These two crystal structures are essentially identical. They represent the first published model for full-length human transferrin and reveal that, in contrast to family members (human lactoferrin and hen ovotransferrin), both lobes are almost equally open: 59.4° and 49.5° rotations are required to open the N- and C-lobes, respectively (compared with closed pig TF). Availability of this structure is critical to a complete understanding of the metal binding properties of each lobe of hTF; the apo-hTF structure suggests that differences in the hinge regions of the N- and C-lobes may influence the rates of iron binding and release. In addition, we evaluate potential interactions between apo-hTF and the human transferrin receptor. The transferrins are a family of bilobal iron-binding proteins that play the crucial role of binding ferric iron and keeping it in solution, thereby controlling the levels of this important metal in the body (1Harris D.C. Aisen P. Loehr T.M. Iron Carriers and Iron Proteins. VCH Publishers, Inc., New York1989: 241-351Google Scholar, 2Aisen P. Leibman A. Zweier J. J. Biol. Chem. 1978; 253: 1930-1937Abstract Full Text PDF PubMed Google Scholar). Human serum transferrin (hTF) 4The abbreviations used are: hTF, human serum transferrin; hTF-NG, recombinant non-glycosylated human serum transferrin; hTF-Gly, commercially available glycosylated human serum transferrin; TF, transferrin; apo-TF, transferrin lacking iron; oTF, ovotransferrin; LTF, lactoferrin; TFR, transferrin receptor; cryo-EM, cryo-electron microscopy; SeMet, selenomethionine; NCS, non-crystallographic symmetry; r.m.s., root mean square. 4The abbreviations used are: hTF, human serum transferrin; hTF-NG, recombinant non-glycosylated human serum transferrin; hTF-Gly, commercially available glycosylated human serum transferrin; TF, transferrin; apo-TF, transferrin lacking iron; oTF, ovotransferrin; LTF, lactoferrin; TFR, transferrin receptor; cryo-EM, cryo-electron microscopy; SeMet, selenomethionine; NCS, non-crystallographic symmetry; r.m.s., root mean square.is synthesized in the liver and secreted into the plasma; it acquires Fe(III) from the gut and delivers it to iron requiring cells by binding to specific transferrin receptors (TFR) on their surface. The entire hTF ·TFR complex is taken up by receptor-mediated endocytosis culminating in iron release within the endosome (3Klausner R.D. Ashwell G. van Renswoude J. Harford J.B. Bridges K.R. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2263-2266Crossref PubMed Scopus (472) Google Scholar). Essential to the re-utilization of hTF, iron-free hTF (apo-hTF) remains bound to the TFR at low pH. When the apo-hTF ·TFR complex is returned to the cell surface, apo-hTF is released to acquire more iron. Strong homologies exist, both between TF family members, and between the two lobes of any given TF (4Baker H.M. Anderson B.F. Baker E.N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3579-3583Crossref PubMed Scopus (208) Google Scholar, 5Lambert L.A. Perri H. Meehan T.J. Comp. Biochem Physiol. B. 2005; 140: 11-25Crossref PubMed Scopus (103) Google Scholar). Each N- and C-lobe is divided into two subdomains (designated N1 and N2, and C1 and C2) connected by a hinge that gives rise to a deep cleft containing the iron-binding ligands. Iron is coordinated by four highly conserved amino acid residues: an aspartic acid (the sole ligand from the N1- or C1-subdomain), a tyrosine in the hinge at the edge of the N2- or C2-subdomain, a second tyrosine within the N2- or C2-subdomain, and a histidine at the hinge bordering the N1- or C1-subdomain. In addition, the iron atom is bound by two oxygen atoms from the synergistic anion (carbonate), which is itself stabilized by a conserved arginine residue (6Lambert L.A. Perri H. Halbrooks P.J. Mason A.B. Comp. Biochem. Physiol. B. 2005; 142: 129-141Crossref PubMed Scopus (147) Google Scholar). A key feature of iron binding and release by TF family members is the large conformational change involving not only opening of the two subdomains in each lobe but also a twist between the N1- and N2-, or C1- and C2-subdomains (7Kurokawa H. Mikami B. Hirose M. J. Mol. Biol. 1995; 254: 196-207Crossref PubMed Scopus (198) Google Scholar, 8Kurokawa H. Dewan J.C. Mikami B. Sacchettini J.C. Hirose M. J. Biol. Chem. 1999; 274: 28445-28452Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Although the N- and C-lobes of hTF share 56% sequence similarity, many studies show that the rate of iron release from the C-lobe is considerably slower than the rate of release from the N-lobe, particularly at the putative endosomal pH of ∼5.6 (9Princiotto J.V. Zapolski E.J. Nature. 1975; 255: 87-88Crossref PubMed Scopus (101) Google Scholar, 10Baldwin D.A. De Sousa D.M.R. Von Wandruszka R.M.A. Biochim. Biophys. Acta. 1982; 719: 140-146Crossref PubMed Scopus (51) Google Scholar, 11Baker E.N. Baker H.M. Kidd R.D. Biochem. Cell Biol. 2002; 80: 27-34Crossref PubMed Scopus (158) Google Scholar, 12Lestas A.N. Br. J. Haematol. 1976; 32: 341-350Crossref PubMed Scopus (98) Google Scholar, 13Mason A.B. He Q.Y. Halbrooks P.J. Everse S.J. Gumerov D.R. Kaltashov I.A. Smith V.C. Hewitt J. MacGillivray R.T.A. Biochemistry. 2002; 41: 9448-9454Crossref PubMed Scopus (48) Google Scholar, 14Halbrooks P.J. He Q.Y. Briggs S.K. Everse S.J. Smith V.C. MacGillivray R.T.A. Mason A.B. Biochemistry. 2003; 42: 3701-3707Crossref PubMed Scopus (55) Google Scholar, 15Mason A.B. Halbrooks P.J. James N.G. Connolly S.A. Larouche J.R. Smith V.C. MacGillivray R.T.A. Chasteen N.D. Biochemistry. 2005; 44: 8013-8021Crossref PubMed Scopus (46) Google Scholar). At least some of the difference is attributed to the presence of a “dilysine trigger” in the N-lobe, which is replaced by a triad of residues in the C-lobe (14Halbrooks P.J. He Q.Y. Briggs S.K. Everse S.J. Smith V.C. MacGillivray R.T.A. Mason A.B. Biochemistry. 2003; 42: 3701-3707Crossref PubMed Scopus (55) Google Scholar). The dilysine trigger is composed of Lys206 in the N2-subdomain and Lys296 in the N1-subdomain, which reside on opposite sides of the iron binding cleft and are oriented with side chains extending toward one another allowing them to share a hydrogen bond in the iron-bound (closed) conformation (16Dewan J.C. Mikami B. Hirose M. Sacchettini J.C. Biochemistry. 1993; 32: 11963-11968Crossref PubMed Scopus (167) Google Scholar, 17He Q.-Y. Mason A.B. Tam B.M. MacGillivray R.T.A. Woodworth R.C. Biochemistry. 1999; 38: 9704-9711Crossref PubMed Scopus (82) Google Scholar). In the C-lobe, a triad of residues (Lys534 in the C2-subdomain, Arg632 and Asp634 in the C1-subdomain) replaces the lysine pair (14Halbrooks P.J. He Q.Y. Briggs S.K. Everse S.J. Smith V.C. MacGillivray R.T.A. Mason A.B. Biochemistry. 2003; 42: 3701-3707Crossref PubMed Scopus (55) Google Scholar, 18Halbrooks P.J. Giannetti A.M. Klein J.S. Bjorkman P.J. Larouche J.R. Smith V.C. MacGillivray R.T.A. Everse S.J. Mason A.B. Biochemistry. 2005; 44: 15451-15460Crossref PubMed Scopus (29) Google Scholar). The release of iron from hTF depends upon a number of factors, including pH, a chelator (physiologically relevant chelators include citrate, pyrophosphate, and ATP), and ionic strength, as well as the specific TFR (19He Q.-Y. Mason A.B. Templeton D.M. Molecular and Cellular Iron Transport. Marcel Dekker, Inc., New York2002: 95-123Google Scholar, 20Baker E.N. Adv. Inorg. Chem. 1994; 41: 389-463Crossref Scopus (213) Google Scholar, 21Zak O. Tam B. MacGillivray R.T.A. Aisen P. Biochemistry. 1997; 36: 11036-11043Crossref PubMed Scopus (54) Google Scholar). Raymond et al.(22Hamilton D.H. Turcot I. Stintzi A. Raymond K.N. J. Biol. Inorg. Chem. 2004; 9: 936-944Crossref PubMed Scopus (37) Google Scholar) suggest that a complete model must explain the differences in the rate of iron release from the two lobes, the observed variable chelator concentration dependence, and the effect of anions, as well as the presence or absence of cooperativity between the lobes. A 7.5-Å cryoelectron microscopy (cryo-EM) model of diferric hTF bound to TFR was created by docking a human TF N-lobe structure and a rabbit TF C-lobe structure into the electron density map of the complex (because there is no full-length human TF structure available) (23Cheng Y. Zak O. Aisen P. Harrison S.C. Walz T. Cell. 2004; 116: 565-576Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). This model offers a preliminary view of the regions of hTF and TFR that interact; it is suggested that both the N1- and N2-subdomains of the hTF N-lobe contact the TFR, whereas only the C1-subdomain of the hTF C-lobe appears to be involved in the interaction. Interestingly, a translation of ∼9 Å of the ferric N-lobe (relative to the ferric C-lobe) is required to dock the two lobes into the cryo-EM density. Of relevance, at pH 7.4, the TFR discriminates between diferric, the two monoferric species, and apo-hTF, although the basis of this discrimination has not been explained (24Young S.P. Bomford A. Williams R. Biochem. J. 1984; 219: 505-510Crossref PubMed Scopus (137) Google Scholar, 25Mason A.B. He Q.-Y. Tam B.M. MacGillivray R.T.A. Woodworth R.C. Biochem. J. 1998; 330: 35-40Crossref PubMed Scopus (27) Google Scholar, 26Evans R.W. Crawley J.B. Garratt R.C. Grossmann J.G. Neu M. Aitken A. Patel K.J. Meilak A. Wong C. Singh J. Bomford A. Hasnain S.S. Biochemistry. 1994; 33: 12512-12520Crossref PubMed Scopus (25) Google Scholar). Significantly, our studies with authentic monoferric hTF constructs established that each lobe contributes equally (and non-additively) to the binding energy of this interaction with the TFR (15Mason A.B. Halbrooks P.J. James N.G. Connolly S.A. Larouche J.R. Smith V.C. MacGillivray R.T.A. Chasteen N.D. Biochemistry. 2005; 44: 8013-8021Crossref PubMed Scopus (46) Google Scholar). Clearly a structure of apo-hTF is required to determine whether a change in orientation of the two lobes could provide both a rationale for discrimination and further insight into the receptor interaction. Here we report the structure of full-length apo-hTF that has been independently determined by two methods; both a nonglycosylated recombinant form of hTF (pH 6.5) and a glycosylated native form of hTF (pH 7.0) were solved to a resolution of 2.7 Å. These two structures, which are identical within the limits of the resolution, find both the N- and C-lobes in the open conformation. This work represents the first mammalian TF structure with an apo-C-lobe, the first published structure of full-length hTF, and the first report of a baby hamster kidney expression system to substitute the methionine residues in hTF with selenomethionine (SeMet). The apo-hTF structure allows comparisons to other relevant structures, including those for diferric pig (2.15 Å, 72% identical) and rabbit TF (2.6 Å, 79% identical) (27Hall D.R. Hadden J.M. Leonard G.A. Bailey S. Neu M. Winn M. Lindley P.F. Acta Crystallogr. D. Biol. Crystallogr. 2002; 58: 70-80Crossref PubMed Scopus (94) Google Scholar), and an unpublished model for an unrefined monoferric hTF with iron in the C-lobe (3.3 Å) (28Zuccola H.J. The Crystal Structure of Monoferric Human Serum Transferrin. Georgia Institute of Technology, Atlanta, GA1993Google Scholar). Production of hTF-NG—To produce recombinant non-glycosylated hTF (hTF-NG) with SeMet substituted for methionine, baby hamster kidney cells transfected with the pNUT plasmid containing the sequence of the N-His-tagged hTF-NG were placed into four expanded surface roller bottles (13Mason A.B. He Q.Y. Halbrooks P.J. Everse S.J. Gumerov D.R. Kaltashov I.A. Smith V.C. Hewitt J. MacGillivray R.T.A. Biochemistry. 2002; 41: 9448-9454Crossref PubMed Scopus (48) Google Scholar). Addition of culture media containing SeMet results in a significant deterioration of the cells within 24-48 h. To maximize the incorporation, medium containing SeMet was added when production of hTF was at a maximum as determined by a competitive immunoassay (29Mason A.B. He Q.-Y. Adams T.E. Gumerov D.R. Kaltashov I.A. Nguyen V. MacGillivray R.T.A. Protein Exp. Purif. 2001; 23: 142-150Crossref PubMed Scopus (33) Google Scholar). Briefly, adherent baby hamster kidney cells were grown in Dulbecco's modified Eagle's medium/F-12 containing 10% fetal bovine serum. This medium was changed twice at 2-day intervals, followed by addition of Dulbecco's modified Eagle's medium/F-12 containing 1% Ultroser G and 1 mm butyric acid. After one or two changes in this medium, 200 ml of SeMet containing Dulbecco's modified Eagle's medium/F-12 (lacking normal methionine), with butyric acid and Ultroser G was added to each roller bottle. Following a 4-h wash-in period this media was discarded and replaced with 250 ml of the same medium for an additional 48 h of incubation. The recombinant hTF-NG was purified from the medium as described in detail (30Mason A.B. Halbrooks P.J. Larouche J.R. Briggs S.K. Moffett M.L. Ramsey J.E. Connolly S.A. Smith V.C. MacGillivray R.T.A. Protein Expr. Purif. 2004; 36: 318-326Crossref PubMed Scopus (40) Google Scholar). In two production runs between 8 and 16 mg of SeMet containing N-His hTF-NG was produced, of which approximately half was recovered. Electrospray mass spectrometry analysis indicated a mass consistent with incorporation of 8-9 SeMet residues (data not shown). Purification of Apo-hTF-Gly—In independent experiments, lyophilized human serum TF lacking iron (apo-hTF-Gly) was obtained from Sigma-Aldrich and reconstituted in 50 mm Tris-HCl, pH 8.0, and 20 mm sodium carbonate at a protein concentration of 5 mg/ml. The protein was applied to a 10-ml Q-Sepharose High Performance column (GE HealthCare) equilibrated with 50 mm Tris-HCl, pH 8.0, and 20 mm sodium carbonate. The apo-hTF was eluted using a linear gradient from 0 to 150 mm NaCl over 3 column volumes. Peak fractions were pooled and dialyzed overnight into 20 mm Tris-HCl, pH 8.0, 20 mm sodium carbonate, and 200 mm sodium chloride. Crystallization—Recombinant diferric hTF-NG (with or without a His tag) at a concentration of 15 mg/ml in 0.1 m ammonium bicarbonate, was mixed with an equal volume of reservoir solution composed of 0.3 m ammonium citrate (pH 6.5) and 16-18% PEG 3350 at 20 °C. The SeMet N-His hTF-NG required slightly lower levels of PEG 3350 and streak seeding using the non-SeMet labeled hTF-NG crystals. Clear crystals (0.2 mm × 0.3 mm × 0.4 mm) formed in 3-5 weeks. Crystals of the SeMet-labeled protein were essentially isomorphous with those of the native protein, showing similar cell dimensions and crystallizing in the orthorhombic space group P212121 with two molecules in the asymmetric unit (Table 1). All crystals were cryoprotected by addition to the hanging drop of 0.5 μl of a solution of 25% PEG 3350/30% ethylene glycol.TABLE 1Data collection, crystallographic refinement, and model statisticsProteinNative 1 hTF-GlyNative 2 hTF-NGInflection hTF-NGPeak hTF-NGRemote hTF-NGCell parametersSpace groupP212121P212121P212121P212121P212121a, b, c (Å)88.3288.9988.4988.2888.47103.26102.16103.35103.39103.55200.36197.04199.17198.94198.38Data collection statisticsLocationAPSChess A1BNLBNLBNL22IDBNL X26X25X26CX26CWavelength (Å)1.00000.935, 1.1000.97940.97900.9641Resolution range (Å)30-2.750-2.750-2.925-3.250-3.3Unique reflections49,87147,96837,49831,12428,343Completeness (%)96.7 (90.5)95.9 (83.6)99.9 (98.9)99.4 (93.8)100 (99.9)Redundancy6.6 (6.5)6.0 (4.5)6.0 (5.3)7.1 (6.5)7.3 (7.5)Rmerge (%)aRmerge = ΣΣI(h)j - <I(h)>/ΣI(h), where I(h)j is the jth measurement of diffraction intensity of reflection h, and <I(h)> is the average intensity of reflection h for all j measurements.6.87.18.211.612.7I/σ22.8 (2.0)41.0 (5.3)12.6 (0.9)23.6 (3.4)22.8 (4.0)DetectorMar300Q210 Q4Q315Q4Q4Integration softwareHKL2000HKL v1.98.2HKL2000HKL2000HKL2000Molecular replacementSearch models1A8E (N-lobe)1H76 (C-lobe)MAD phasesFigure of merit (FOM)100-2.9 ÅAcentrics0.41Centrics0.38Density-mod. mean FOM0.80Model statisticsResolution (Å)15-2.715-2.7RworkbRwork = Σ(|Fo| - |Fc|)/Σ|Fo|.23.227.3RfreecRfree is calculated using a test set of 5% of the reflection excluded from refinement.29.332.3Refined modelNo. amino acid residues1,3521,352No. citrate molecules74No. glycerol molecules42No. atoms, non-hydrogenProtein10,48610,488Ligand11564r.m.s. deviation, bonds (Å)0.0090.009r.m.s. deviation, angles (°)1.571.33Ramachandran plot (%)Most favored regions77.078.2Additionally allowed20.619.4Generously allowed1.91.9Disallowed regions0.50.5Average B factor (Å2)75.267.4Range of B factors (Å2)32-12514-135a Rmerge = ΣΣI(h)j - <I(h)>/ΣI(h), where I(h)j is the jth measurement of diffraction intensity of reflection h, and <I(h)> is the average intensity of reflection h for all j measurements.b Rwork = Σ(|Fo| - |Fc|)/Σ|Fo|.c Rfree is calculated using a test set of 5% of the reflection excluded from refinement. Open table in a new tab Native apo-hTF-Gly was concentrated in a Centriprep 30 concentrator (Millipore) to 30 mg/ml and screened against commercially available 96-condition screens using a Mosquito crystallization robot (TTP Labtech) with a hanging drop format (drop size, 200 nl of protein plus 200-nl well solution). Conditions yielding the best crystals were then further refined using 24-well VDX plates (Hampton Research). The crystals used for native data collection were obtained from hanging drops with a well solution of 0.2 m ammonium citrate, pH 7.0, 20% PEG 3350, and 15% glycerol, incubated at 21 °C. Drop sizes varied from 2to 16 μl and consisted of equal parts of protein and well solution. Crystals grew in ∼24 h and were flash frozen in propane cooled to -170 °C. Data Collection and Refinement—For the apo-hTF-NG crystals that contained SeMet, multiple wavelength anomalous dispersion data were collected at -170 °C on beamlines X26C (peak and remote) and X25 (inflection) at Brookhaven National Laboratory. The data were reduced, scaled, and merged using DENZO/SCALEPACK (31Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38436) Google Scholar) (Table 1). To find the selenium sites, multiple wavelength anomalous dispersion data were prepared with XPREP and analyzed with ShelxD (32Schneider T.R. Sheldrick G.M. Acta Crystallogr. D. Biol. Crystallogr. 2002; 58: 1772-1779Crossref PubMed Scopus (1575) Google Scholar). The data sets were combined, and refinement of the selenium sites was carried out using autoSHARP (33La Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1797) Google Scholar, 34Vonrhein C. Blanc E. Roversi P. Bricogne G. Doublie S. Crystallographic Methods. Humana Press, Totowa, NJ2006: 215-230Google Scholar). Profess (35Collaborative Computational Project, No. 4.Acta Crystallogr. D. Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19728) Google Scholar) was used to find NCS, which revealed two molecules in the asymmetric unit. Following a round of density modification, the structure of apo-hTF N-lobe (Protein Data Bank (PDB) 1BP5) was used as a search model for a phased translation and rotation function using MOLREP (36Vagin A.A. Isupov M.N. Acta. Crystallogr. D. Biol. Crystallogr. 2001; 57: 1451-1456Crossref PubMed Scopus (186) Google Scholar). Subsequent model building of the C-lobe was done using O (37Jones T.A. J. Appl. Crystallogr. 1978; 11: 268-272Crossref Google Scholar) in a stepwise manner by incorporating fragments of pig holo-TF (converted to the human sequence). Iterative rounds of density modification and phase recombination were performed with SOLOMON (38Abrahams J.P. Leslie A.G. Acta Crystallogr. D. Biol. Crystallogr. 1996; 52: 30-42Crossref PubMed Scopus (1141) Google Scholar). Refinement was accomplished using both CNS (39Brunger 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. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16946) Google Scholar) and REFMAC (35Collaborative Computational Project, No. 4.Acta Crystallogr. D. Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19728) Google Scholar, 40Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. D. Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1650) Google Scholar). Native diffraction data from an apo-hTF-Gly crystal were collected at the Advanced Photon Source on SER-CAT beamline 22ID at -170 °C. The crystal belonged to the orthorhombic space group P2B1B2B1B2B1B, with two molecules in the asymmetric unit and a solvent content of 59.3%. The images were reduced, scaled, and merged using HKL2000 (31Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38436) Google Scholar) (Table 1), and the structure was solved using the molecular replacement program Phaser (41McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. D. Biol. Crystallogr. 2005; 61: 458-464Crossref PubMed Scopus (1596) Google Scholar). A search model consisting of the human apo-N-lobe structure (1BP5) and the rabbit holo-C1-subdomain (residues 342-424 and 579-676 from 1JNF) was used, leading to a single solution containing two copies of each component. After a single round of rigid body refinement using the program REFMAC (35Collaborative Computational Project, No. 4.Acta Crystallogr. D. Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19728) Google Scholar, 40Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. D. Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1650) Google Scholar) the rabbit C2-subdomain was fit into the electron density. The C1- and C2-subdomains were then mutated to the human sequence, and the linker regions between the N- and C-lobes and between the C1- and C2-subdomains were built into the electron density. This structure was further refined with REFMAC using medium NCS restraints initially, followed by release of NCS restraints. After building in the glycerol and citrate molecules, final rounds of refinement were performed in CNS (39Brunger 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. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16946) Google Scholar). Comparison of (Recombinant) Apo-hTF-NG and (Native) Apo-hTF-Gly—Both protein preparations crystallized in the orthorhombic space group P212121, having almost identical unit cell dimensions and two molecules per asymmetric unit (Table 1). The recombinant and native structures had an r.m.s. deviation of only 0.73 Å for 1352 equivalent CPαP positions, and therefore we are, for the most part, presenting and discussing them as a single structure (apo-hTF). As in all other serum TF structures, no electron density for residues 1-3 is visible. Even the His-tagged recombinant apo-hTF-NG, with 14 extra amino acids at the N terminus, showed no density in this region, implying that the amino terminus is very flexible. Additionally, no density is found in the vicinity of Asn413 and Asn611 in the native apo-hTF-Gly, indicating that the glycan moieties are flexible and/or present in multiple conformations. Clearly our recombinant hTF lacks carbohydrate due to the mutation of the asparagine linkage sites to aspartic acid residues. Significantly, the presence or absence of carbohydrate has no effect on either iron release or interaction with the TFR (42Mason A.B. Miller M.K. Funk W.D. Banfield D.K. Savage K.J. Oliver R.W.A. Green B.N. MacGillivray R.T.A. Woodworth R.C. Biochemistry. 1993; 32: 5472-5479Crossref PubMed Scopus (102) Google Scholar, 43Gumerov D.R. Mason A.B. Kaltashov I.A. Biochemistry. 2003; 42: 5421-5428Crossref PubMed Scopus (40) Google Scholar). Quality of Final Model—Data collection, refinement, and model statistics are summarized in Table 1. No breaks in the main-chain density were observed in apo-hTF and, as shown in Table 1, the geometry is good. The Ramachandran plot of the mainchain torsion angles shows that 97.6% of the residues lie in most favored or allowed regions and that only 6 of the 1352 residues of the two non-crystallographic symmetry-related molecules reside in the disallowed region. Four of these residues are Leu294 and Leu630 in each of the molecules, comprising the central residues in classic γ-turns (Leu-Leu-Phe) with phi and psi angles of ∼77° and -46°, respectively (44Baker E.N. Hubbard R.E. Prog. Biophys. Mol. Biol. 1984; 44: 97-179Crossref PubMed Scopus (1640) Google Scholar). This structural feature was first noted in the hTF N-lobe (45MacGillivray 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: 7919-7928Crossref PubMed Scopus (226) Google Scholar) and subsequently has been observed in each lobe of all mammalian and avian TF molecules and in the C-lobe of all fish TFs (5Lambert L.A. Perri H. Meehan T.J. Comp. Biochem Physiol. B. 2005; 140: 11-25Crossref PubMed Scopus (103) Google Scholar, 6Lambert L.A. Perri H. Halbrooks P.J. Mason A.B. Comp. Biochem. Physiol. B. 2005; 142: 129-141Crossref PubMed Scopus (147) Google Scholar). Interestingly, the Leu-Leu-Phe sequence is not conserved in any insect TF N-lobe and is only partially conserved in the insect C-lobe sequences (5Lambert L.A. Perri H. Meehan T.J. Comp. Biochem Physiol. B. 2005; 140: 11-25Crossref PubMed Scopus (103) Google Scholar, 6Lambert L.A. Perri H. Halbrooks P.J. Mason A.B. Comp. Biochem. Physiol. B. 2005; 142: 129-141Crossref PubMed Scopus (147) Google Scholar). However, because the function of insect TF remains unknown, the importance of this finding is unclear. Although the role of the γ-turn in mammalian TFs has not been definitively established, we believe that it is significant that it immediately precedes the dilysine trigger residue Lys296 in the N-lobe and triad residue Arg632 in the C-lobe (45MacGillivray 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: 7919-7928Crossref PubMed Scopus (226) Google Scholar). Within this context, the γ-turn may help to stabilize the orientation of these two residues to provide better repulsion or a triggering action, aiding in opening of the cleft. Overall Organization—As with all TF family members, the structure of hTF illustrates the bilobal nature of the molecule (Fig. 1) with an amino-terminal lobe (N-lobe, residues 1-331) and a carboxyl-terminal lobe (C-lobe, residues 339-679). The lobes are connected by a linker peptide (residues 332-338) that is unstructured, but visible in our model (although missing in the diferric pig TF structure (27Hall D.R. Hadden J.M. Leonard G.A. Bailey S. Neu M. Winn M. Lindley P.F. Acta Crystallogr. D. Biol. Crystallogr. 2002; 58: 70-80Crossref PubMed Scopus (94) Google Scholar)). Each of the lobes is further separated into two subdomains: the N1-(1-92 and 247-331) and C1-(339-425 and 573-679) subdomains are each composed of two discontinuous sections of the polypeptide chain, whereas the N2-(93-246) and C2-(426-572) subdomains are each composed of a single region of continuous polypeptide (Fig. 1 and Supplemental Fig. S1). Though kinetically distinct, the fold" @default.
• W2090296940 created "2016-06-24" @default.
• W2090296940 creator A5004512319 @default.
• W2090296940 creator A5013783603 @default.
• W2090296940 creator A5017946236 @default.
• W2090296940 creator A5036533962 @default.
• W2090296940 creator A5056931515 @default.
• W2090296940 creator A5057559845 @default.
• W2090296940 creator A5066067174 @default.
• W2090296940 date "2006-08-01" @default.
• W2090296940 modified "2023-10-12" @default.
• W2090296940 title "The Crystal Structure of Iron-free Human Serum Transferrin Provides Insight into Inter-lobe Communication and Receptor Binding" @default.
• W2090296940 cites W119403069 @default.
• W2090296940 cites W1442483700 @default.
• W2090296940 cites W1518065048 @default.
• W2090296940 cites W1535633114 @default.
• W2090296940 cites W1539796472 @default.
• W2090296940 cites W1551307287 @default.
• W2090296940 cites W1587187427 @default.
• W2090296940 cites W1591680814 @default.
• W2090296940 cites W1603905705 @default.
• W2090296940 cites W1872922503 @default.
• W2090296940 cites W1967684793 @default.
• W2090296940 cites W1969222787 @default.
• W2090296940 cites W1971251199 @default.
• W2090296940 cites W1972584945 @default.
• W2090296940 cites W1972697551 @default.
• W2090296940 cites W1973551683 @default.
• W2090296940 cites W1974021683 @default.
• W2090296940 cites W1974704062 @default.
• W2090296940 cites W1975380286 @default.
• W2090296940 cites W1976352074 @default.
• W2090296940 cites W1980368334 @default.
• W2090296940 cites W1982831087 @default.
• W2090296940 cites W1983732912 @default.
• W2090296940 cites W1983891046 @default.
• W2090296940 cites W1986191025 @default.
• W2090296940 cites W1987535606 @default.
• W2090296940 cites W1988811065 @default.
• W2090296940 cites W1991010641 @default.
• W2090296940 cites W1991967206 @default.
• W2090296940 cites W1994286350 @default.
• W2090296940 cites W1995017064 @default.
• W2090296940 cites W2001263732 @default.
• W2090296940 cites W2001641653 @default.
• W2090296940 cites W2001776577 @default.
• W2090296940 cites W2004067151 @default.
• W2090296940 cites W2004417760 @default.
• W2090296940 cites W2006756004 @default.
• W2090296940 cites W2012014050 @default.
• W2090296940 cites W2015642465 @default.
• W2090296940 cites W2016900056 @default.
• W2090296940 cites W2019296008 @default.
• W2090296940 cites W2020412283 @default.
• W2090296940 cites W2022683706 @default.
• W2090296940 cites W2025812024 @default.
• W2090296940 cites W2026455613 @default.
• W2090296940 cites W2029683748 @default.
• W2090296940 cites W2031856351 @default.
• W2090296940 cites W2032703413 @default.
• W2090296940 cites W2033249426 @default.
• W2090296940 cites W2034959038 @default.
• W2090296940 cites W2043031923 @default.
• W2090296940 cites W2044769662 @default.
• W2090296940 cites W2046456805 @default.
• W2090296940 cites W2046818843 @default.
• W2090296940 cites W2048285411 @default.
• W2090296940 cites W2054040797 @default.
• W2090296940 cites W2056725846 @default.
• W2090296940 cites W2058310290 @default.
• W2090296940 cites W2062090518 @default.
• W2090296940 cites W2066548059 @default.
• W2090296940 cites W2067435689 @default.
• W2090296940 cites W2074840065 @default.
• W2090296940 cites W2075491845 @default.
• W2090296940 cites W2076199852 @default.
• W2090296940 cites W2084429843 @default.
• W2090296940 cites W2087517456 @default.
• W2090296940 cites W2094737738 @default.
• W2090296940 cites W2101446086 @default.
• W2090296940 cites W2108156627 @default.
• W2090296940 cites W2112200653 @default.
• W2090296940 cites W2113952988 @default.
• W2090296940 cites W2127742502 @default.
• W2090296940 cites W2128420125 @default.
• W2090296940 cites W2130463690 @default.
• W2090296940 cites W2131184459 @default.
• W2090296940 cites W2141966916 @default.
• W2090296940 cites W2148777613 @default.
• W2090296940 cites W2153994323 @default.
• W2090296940 cites W2158630348 @default.
• W2090296940 cites W2162868807 @default.
• W2090296940 cites W2164466007 @default.
• W2090296940 cites W2336969242 @default.
• W2090296940 cites W2344346124 @default.
• W2090296940 cites W2413184859 @default.
• W2090296940 doi "https://doi.org/10.1074/jbc.m604592200" @default.
• W2090296940 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1895924" @default.

• next