Alternative Structural State of Transferrin

Abstract

Transferrins bind Fe3+ very tightly in a closed interdomain cleft by the coordination of four protein ligands (Asp60, Tyr92, Tyr191, and His250 in ovotransferrin N-lobe) and of a synergistic anion, physiologically bidentate CO32−. Upon Fe3+uptake, transferrins undergo a large scale conformational transition: the apo structure with an opening of the interdomain cleft is transformed into the closed holo structure, implying initial Fe3+ binding in the open form. To solve the Fe3+-loaded, domain-opened structure, an ovotransferrin N-lobe crystal that had been grown as the apo form was soaked with Fe3+-nitrilotriacetate, and its structure was solved at 2.1 Å resolution. The Fe3+-soaked form showed almost exactly the same overall open structure as the iron-free apo form. The electron density map unequivocally proved the presence of an iron atom with the coordination by the two protein ligands of Tyr92-OH and Tyr191-OH. Other Fe3+ coordination sites are occupied by a nitrilotriacetate anion, which is stabilized through the hydrogen bonds with the peptide NH groups of Ser122, Ala123, and Gly124 and a side chain group of Thr117. There is, however, no clear interaction between the nitrilotriacetate anion and the synergistic anion binding site, Arg121. Transferrins bind Fe3+ very tightly in a closed interdomain cleft by the coordination of four protein ligands (Asp60, Tyr92, Tyr191, and His250 in ovotransferrin N-lobe) and of a synergistic anion, physiologically bidentate CO32−. Upon Fe3+uptake, transferrins undergo a large scale conformational transition: the apo structure with an opening of the interdomain cleft is transformed into the closed holo structure, implying initial Fe3+ binding in the open form. To solve the Fe3+-loaded, domain-opened structure, an ovotransferrin N-lobe crystal that had been grown as the apo form was soaked with Fe3+-nitrilotriacetate, and its structure was solved at 2.1 Å resolution. The Fe3+-soaked form showed almost exactly the same overall open structure as the iron-free apo form. The electron density map unequivocally proved the presence of an iron atom with the coordination by the two protein ligands of Tyr92-OH and Tyr191-OH. Other Fe3+ coordination sites are occupied by a nitrilotriacetate anion, which is stabilized through the hydrogen bonds with the peptide NH groups of Ser122, Ala123, and Gly124 and a side chain group of Thr117. There is, however, no clear interaction between the nitrilotriacetate anion and the synergistic anion binding site, Arg121. nitrilotriacetate 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol transferrin Transferrins are a group of iron-binding proteins which includes serum transferrin, lactoferrin, and ovotransferrin (1Aisen P. Listowsky I. Annu. Rev. Biochem. 1980; 49: 357-393Crossref PubMed Scopus (938) Google Scholar). The proteins serve to control the iron level in the body fluid of vertebrates by their ability to bind very tightly two Fe3+ ions (1Aisen P. Listowsky I. Annu. Rev. Biochem. 1980; 49: 357-393Crossref PubMed Scopus (938) Google Scholar). They are ∼80-kDa single-chain proteins and consist of two similarly sized homologous N- and C-lobes, which are further divided into two similarly sized domains (domains N1 and N2 in the N-lobe and domains C1 and C2 in the C-lobe). The two iron binding sites are located within the interdomain cleft of each lobe. Crystal structures of the diferric forms (2Bailey S. Evans R.W. Garratt R.C. Gorinsky B. Hasnain S. Horsburgh C. Jhoti H. Lindley P.F. Mydin A. Sarra R. Watson J.L. Biochemistry. 1988; 27: 5804-5812Crossref PubMed Scopus (369) Google Scholar, 3Anderson B.F. Baker H.M. Norris G.E. Rice D.W. Baker E.N. J. Mol. Biol. 1989; 209: 711-734Crossref PubMed Scopus (546) Google Scholar, 4Kurokawa H. Mikami B. Hirose M. J. Mol. Biol. 1995; 254: 196-207Crossref PubMed Scopus (200) Google Scholar, 5Rawas A. Muirhead H. Williams J. Acta Crystallogr. Sec. D. 1996; 52: 631-640Crossref PubMed Scopus (55) Google Scholar, 6Moore S.A. Anderson B.F. Groom C.R. Haridas M. Baker E.N. J. Mol. Biol. 1997; 274: 222-236Crossref PubMed Scopus (338) Google Scholar) and the monoferric N-lobes (7Sarra R. Garratt R. Gorinsky B. Jhoti H. Lindley P. Acta Crystallogr. Sec. B. 1990; 46: 763-771Crossref Scopus (93) Google Scholar, 8Dewan J.C. Mikami B. Hirose M. Sacchettini J.C. Biochemistry. 1993; 32: 11963-11968Crossref PubMed Scopus (167) Google Scholar, 9Day C.L. Anderson B.F. Tweedie J.W. Baker E.N. J. Mol. Biol. 1993; 232: 1084-1100Crossref PubMed Scopus (76) Google Scholar, 10MacGillivray R.T.A. Moore S.A. Chen J. Anderson B.F. Baker H. Luo Y. Bewley M. Smith C.A. Murphy M.E.P. Wang Y. Mason A.B. Woodworth R.C. Brayer G.D. Baker E.N. Biochemistry. 1998; 37: 7919-7928Crossref PubMed Scopus (228) Google Scholar) of several transferrins reveal that the two domains are closed over an Fe3+ ion. Four of the six Fe3+ coordination sites are occupied by the protein ligands of two tyrosine residues, one aspartic acid residue, and one histidine residue (Asp60, Tyr92, Tyr191, and His250 in ovotransferrin N-lobe) and the other two, by a synergistic anion, physiologically bidentate CO32− (2Bailey S. Evans R.W. Garratt R.C. Gorinsky B. Hasnain S. Horsburgh C. Jhoti H. Lindley P.F. Mydin A. Sarra R. Watson J.L. Biochemistry. 1988; 27: 5804-5812Crossref PubMed Scopus (369) Google Scholar, 3Anderson B.F. Baker H.M. Norris G.E. Rice D.W. Baker E.N. J. Mol. Biol. 1989; 209: 711-734Crossref PubMed Scopus (546) Google Scholar, 4Kurokawa H. Mikami B. Hirose M. J. Mol. Biol. 1995; 254: 196-207Crossref PubMed Scopus (200) Google Scholar, 5Rawas A. Muirhead H. Williams J. Acta Crystallogr. Sec. D. 1996; 52: 631-640Crossref PubMed Scopus (55) Google Scholar, 6Moore S.A. Anderson B.F. Groom C.R. Haridas M. Baker E.N. J. Mol. Biol. 1997; 274: 222-236Crossref PubMed Scopus (338) Google Scholar, 7Sarra R. Garratt R. Gorinsky B. Jhoti H. Lindley P. Acta Crystallogr. Sec. B. 1990; 46: 763-771Crossref Scopus (93) Google Scholar, 8Dewan J.C. Mikami B. Hirose M. Sacchettini J.C. Biochemistry. 1993; 32: 11963-11968Crossref PubMed Scopus (167) Google Scholar, 9Day C.L. Anderson B.F. Tweedie J.W. Baker E.N. J. Mol. Biol. 1993; 232: 1084-1100Crossref PubMed Scopus (76) Google Scholar, 10MacGillivray R.T.A. Moore S.A. Chen J. Anderson B.F. Baker H. Luo Y. Bewley M. Smith C.A. Murphy M.E.P. Wang Y. Mason A.B. Woodworth R.C. Brayer G.D. Baker E.N. Biochemistry. 1998; 37: 7919-7928Crossref PubMed Scopus (228) Google Scholar). For the iron-free apo form, however, x-ray crystallographic (11Anderson B.F. Baker H.M. Norris G.E. Rumball S.V. Baker E.N. Nature. 1990; 344: 784-787Crossref PubMed Scopus (351) Google Scholar, 12Rawas A. Muirhead H. Williams J. Acta Crystallogr. Sec. D. 1997; 53: 464-468Crossref PubMed Scopus (25) Google Scholar, 13Jeffrey P.D. Bewley M.C. MacGillivray T.A. Mason A.B. Woodworth R.C. Baker E.N. Biochemistry. 1998; 37: 13978-13986Crossref PubMed Scopus (171) Google Scholar) and solution scattering (14Grossmann J.G. Neu M. Pantos E. Schwab F.J. Evans R.W. Townes-Andrews E. Lindley P.F. Appel H. Thies W.-G. Hasnain S.S. J. Mol. Biol. 1992; 225: 811-819Crossref PubMed Scopus (149) Google Scholar, 15Grossmann J.G. Neu M. Evans R.W. Lindley P.F. Appel H. Hasnain S.S. J. Mol. Biol. 1993; 229: 585-590Crossref PubMed Scopus (66) Google Scholar, 16Mecklenburg S.L. Donohoe R.J. Olah G.A. J. Mol. Biol. 1997; 270: 739-750Crossref PubMed Scopus (35) Google Scholar, 17Grossmann J.G. Crawley J.B. Strange R.W. Patel K.J. Murphy L.M. Neu M. Evans R.W. Hasnain S.S. J. Mol. Biol. 1998; 279: 461-472Crossref PubMed Scopus (53) Google Scholar) analyses have revealed that all of the transferrin lobes, except for the lactoferrin C-lobe in crystal, assume a conformation with an opening of the interdomain cleft. This implies that transferrin initially binds the Fe3+ ion in the open form before being transformed into the closed holo form (18Baker E.N. Rumball S.V. Anderson B.F. Trends Biochem. Sci. 1987; 12: 350-353Abstract Full Text PDF Scopus (168) Google Scholar, 19Baker E.N. Lindley P.F. J. Inorg. Biochem. 1992; 47: 147-160Crossref PubMed Scopus (183) Google Scholar). Differential domain and hinge locations of the four protein ligands (Asp60 in the domain 1, Tyr191 in the domain 2, and Tyr92 and His250 in different hinges) (2Bailey S. Evans R.W. Garratt R.C. Gorinsky B. Hasnain S. Horsburgh C. Jhoti H. Lindley P.F. Mydin A. Sarra R. Watson J.L. Biochemistry. 1988; 27: 5804-5812Crossref PubMed Scopus (369) Google Scholar, 3Anderson B.F. Baker H.M. Norris G.E. Rice D.W. Baker E.N. J. Mol. Biol. 1989; 209: 711-734Crossref PubMed Scopus (546) Google Scholar, 4Kurokawa H. Mikami B. Hirose M. J. Mol. Biol. 1995; 254: 196-207Crossref PubMed Scopus (200) Google Scholar, 5Rawas A. Muirhead H. Williams J. Acta Crystallogr. Sec. D. 1996; 52: 631-640Crossref PubMed Scopus (55) Google Scholar, 6Moore S.A. Anderson B.F. Groom C.R. Haridas M. Baker E.N. J. Mol. Biol. 1997; 274: 222-236Crossref PubMed Scopus (338) Google Scholar, 7Sarra R. Garratt R. Gorinsky B. Jhoti H. Lindley P. Acta Crystallogr. Sec. B. 1990; 46: 763-771Crossref Scopus (93) Google Scholar, 8Dewan J.C. Mikami B. Hirose M. Sacchettini J.C. Biochemistry. 1993; 32: 11963-11968Crossref PubMed Scopus (167) Google Scholar, 9Day C.L. Anderson B.F. Tweedie J.W. Baker E.N. J. Mol. Biol. 1993; 232: 1084-1100Crossref PubMed Scopus (76) Google Scholar, 10MacGillivray R.T.A. Moore S.A. Chen J. Anderson B.F. Baker H. Luo Y. Bewley M. Smith C.A. Murphy M.E.P. Wang Y. Mason A.B. Woodworth R.C. Brayer G.D. Baker E.N. Biochemistry. 1998; 37: 7919-7928Crossref PubMed Scopus (228) Google Scholar) inevitably require an alternative Fe3+ coordination structure for the Fe3+-loaded, domain-opened intermediate. Such an alternative structural state has been a central question to be solved for the understanding of the Fe3+ binding pathway in transferrin. A major difficulty encountered in the structural analysis for the intermediate is to prepare a stable protein form that reasonably mimics it. One of the most promising ways may be the site-directed mutagenesis approach for the amino acid residues that are implicated in the Fe3+ coordination. An Fe3+-loaded, domain-opened transferrin form, however, has not been obtained so far by site-directed mutagenesis; either the Asp- or His-ligand mutant of the lactoferrin N-lobe assumes the holo-like closed conformation (20Faber H.R. Bland T. Day C.L. Norris G.E. Tweedie J.W. Baker E.N. J. Mol. Biol. 1996; 256: 352-363Crossref PubMed Scopus (51) Google Scholar,21Nicholson H. Anderson B.F. Bland T. Shewry S.C. Tweedie J.W. Baker E.N. Biochemistry. 1997; 36: 341-346Crossref PubMed Scopus (44) Google Scholar). The mutant lactoferrin N-lobe in which the synergistic anion-binding residue, Arg121, is replaced by the serine or glutamic acid residue also assumes the closed conformation (22Faber H.R. Baker C.J. Day C.L. Tweedie J.W. Baker E.N. Biochemistry. 1996; 35: 14473-14479Crossref PubMed Scopus (29) Google Scholar). In the present study, we employed an alternative strategy using an apo crystal: the Fe3+ soaking conditions in which the colorless crystal turns red without any collapse were searched. As a successful condition, an apo crystal of ovotransferrin N-lobe was soaked with the Fe3+·NTA1complex in the absence of CO32−, and then its structure was solved at a 2.1 Å resolution. We report here a novel structural state of transferrin: the Fe3+-loaded structure of ovotransferrin N-lobe with essentially the same open conformation as the apo form. In this structure, the bound iron atom is coordinated by the two protein ligands of Tyr92-OH and Tyr191-OH. Other Fe3+ coordination sites are occupied by a NTA anion, which is stabilized through the hydrogen bonds with protein groups. The observation strongly suggests that the two tyrosine residues are the initial Fe3+-binding ligands in the open transferrin. The isolated N-lobe (N-terminal half-molecule) of hen ovotransferrin was purified as described (23Oe H. Doi E. Hirose M. J. Biochem. 1988; 103: 1066-1072Crossref PubMed Scopus (51) Google Scholar). The apo form of the protein was crystallized using the hanging drop vapor diffusion method. A solution of a crystallization droplet was prepared on a siliconized coverslip by mixing 5 μl of protein solution (44.4 mg/ml in 0.05 m BisTris-HCl buffer, pH 6.0) with 5 μl of precipitant solution (0.05 m BisTris buffer, pH 6.0, 52% ammonium sulfate). The droplets were equilibrated against 1 ml of the precipitant solution at 20 °C. Hexagonal apo crystals were obtained within 1 month. For Fe3+ soaking, the apo crystals were first transferred to a precipitant solution at higher pH (0.05 m BisTris-HCl buffer, pH 7.5, 52% ammonium sulfate) and then stepwise to raised precipitant concentrations (56, 62, 68, 74, and then 80% ammonium sulfate). The colorless crystals were incubated at 20 °C with 3.0 mm Fe3+·NTA for 4 h, thereby being transformed into red ones. Diffraction data were collected using CuKα radiation (= 1.5418 Å) with a Siemens Hi-Star area detector coupled to a rotating anode generator (Mac Science M18XHF). The crystal of the Fe3+-soaked form was found to belong to the same space group of P6322 (TableI) as that of the apo form. 188,024 reflections were collected to 2.09 Å. The data were processed, merged, and scaled with the SAINT program (Siemens Analytical x-ray Instruments, Inc., Madison, WI).Table ISummary of data collection and refinementCrystal dataSpace groupP6322Cell dimensionsa (Å)125.29b (Å)125.29c (Å)87.53V m(Å3/Da)2.7Molecules/asymmetric unit1Observed reflections188,024Resolution (Å)2.09Independent of reflections of protein of in a As the structure, we employed the apo structure of ovotransferrin N-lobe H. B. and M. in that had been solved at Å by the using the apo the apo structure and the data of the Fe3+-soaked form, were by A for and Scholar). One NTA and one iron which were a clear density map of the first were in the by of and The and of NTA for A for and Scholar) were prepared and of NTA by and Inc., The at 1 and at were obtained using the data of the Fe3+-soaked form at Å of the in which the NTA was An density map at was with a data of the Fe3+-soaked form at Å with of by the program The were the without an iron atom by and with the data by The N-lobe of ovotransferrin amino acid residues (23Oe H. Doi E. Hirose M. J. Biochem. 1988; 103: 1066-1072Crossref PubMed Scopus (51) Google Scholar). however, are not in the no electron density be for In the electron density is no in the chain density at the 1 are in I. The overall and were and for the data For the the was and the and and a the in is to be Å. A C. J. Full Text PDF PubMed Scopus Google Scholar) of the chain is in of the residues are in the with of the residues within the as in the program J. Crystallogr. 1993; Google Scholar). As a residue, the This residue is the central residue in a The of the residue is the one in all of the N- and of serum transferrin (2Bailey S. Evans R.W. Garratt R.C. Gorinsky B. Hasnain S. Horsburgh C. Jhoti H. Lindley P.F. Mydin A. Sarra R. Watson J.L. Biochemistry. 1988; 27: 5804-5812Crossref PubMed Scopus (369) Google ovotransferrin H. Mikami B. Hirose M. J. Mol. Biol. 1995; 254: 196-207Crossref PubMed Scopus (200) Google and lactoferrin S.A. Anderson B.F. Groom C.R. Haridas M. Baker E.N. J. Mol. Biol. 1997; 274: 222-236Crossref PubMed Scopus (338) Google Scholar). the overall structure of ovotransferrin N-lobe as a The overall structure of the Fe3+-soaked form was almost exactly the same as that of the apo form. The for was Å. with the holo and structure of ovotransferrin N-lobe J.C. Mikami B. Hirose M. Sacchettini J.C. Biochemistry. 1993; 32: 11963-11968Crossref PubMed Scopus (167) Google a domain-opened conformation The and of the opening were almost the same as in the N-lobes of the of lactoferrin (11Anderson B.F. Baker H.M. Norris G.E. Rumball S.V. Baker E.N. Nature. 1990; 344: 784-787Crossref PubMed Scopus (351) Google Scholar) and A. Muirhead H. Williams J. Acta Crystallogr. Sec. D. 1997; 53: 464-468Crossref PubMed Scopus (25) Google Scholar) and hen J. C. B. J. C. and M. in as by the body M. Anderson B.F. Norris G.E. Baker E.N. C. J. Mol. Biol. 1993; PubMed Scopus Google the domains a through the two the observation in is that an iron atom in the interdomain cleft of the Fe3+-soaked form. The two tyrosine residues in the holo form and also to in the iron coordination in the Fe3+-soaked form, the other two protein ligands of and His250 are located far the iron The Fe3+ binding structure was in for the Fe3+-soaked form. a is a of the electron density map with the of the NTA for the Fe3+-soaked form. The the of iron and NTA to the Tyr92 and Tyr191 To the of an iron atom by an alternative we the density map with of an iron As in the of iron atom is by the in the density 4 a is a the iron coordination and hydrogen structure in the Fe3+-soaked form. As in the the iron of Tyr92-OH and are Å and the Fe3+ coordination by two tyrosine The other two protein ligands of however, are not in the coordination of the iron the of and iron are Å and at of the other four Fe3+ coordination sites are occupied by the iron of and are and The coordination may be coordinated by or almost the of this iron is a of with the other coordination The and or are also the of the iron binding in a The binding of NTA is stabilized through the with the protein is hydrogen to and to and protein groups are the that form the hydrogen bonds with CO32− anion in the holo form As a however, NTA has no interaction with the synergistic anion-binding residue, Arg121. in the is that the hydrogen of to in the Fe3+-soaked form is replaced by that of to in the holo form. x-ray crystallographic (2Bailey S. Evans R.W. Garratt R.C. Gorinsky B. Hasnain S. Horsburgh C. Jhoti H. Lindley P.F. Mydin A. Sarra R. Watson J.L. Biochemistry. 1988; 27: 5804-5812Crossref PubMed Scopus (369) Google Scholar, 3Anderson B.F. Baker H.M. Norris G.E. Rice D.W. Baker E.N. J. Mol. Biol. 1989; 209: 711-734Crossref PubMed Scopus (546) Google Scholar, 4Kurokawa H. Mikami B. Hirose M. J. Mol. Biol. 1995; 254: 196-207Crossref PubMed Scopus (200) Google Scholar, 5Rawas A. Muirhead H. Williams J. Acta Crystallogr. Sec. D. 1996; 52: 631-640Crossref PubMed Scopus (55) Google Scholar, 6Moore S.A. Anderson B.F. Groom C.R. Haridas M. Baker E.N. J. Mol. Biol. 1997; 274: 222-236Crossref PubMed Scopus (338) Google Scholar, 7Sarra R. Garratt R. Gorinsky B. Jhoti H. Lindley P. Acta Crystallogr. Sec. B. 1990; 46: 763-771Crossref Scopus (93) Google Scholar, 8Dewan J.C. Mikami B. Hirose M. Sacchettini J.C. Biochemistry. 1993; 32: 11963-11968Crossref PubMed Scopus (167) Google Scholar, 9Day C.L. Anderson B.F. Tweedie J.W. Baker E.N. J. Mol. Biol. 1993; 232: 1084-1100Crossref PubMed Scopus (76) Google Scholar, 10MacGillivray R.T.A. Moore S.A. Chen J. Anderson B.F. Baker H. Luo Y. Bewley M. Smith C.A. Murphy M.E.P. Wang Y. Mason A.B. Woodworth R.C. Brayer G.D. Baker E.N. Biochemistry. 1998; 37: 7919-7928Crossref PubMed Scopus (228) Google Scholar, B.F. Baker H.M. Norris G.E. Rumball S.V. Baker E.N. Nature. 1990; 344: 784-787Crossref PubMed Scopus (351) Google Scholar, 12Rawas A. Muirhead H. Williams J. Acta Crystallogr. Sec. D. 1997; 53: 464-468Crossref PubMed Scopus (25) Google Scholar, 13Jeffrey P.D. Bewley M.C. MacGillivray T.A. Mason A.B. Woodworth R.C. Baker E.N. Biochemistry. 1998; 37: 13978-13986Crossref PubMed Scopus (171) Google Scholar) and solution scattering (14Grossmann J.G. Neu M. Pantos E. Schwab F.J. Evans R.W. Townes-Andrews E. Lindley P.F. Appel H. Thies W.-G. Hasnain S.S. J. Mol. Biol. 1992; 225: 811-819Crossref PubMed Scopus (149) Google Scholar, 15Grossmann J.G. Neu M. Evans R.W. Lindley P.F. Appel H. Hasnain S.S. J. Mol. Biol. 1993; 229: 585-590Crossref PubMed Scopus (66) Google Scholar, 16Mecklenburg S.L. Donohoe R.J. Olah G.A. J. Mol. Biol. 1997; 270: 739-750Crossref PubMed Scopus (35) Google Scholar, 17Grossmann J.G. Crawley J.B. Strange R.W. Patel K.J. Murphy L.M. Neu M. Evans R.W. Hasnain S.S. J. Mol. Biol. 1998; 279: 461-472Crossref PubMed Scopus (53) Google Scholar) analyses have revealed that Fe3+ transferrins undergo a large scale conformational the domain-opened apo structure into the closed holo The structural pathway for the large conformational however, has not been The Fe3+-soaked structure the structural for the Fe3+ For Fe3+ to a most NTA is employed (1Aisen P. Listowsky I. Annu. Rev. Biochem. 1980; 49: 357-393Crossref PubMed Scopus (938) Google Scholar). The binding a of transferrin, and NTA (1Aisen P. Listowsky I. Annu. Rev. Biochem. 1980; 49: 357-393Crossref PubMed Scopus (938) Google Scholar, J. Biol. Full Text PDF PubMed Google Scholar). The is a stable form in the absence of other synergistic In the presence of a of however, NTA is replaced by this the holo form of transferrin, and (1Aisen P. Listowsky I. Annu. Rev. Biochem. 1980; 49: 357-393Crossref PubMed Scopus (938) Google J. Biol. Full Text PDF PubMed Google Scholar). The crystal structure of the Fe3+-soaked form essentially the same open conformation as assumes the closed one the of the Fe3+-soaked structure for the iron binding two different may be In the first assumes the same conformation in solution as the Fe3+-soaked structure, and the domain in In this the domain on the anion The the two protein ligands and Tyr191 with structural in the iron binding site, other the protein to be to the structural in As in CO32− forms hydrogen bonds with and in the holo protein groups are all hydrogen to groups of NTA in the Fe3+-soaked form 4 The protein group that forms a hydrogen with in the holo form also forms a hydrogen with in the Fe3+-soaked form. and which are the sites for CO32− in the holo form, are in the Fe3+-soaked form. Such an open be for the of CO32− in pathway for includes an initial of CO32− into the sites and then the of NTA by CO32−. This a with the open in which four of the six Fe3+ coordination sites are occupied by the protein side and and bidentate CO32−. As a structural the crystal structure of a domain in which all of domain 1 as as the aspartic acid and histidine ligands are by the presence of an Fe3+ coordination structure by the two tyrosine residues and CO32− P.F. M. Evans R.W. Garratt R.C. Hasnain S.S. Jhoti H. P. Neu M. Patel Sarra R. Strange R. A. Acta Crystallogr. Sec. D. 1993; 49: PubMed Google Scholar). The of the holo structure is then by the coordination of and His250 ligands to the two Fe3+ sites and by the domain In the the Fe3+-soaked form is initially transformed into a closed conformation by the of and His250 before the bound NTA is replaced by a This however, as a the in of the with the closed conformation in a the of the open conformation in the Fe3+-soaked form is for by the to a crystal in into the closed The also require a coordination and hydrogen structure for NTA in the the and His250 coordination sites are occupied by NTA in the Fe3+-soaked form and As a observation to the no clear has been for the apo crystal is soaked with Fe3+·NTA in the presence of a of This strongly suggests that the apo crystal the of the open form into the closed The crystal may not be to the of the open to closed to the the present however, the which includes a with a holo-like closed conformation in solution be the structure of the in solution has not been In the crystallographic structures of transferrins have been essentially to the two structural one is the iron-free apo form and the the Fe3+-loaded holo form (2Bailey S. Evans R.W. Garratt R.C. Gorinsky B. Hasnain S. Horsburgh C. Jhoti H. Lindley P.F. Mydin A. Sarra R. Watson J.L. Biochemistry. 1988; 27: 5804-5812Crossref PubMed Scopus (369) Google Scholar, 3Anderson B.F. Baker H.M. Norris G.E. Rice D.W. Baker E.N. J. Mol. Biol. 1989; 209: 711-734Crossref PubMed Scopus (546) Google Scholar, 4Kurokawa H. Mikami B. Hirose M. J. Mol. Biol. 1995; 254: 196-207Crossref PubMed Scopus (200) Google Scholar, 5Rawas A. Muirhead H. Williams J. Acta Crystallogr. Sec. D. 1996; 52: 631-640Crossref PubMed Scopus (55) Google Scholar, 6Moore S.A. Anderson B.F. Groom C.R. Haridas M. Baker E.N. J. Mol. Biol. 1997; 274: 222-236Crossref PubMed Scopus (338) Google Scholar, 7Sarra R. Garratt R. Gorinsky B. Jhoti H. Lindley P. Acta Crystallogr. Sec. B. 1990; 46: 763-771Crossref Scopus (93) Google Scholar, 8Dewan J.C. Mikami B. Hirose M. Sacchettini J.C. Biochemistry. 1993; 32: 11963-11968Crossref PubMed Scopus (167) Google Scholar, 9Day C.L. Anderson B.F. Tweedie J.W. Baker E.N. J. Mol. Biol. 1993; 232: 1084-1100Crossref PubMed Scopus (76) Google Scholar, 10MacGillivray R.T.A. Moore S.A. Chen J. Anderson B.F. Baker H. Luo Y. Bewley M. Smith C.A. Murphy M.E.P. Wang Y. Mason A.B. Woodworth R.C. Brayer G.D. Baker E.N. Biochemistry. 1998; 37: 7919-7928Crossref PubMed Scopus (228) Google Scholar, B.F. Baker H.M. Norris G.E. Rumball S.V. Baker E.N. Nature. 1990; 344: 784-787Crossref PubMed Scopus (351) Google Scholar, 12Rawas A. Muirhead H. Williams J. Acta Crystallogr. Sec. D. 1997; 53: 464-468Crossref PubMed Scopus (25) Google Scholar, 13Jeffrey P.D. Bewley M.C. MacGillivray T.A. Mason A.B. Woodworth R.C. Baker E.N. Biochemistry. 1998; 37: 13978-13986Crossref PubMed Scopus (171) Google Scholar). As an alternative structural the Fe3+-soaked structure of the ovotransferrin N-lobe is the first of an Fe3+-loaded, open transferrin form. One of the most in the structure is that the two tyrosine residues and in the Fe3+ coordination as protein ligands and 4 and The coordination structure by the two tyrosine residues is with the structure for the Fe3+-loaded, domain-opened transferrin intermediate E.N. Lindley P.F. J. Inorg. Biochem. 1992; 47: 147-160Crossref PubMed Scopus (183) Google the structure has been the crystallographic data of the domain N2 of ovotransferrin in which the and not the two are by P.F. M. Evans R.W. Garratt R.C. Hasnain S.S. Jhoti H. P. Neu M. Patel Sarra R. Strange R. A. Acta Crystallogr. Sec. D. 1993; 49: PubMed Google Scholar). of the assumes the open or closed conformation in the that the overall structure of the Fe3+-soaked form is almost that of the apo form is with the that the two tyrosine residues are the protein ligands for the Fe3+ in the transferrin with the domain-opened was by the for