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• W1973551683 abstract "The three-dimensional crystal structure of hen apo-ovotransferrin has been solved by molecular replacement and refined by simulated annealing and restrained least squares to a 3.0-Å resolution. The final model, which comprises 5312 protein atoms (residues 1 to 686) and 28 carbohydrate atoms (from two monosaccharides attached to Asn473), gives an R-factor of 0.231 for the 11,989 observed reflections between 20.0- and 3.0-Å resolution. In the structure, both empty iron binding clefts are in the open conformation, lending weight to the theory that Fe3+binding or release in transferrin proceeds via a mechanism that involves domain opening and closure. Upon opening, the domains rotate essentially as rigid bodies. The two domains of the N-lobe rotate away from one another by 53°, whereas the C-lobe domains rotate away each another by 35°. These rotations take place about an axis that passes through the two β-strands, linking the domains. The domains of each lobe make different contacts with one another in the open and closed forms. These contacts form two interdomain interfaces on either side of the rotation axis, and domain opening or closing produces a see-saw motion between these two alternative close-packed interfaces. The interdomain disulfide bridge (Cys478-Cys671), found only in the C-lobe, may restrict domain opening but does not completely prevent it. The three-dimensional crystal structure of hen apo-ovotransferrin has been solved by molecular replacement and refined by simulated annealing and restrained least squares to a 3.0-Å resolution. The final model, which comprises 5312 protein atoms (residues 1 to 686) and 28 carbohydrate atoms (from two monosaccharides attached to Asn473), gives an R-factor of 0.231 for the 11,989 observed reflections between 20.0- and 3.0-Å resolution. In the structure, both empty iron binding clefts are in the open conformation, lending weight to the theory that Fe3+binding or release in transferrin proceeds via a mechanism that involves domain opening and closure. Upon opening, the domains rotate essentially as rigid bodies. The two domains of the N-lobe rotate away from one another by 53°, whereas the C-lobe domains rotate away each another by 35°. These rotations take place about an axis that passes through the two β-strands, linking the domains. The domains of each lobe make different contacts with one another in the open and closed forms. These contacts form two interdomain interfaces on either side of the rotation axis, and domain opening or closing produces a see-saw motion between these two alternative close-packed interfaces. The interdomain disulfide bridge (Cys478-Cys671), found only in the C-lobe, may restrict domain opening but does not completely prevent it. Transferrins are a group of iron-binding proteins that includes serum transferrin, ovotransferrin, and lactoferrin (1Aisen P. Listowsky I. Annu. Rev. Biochem. 1980; 49: 357-393Crossref PubMed Scopus (938) Google Scholar). These proteins serve to control the levels of iron in the body fluids of vertebrates by their ability to bind very tightly two Fe3+ ions together with two CO32−ions. Serum transferrins and ovotransferrin can act as iron transporters, whereas the lactoferrins possess an antimicrobial activity (2Arnold R.R. Cole M.F. McGhee J.R. Science. 1977; 197: 263-265Crossref PubMed Scopus (488) Google Scholar) and a sequence-specific DNA binding capacity (3He J. Furmanski P. Nature. 1995; 373: 721-724Crossref PubMed Scopus (325) Google Scholar). The ∼80-kDa transferrin molecule consists of two similarly sized homologous N- and C-lobes, which are further divided into two similarly sized domains (N1 and N2 in the N-lobe; 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 fully iron-loaded diferric forms (4Bailey 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, 5Anderson B.F. Baker H.M. Norris G.E. Rice D.W. Baker E.N. J. Mol. Biol. 1989; 209: 711-734Crossref PubMed Scopus (548) Google Scholar, 6Kurokawa H. Mikami B. Hirose M. J. Mol. Biol. 1995; 254: 196-207Crossref PubMed Scopus (200) Google Scholar, 7Rawas A. Muirhead H. Williams J. Acta Crystallogr. Sec. D. 1996; 52: 631-640Crossref PubMed Scopus (55) Google Scholar) and the monoferric N-lobes (8Sarra R. Garratt R. Gorinsky B. Jhoti H. Lindley P. Acta Crystallogr. Sec. B. 1990; 46: 763-771Crossref Scopus (93) Google Scholar, 9Dewan J.C. Mikami B. Hirose M. Sacchettini J.C. Biochemistry. 1993; 32: 11963-11968Crossref PubMed Scopus (167) Google Scholar, 10Day C.L. Anderson B.F. Tweedie J.W. Baker E.N. J. Mol. Biol. 1993; 232: 1084-1100Crossref PubMed Scopus (76) Google Scholar, 11MacGillivray 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 of each lobe are closed over an Fe3+ ion. Four of the six Fe3+ coordination sites are occupied by protein ligands (2 Tyr residues, 1 Asp, and 1 His) and the other two by a bidentate carbonate anion. The recent x-ray structure of theHemophilus influenzae Fe3+-binding protein has revealed that its overall structure, apart from consisting of a single lobe, is remarkably similar to that of the transferrin family (12Bruns C.M. Nowalk A.J. Arvai A.S. McTigue M.A. Vaughan K.G. Mietzner T.A. McRee D.E. Nat. Struct. Biol. 1997; 4: 919-924Crossref PubMed Scopus (162) Google Scholar). The ligands to iron are, however, a phosphate oxygen, a water molecule, and four protein ligands (2 Tyr residues, 1 Glu, and 1 His). Except for the carboxylate ligand, the other three protein ligands of the Fe3+-binding protein are all located in different parts of the protein fold from those of the transferrins, indicating convergent evolution for the development of the Fe3+ binding function (12Bruns C.M. Nowalk A.J. Arvai A.S. McTigue M.A. Vaughan K.G. Mietzner T.A. McRee D.E. Nat. Struct. Biol. 1997; 4: 919-924Crossref PubMed Scopus (162) Google Scholar). It is generally believed that the two domains of each transferrin lobe must open to allow entry or release of Fe3+ (13Baker E.N. Rumball S.V. Anderson B.F. Trends Biochem. Sci. 1987; 12: 350-353Abstract Full Text PDF Scopus (168) Google Scholar, 14Anderson B.F. Baker H.M. Norris G.E. Rumball S.V. Baker E.N. Nature. 1990; 344: 784-787Crossref PubMed Scopus (352) Google Scholar, 15Lindley P.F. Bajaj M. Evans R.W. Garratt R.C. Hasnain S.S. Jhoti H. Kuser P. Neu M. Patel K. Sarra R. Strange R. Walton A. Acta Crystallogr. Sec. D. 1993; 49: 292-304Crossref PubMed Google Scholar); indeed, an x-ray solution scattering study has revealed iron-induced conformational changes in both lobes of several transferrins (16Grossmann 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). The iron-loaded holo form shows a higher affinity for the transferrin receptor than does the apo form, indicating that iron-dependent conformational changes may be important in the cellular uptake of iron (17Young S.P. Bomford A. Williams R. Biochem. J. 1984; 219: 505-510Crossref PubMed Scopus (138) Google Scholar, 18Mason A.B. Brown S.A. Church W.R. J. Biol. Chem. 1987; 262: 9011-9015Abstract Full Text PDF PubMed Google Scholar, 19Mason A.B. Woodworth R.C. Oliver R.W.A. Green B.N. Lin L.-N. Brandts J.F. Savage K.J. Tam B.M. MacGillivray R.T.A. Biochem. J. 1996; 319: 361-368Crossref PubMed Scopus (30) Google Scholar, 20Mason A.B. Tam B.M. Woodworth R.C. Oliver R.W.A. Green B.N. Lin L.-N. Brandts J.F. Savage K.J. Lineback J.A. MacGillivray R.T.A. Biochem. J. 1997; 326: 77-85Crossref PubMed Scopus (50) Google Scholar). Although the x-ray structures of the diferric forms of several transferrins are known (4Bailey 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, 5Anderson B.F. Baker H.M. Norris G.E. Rice D.W. Baker E.N. J. Mol. Biol. 1989; 209: 711-734Crossref PubMed Scopus (548) Google Scholar, 6Kurokawa H. Mikami B. Hirose M. J. Mol. Biol. 1995; 254: 196-207Crossref PubMed Scopus (200) Google Scholar, 7Rawas A. Muirhead H. Williams J. Acta Crystallogr. Sec. D. 1996; 52: 631-640Crossref PubMed Scopus (55) Google Scholar), data for the bilobal apo form is limited. The crystal structure of the apo form of human lactoferrin has been determined at 2.8-Å resolution by Anderson et al. (14Anderson B.F. Baker H.M. Norris G.E. Rumball S.V. Baker E.N. Nature. 1990; 344: 784-787Crossref PubMed Scopus (352) Google Scholar) and recently at 2.0-Å resolution by Jameson et al. (21Jameson G.B. Anderson B.F. Norris G.E. Thomas D.H. Baker E.N. Acta Crystallogr. Sec. D. 1998; 54: 1319-1335Crossref PubMed Scopus (0) Google Scholar). In that structure the iron binding cleft in the N-lobe is wide open when compared with the closed holo structure. Upon uptake or release of iron, domains N1 and N2 rotate 54° away from one another as rigid bodies around a rotation axis that passes through the two antiparallel β-strands linking the domains. By contrast, the C-lobe is found in the closed conformation in both the holo and apo forms. This unexpected lack of conformational change may be related to the presence of an interdomain disulfide bridge (Cys483-Cys677) in the C-lobe of lactoferrin. It is also possible that lactoferrin might lack, because of its non-iron-transporting nature, an open/close mechanism that might be important for the selective recognition of the holo form by the receptor. The absence in human (5Anderson B.F. Baker H.M. Norris G.E. Rice D.W. Baker E.N. J. Mol. Biol. 1989; 209: 711-734Crossref PubMed Scopus (548) Google Scholar) and bovine (22Moore S.A. Anderson B.F. Groom C.R. Haridas M. Baker E.N. J. Mol. Biol. 1997; 274: 222-236Crossref PubMed Scopus (340) Google Scholar) lactoferrins of the N-lobe dilysine trigger, which has been proposed as a pH-sensitive molecular switch for the opening of that domain and subsequent iron release (9Dewan J.C. Mikami B. Hirose M. Sacchettini J.C. Biochemistry. 1993; 32: 11963-11968Crossref PubMed Scopus (167) Google Scholar), is another structural point that differentiates the lactoferrins from the iron-transporting transferrins. Ovotransferrin, a major egg white protein, should share the same structural characteristics as hen serum transferrin, which functions as an iron transporter, since these proteins are derived from the same gene and differ only in their attached carbohydrate (23Thibodeau S.N. Lee D.C. Palmiter R.D. J. Biol. Chem. 1978; 253: 3771-3774Abstract Full Text PDF PubMed Google Scholar). Although anin vivo iron transport function for ovotransferrin has not been proved, specific transferrin receptor interactions have been demonstrated for the protein (18Mason A.B. Brown S.A. Church W.R. J. Biol. Chem. 1987; 262: 9011-9015Abstract Full Text PDF PubMed Google Scholar, 19Mason A.B. Woodworth R.C. Oliver R.W.A. Green B.N. Lin L.-N. Brandts J.F. Savage K.J. Tam B.M. MacGillivray R.T.A. Biochem. J. 1996; 319: 361-368Crossref PubMed Scopus (30) Google Scholar, 20Mason A.B. Tam B.M. Woodworth R.C. Oliver R.W.A. Green B.N. Lin L.-N. Brandts J.F. Savage K.J. Lineback J.A. MacGillivray R.T.A. Biochem. J. 1997; 326: 77-85Crossref PubMed Scopus (50) Google Scholar, 24Brown-Mason A. Woodworth R.C. J. Biol. Chem. 1984; 259: 1866-1873Abstract Full Text PDF PubMed Google Scholar). Hen ovotransferrin possesses the same six disulfide bridge pattern in the N-lobe as that observed in human lactoferrin: two bridges in the N1-domain and four in the N2-domain. There is, therefore, no interdomain disulfide bridge in the N-lobe of either protein (25Williams J. Elleman T.C. Kingston I.B. Wilkins A.G. Kuhn K.A. Eur. J. Biochem. 1982; 122: 297-303Crossref PubMed Scopus (181) Google Scholar, 26Williams J. Trends Biochem. Sci. 1982; 7: 394-400Abstract Full Text PDF Scopus (99) Google Scholar, 27Crichton R.R. Charloteaux-Wauters M. Eur. J. Biochem. 1987; 164: 485-506Crossref PubMed Scopus (405) Google Scholar). This six-disulfide motif is also observed in the C-lobes of ovotransferrin and lactoferrin. There are, however, three and four additional disulfides, respectively, in the ovotransferrin and lactoferrin C-lobes. One of the disulfides of the ovotransferrin C-lobe exists as the interdomain disulfide Cys478-Cys671 (6Kurokawa H. Mikami B. Hirose M. J. Mol. Biol. 1995; 254: 196-207Crossref PubMed Scopus (200) Google Scholar) and is found in an equivalent site to the lactoferrin interdomain disulfide Cys483-Cys677 (5Anderson B.F. Baker H.M. Norris G.E. Rice D.W. Baker E.N. J. Mol. Biol. 1989; 209: 711-734Crossref PubMed Scopus (548) Google Scholar). This suggests that if a restriction of domain opening is caused by the interdomain cross-link, it may operate in a similar manner in the two proteins. We report here the x-ray crystal structure of hen apo-ovotransferrin determined at 3.0-Å resolution. Comparison of the current apo structure with the previous holo structure (6Kurokawa H. Mikami B. Hirose M. J. Mol. Biol. 1995; 254: 196-207Crossref PubMed Scopus (200) Google Scholar) shows that both lobes adopt an open conformation upon iron release. Apo form of hen ovotransferrin was purified as described previously (28Hirose M. Oe H. Doi E. Agric. Biol. Chem. 1986; 50: 59-64Google Scholar). Colorless apo-ovotransferrin crystals were grown at 4 °C by the hanging drop vapor diffusion method where 5 μl of protein solution (85 mg/ml, pH 6.0, 0.02 m acetate buffer) was mixed with 5 μl of precipitant solution (4–6% polyethylene glycol 6000, pH 6.0, 0.02 m acetate buffer) on a silanized coverslip that was inverted and sealed above 0.7 ml of the precipitant solution. The crystal used for data collection was the largest available and had approximate dimensions of 2.5 x 0.5 x 0.4 mm. Many crystals were examined, but the data set employed herein was the best obtained to date. X-ray data were collected at −150 °C (29Dewan J.C. Tilton R.F. J. Appl. Crystallogr. 1987; 20: 130-132Crossref Scopus (17) Google Scholar) with CuKαradiation (λ=1.5418 Å) using a Siemens Hi-Star area detector coupled to a MacScience M18XHF rotating-anode generator. A total of 31,370 reflections was collected and averaged (Rsymm = 0.069), yielding 12,920 unique data (80% complete). The meanI/ς(I) for the data set was 6.5. The structure was solved by molecular replacement using X-PLOR (30Brünger A.T. X-PLOR , Version 3.85. Yale University, New Haven, CT1996Google Scholar). The only search model to give the correct solution was the diferric ovotransferrin (6Kurokawa H. Mikami B. Hirose M. J. Mol. Biol. 1995; 254: 196-207Crossref PubMed Scopus (200) Google Scholar) C-lobe, with the domains opened by 40°. The N1-domain was then placed in the same relative orientation as domains N1/C1 of the diferric structure. After rigid body refinement, the N2-domain could be fitted into its electron density. Rigid body refinement, simulated annealing, and positional and B-factor refinement (30Brünger A.T. X-PLOR , Version 3.85. Yale University, New Haven, CT1996Google Scholar) have resulted in a finalr = 0.231 (Rfree = 0.265) for the 11,989 reflections (75% cimoleteness) with F > 2ς(F) between 20.0 and 3.0-Å resolution. A bulk solvent correction was applied to the data. The final model consists of 5312 non-hydrogen protein atoms (residues 1–686) and 28 carbohydrate atoms (two residues attached to Asn473). Further experimental details appear in Table I. Model building and superposition of the structures were performed with TURBO-FRODO (Biographics).Table IStatistics for data collection and refinement of hen apo-ovotransferrinCrystal systemTetragonalSpace groupP43212Unit cell (Å)a = b = 92.26, c = 178.19Molecules/asymetric unit1DetectorSiemens Hi-StarCrystal-detector distance (cm)15No. crystals1Resolution limit (Å)3.0Scan width (°/frame)0.25Scan speed (°/min)0.25Scan range φ-scan0–45° (2θ = 15°, χ = 0°) ω-scan0–45° (2θ = 15°, χ = 30°)Measured reflections31,370Unique reflections12,920 [‖I‖ > ς (I)]Completeness (%)80.0Rsymm on I (%)6.9Final model1–686 residues, 2 sugarsResolution range (Å)20.0–3.0Reflections11,989 [‖F‖ > 2ς(F)]Completeness (%)75.0R-factor (%)23.1Rfree(%)26.5Root mean square bond length (Å)0.010 angle (°)1.70 Open table in a new tab The final model is comprised of the entire polypeptide chain (residues 1 to 686). In the final 2Fo − Fc electron density map, there are no breaks in the main-chain density when contoured at the 1ς level. Two sugar residues are also included in the model attached to Asn473. The root mean square deviations from standard bond lengths and angles are 0.010 Å and 1.70°, respectively (TableI). A Ramachandran plot of the main-chain torsion angles shows that 60% of the residues are in the core regions, with 95% lying within the allowed regions as defined by the program PROCHECK (31Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Two non-glycine residues (Leu299 and Leu636) lie outside the allowed regions of conformational space (ψ = 64°, φ = −48°, and ψ = 69°, φ = −55°, respectively); both are the central residues of two γ-turns in equivalent positions in each ovotransferrin lobe. The apo-ovotransferrin structure, superimposed on that of diferric ovotransferrin (Protein Data Bank code 1OVT) (6Kurokawa H. Mikami B. Hirose M. J. Mol. Biol. 1995; 254: 196-207Crossref PubMed Scopus (200) Google Scholar), is shown as a ribbon trace in Fig.1. There is no difference in the secondary structure assignment (β-strands a-k and α-helices 1–12) between the holo and apo forms (6Kurokawa H. Mikami B. Hirose M. J. Mol. Biol. 1995; 254: 196-207Crossref PubMed Scopus (200) Google Scholar). As in the diferric ovotransferrin structure, hen apo-ovotransferrin is folded into two homologous N- and C-lobes, and each of the lobes is divided into two distinct and similarly sized α/β domains (N-lobe: N1- and N2-domains; C- lobe: C1- and C2-domains). The two empty iron binding sites are located between the two domains in each lobe, and the two domains are linked by two antiparallel β-strands (e and j) that run behind the iron binding site of each lobe. Both iron binding clefts of apo-ovotransferrin are in the open conformation. The N2- and C2-domains move away from the N1- and C1-domains, respectively, almost as rigid bodies. Between the open and closed forms, the root mean square deviation of equivalent C-α atoms that are within a distance of 2.0 Å of one another is 1.02 Å for the N1-domain (186 C-α atoms) and 1.12 Å for the N2-domain (160 C-α atoms). For the C-lobe, these values are 1.06 Å for the C1-domain (204 C-α atoms) and 0.87 Å for the C2-domain (180 C-α atoms). The major conformational changes occur in the hinge regions on the two β-strands that link the domains of each lobe. In addition to the domain opening in the two lobes, there is a small rotation of the C1-domain relative to the N1-domain. The value of the rotation is 7°, suggesting that the N1- and C1-domains remain relatively fixed with respect to one another. Two GlcNAc residues attached to Asn473 reside between the C1- and C2-domains. To characterize the conformational changes that have occurred upon domain opening, the domain motion in each lobe was considered separately as a rotation and a translation (32Gerstein M. Anderson B.F. Norris G.E. Baker E.N. Lesk A.M. Chothia C. J. Mol. Biol. 1993; 234: 357-372Crossref PubMed Scopus (143) Google Scholar). In the N-lobe the N1-domains of the open and closed forms were superimposed, and the rotation and translation required to superimpose the N2-domains were determined to be 53° and 1.8 Å, respectively (Fig. 2 a). The rotation axis passes close to residues Ser91 and Val247 and is nearly parallel to a line through the C-α atoms of these two residues (Fig. 3). The same analysis was applied to the motion in the C-lobe. A 35° rotation, with a 0.2-Å translation, was required to superimpose the C2-domains of the open and closed forms (Fig. 2 b). The rotation axis passes close to residues Ala429 and Val589 and is nearly parallel to a line through the two C-α atoms of these residues (Fig. 3). The C-α atoms of Cys478 and Cys671, which form the C-lobe interdomain disulfide bridge, also lie close to this axis (Fig. 3). In either the N- or C-lobe, domain 2 translates only 1.8 or 0.2 Å with respect to domain 1, and thus, the motions of the N2- or C2-domains relative to the N1- or C1-domains are almost a pure rotation about the two central residues in each hinge.Figure 3The two hinges in the open apo-ovotransferrin N-lobe (left) and C-lobe (right). The views are as shown in Fig. 2, but rotated by 90°. The rotation axes, indicated by horizontal lines, are parallel to the plane of the page and perpendicular to the viewing direction. The C-α atoms of Ser91 and Val247 for the N-lobe and of Ala429 and Val589 for the C-lobe are indicated by open spheres. The shaded spheres in the C-lobe are the C-α atoms of Cys478 and Cys671. The carbohydrate moiety in the apo-ovotransferrin C-lobe is drawn as aball-and-stick representation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The two lobes of ovotransferrin make different interdomain contacts when in the open or closed form (Fig. 2). Most of the contacting residues are grouped into two categories: 1) those that make contact only in the closed form and 2) those that make contact only in the open form. Fig.4 shows the positions of the contacting residues in the open and closed forms. In the closed form there are 22 contacting residues observed for the N-lobe and 16 for the C-lobe. The differences in the accessible surface areas of these residues, between the open and closed forms, are listed in TableII. Almost all of the residues that make contact with the other domain in the closed form are exposed in the open form. In the open form there are 13 contacting residues for the N-lobe and 9 for the C-lobe, and most of them are exposed in the closed form (Table II). The contacting residues in the closed form are found mostly in the interdomain binding cleft and form the large interface. The contacting residues in the open form are located on the opposite side of the rotation axis and constitute the small interface. The alternating exposure and burying of the interfaces on either side of the rotation axis constitutes a “see-saw” motion that has been noted before by Gerstein et al. (32Gerstein M. Anderson B.F. Norris G.E. Baker E.N. Lesk A.M. Chothia C. J. Mol. Biol. 1993; 234: 357-372Crossref PubMed Scopus (143) Google Scholar) in the case of lactoferrin.Table IIResidues making interdomain contacts and their accessible surface areasInterfaceResidueContact inSurface area (Å2)InterfaceResidueContact inSurface area (Å2)OpenClosedDiff.OpenClosedDiff.L/N1D60C51.63.0−48.6L/C1K352C154.3150.3−4.0I184C93.717.3−76.4D395C41.47.8−33.6T85C126.0100.2−25.8G397C6.33.7−2.6Y92C58.40.3−58.1S430C53.148.9−4.2E215C143.17.5−135.6V589C/O1.82.70.9V247C/O25.86.6−19.2H592C34.613.7−20.9H250C39.614.7−24.9D634C65.136.1−29.0P293C109.623.2−86.4K638C39.34.5−34.8V294C104.658.8−45.8L640C88.027.9−60.1K296C95.312.8−82.5L668C17.15.3−11.8K301C27.56.7−20.8C671C/O32.67.8−24.8Y324C17.522.24.7L/C2R460C97.428.6−68.8L/N2Y93C29.211.4−17.8T461C10.70.1−10.6R121C132.139.2−92.9G469C14.80.5−14.3S122C16.00.5−15.5H472C/O33.434.71.3N126C31.510.3−21.2H518C121.837.0−84.8I127C1.619.017.4Y524C54.35.5−48.8L133C/O20.328.98.6Q541C35.33.4−31.9W140C/O31.625.5−6.1S543C33.011.2−21.8P187C130.641.6−89.0S/C1F661C/O0.89.68.8Y191C95.44.7−90.7I665O1.22.71.5H196C61.822.0−39.8S667O35.447.612.2K209C46.21.3−44.9L668O17.15.3−11.8E215C143.17.5−135.6S/C2F432O10.015.25.2K240C109.069.7−39.3T461O69.474.45.0S/N1L320O1.78.56.8V465O18.21.2−17.0E323O30.3128.898.5G469O0.514.814.3S326O18.343.725.4H472O34.733.4−1.0Q329O25.348.523.2T477O73.757.6−16.1S330O12.740.627.9M331C/O22.723.20.5R332O149.1124.1−25.0S/N2G130O013.313.3H134O92.6156.263.6I1e43O63.9100.336.5V148O18.919.80.9W244O7.818.710.9A245O024.024.0R246O78.6103.825.2Each residue listed makes at least one contact between the domains. The interfaces L/N1, L/N2, L/C1, and L/C2 represent the large interfaces of the N1, N2, C1, and C2 domains, respectively, whereas S/N1, S/N2, S/C1, and S/C2 represent the small interfaces of the N1, N2, C1, and C2 domains, respectively. Contacts in the closed (C), open (O), or closed and open forms (C/O) are defined as atoms separated by less than the sum of their van der Waals radii. Diff. is the difference in the accessible surface area for each contacting residue calculated for the closed form minus the open form. Surface areas were calculated by NACCESS (51Hubbard S.J. Thornton J.M. NACCESS Computer Program. Department of Biochemistry and Molecular Biology, University College, London1993Google Scholar). Open table in a new tab Each residue listed makes at least one contact between the domains. The interfaces L/N1, L/N2, L/C1, and L/C2 represent the large interfaces of the N1, N2, C1, and C2 domains, respectively, whereas S/N1, S/N2, S/C1, and S/C2 represent the small interfaces of the N1, N2, C1, and C2 domains, respectively. Contacts in the closed (C), open (O), or closed and open forms (C/O) are defined as atoms separated by less than the sum of their van der Waals radii. Diff. is the difference in the accessible surface area for each contacting residue calculated for the closed form minus the open form. Surface areas were calculated by NACCESS (51Hubbard S.J. Thornton J.M. NACCESS Computer Program. Department of Biochemistry and Molecular Biology, University College, London1993Google Scholar). The different interdomain contacts in the apo and holo forms play a role in Fe3+ binding stability. For example, helix 11 (residues 321–332 in the N-lobe; 658–669 in the C-lobe) is located in the small interface, and 6 and 4 residues of the helix, respectively, in the N- and C-lobes participate in interdomain contacts (Table II). Previous observations have stressed the importance of the helix in Fe3+ binding stability. The removal of residues 280 to 332 from the ovotransferrin N-lobe results in a greatly reduced Fe3+ binding stability (33Kurokawa H. Mikami B. Hirose M. J. Biol. Chem. 1994; 269: 6671-6676Abstract Full Text PDF PubMed Google Scholar). Likewise, helix 11 occurs in both lobes of lactoferrin and is disordered in the isolated N-lobe crystal structure. This half molecule displays a much lower Fe3+ binding affinity (10Day C.L. Anderson B.F. Tweedie J.W. Baker E.N. J. Mol. Biol. 1993; 232: 1084-1100Crossref PubMed Scopus (76) Google Scholar). Fig.5 shows the superposition, on domain 1 in each case, of three open apo-transferrin lobes, namely the N- and C-lobes of apo-ovotransferrin and the apo-lactoferrin N-lobe (Protein Data Bank code 1LFH). The root mean square deviation of equivalent C-α atoms that are within 2 Å of one another is 1.09 Å between the N1-domain of apo-ovotransferrin and the N1-domain of apo-lactoferrin (199 C-α atoms) and 1.10 Å between the N1-domain and C1-domain of apo-ovotransferrin (161 C-α atoms). The extent and orientation of the opening in each lobe, however, are all different. When the different modes of domain opening are expressed as the relative orientations of the rotation axes of domain, the axis of the apo-ovotransferrin N-lobe intersects those of the apo-ovotransferrin C-lobe and of opening, the apo-lactoferrin N-lobe with angles of 17.5° and 12.3°, respectively. The rotation axes of the apo-ovotransferrin C-lobe and the apo-lactoferrin N-lobe can be related to one another by a rotation of 12.4° and a 1.0-Å translation. Such differences may be accounted for, at least in part, by different hinge sequences. Table III shows a sequence alignment of the hinge regions (part of β-strands e and j) in vertebrate transferrins. Other than the iron-coordinating ligands (Tyr92, His250, Tyr431, and His592 in the ovotransferrin sequence), several highly conserved residues are observed. Ala94 (Ala433in the C-lobe) and Ala245 (Ala587 in the C-lobe) are conserved in both the N- and C-lobes of all the vertebrate transferrins except for the substitution of a Ser residue in the rabbit transferrin C-lobe. Thr90, Tyr93, and Val247 are also conserved in all the N-lobes of the transferrins. Ser88 (Arg427 in the C-lobe), Ser91 (Ser430 in the C-lobe), and Ala249 (Thr591 in the C-lobe) are, however, unique residues in the avian transferrin as compared with the mammalian transferrins. Another unique residue to the avian protein is Val589 in the C-lobe; this residue is located at the equivalent position of Val247, that is conserved in the N-lobes of all the transferrins. The hinge residues that are unique to the ovotransferrin N-lobe are Trp244 and Ala248; at positions 244 and 248 (positions 586 and 590 in the C-lobe) Leu and Pro residues are, respectively, conserved in both lobes of all the other transferrins, including the C-lobe of ovotransferrin.Table IIISequence alignment of vertebrate transferrins in the hinge regionsN-Lobe88 91 94244 247 250 Hen ovotransferrin S T T S Y Y A W A R V A A H Human lactoferrin P R T H Y Y A L A R V P S H Bovine lactoferrin P Q T H Y Y A L A Q V P S H Human transferrin P Q T F Y Y A L A Q V P S H Rabbit t" @default.
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• W1973551683 title "Crystal Structure of Hen Apo-ovotransferrin" @default.
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