LACTOFERRIN: ANALYSIS OF THE STRUCTURE PROFILE (original) (raw)
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Journal of Molecular Biology, 1989
The structure of human lactoferrin has been refined crystallographically at 2.8 A (1 A = 0.1 nm) resolution using restrained least squares methods. The starting model was derived from a 3.2 A map phased by multiple isomorphous replacement with solvent flattening. Rebuilding during refinement made extensive use of these experimental phases, in combination with phases calculated from the partial model. The present model, which includes 681 of the 691 amino acid residues, two Fe3+, and two CO',-, gives an R factor of 0.206 for 17,266 observed reflections between 10 and 2.8 A resolution, with a root-meansquare deviation from standard bond lengths of 903 A.
Structure of human lactoferrin at 3.2-A resolution
Proceedings of the National Academy of Sciences of the United States of America, 1987
The three-dimensional structure of human milk lactoferrin, a member of the transferrin family, has been determined crystallographically at 3.2-A resolution. The molecule has two-fold internal homology. The N- and C-terminal halves form two separate globular lobes, connected by a short alpha-helix, and carry one iron-binding site each. Each lobe has the same folding, based on two domains of similar supersecondary structure, with the iron site at the domain interface. Each iron atom is coordinated by four protein ligands: two tyrosines, one histidine, and one aspartate. A probable CO3(2-) (or HCO3-) ion is suggested by the electron density, bound to iron and adjacent to an arginine side chain and a helix N terminus. The protein folding and location of the binding sites show marked similarities with those of other binding proteins, notably the sulfate-binding protein from Salmonella typhimurium.
Iron-binding fragments from the N-terminal and C-terminal regions of human lactoferrin
Biochemical Journal, 1978
Digestion of lactoferrin with pepsin at pH3.0 gave an iron-binding half-molecule that represents the C-terminal part of the native protein. Tryptic or chymotryptic digestion of 30%-iron-saturated lactoferrin yielded the N- and C-terminal half molecules, which could be separated by DEAE-Sephadex chromatography. The N- and C-terminal fragments did not show any immunological cross-reaction. The carbohydrate of lactoferrin was distributed equally between the two fragments.
Proteins, 2016
The bilobal lactoferrin is a ∼76kDa glycoprotein. It sequesters two Fe(3+) ions together with two CO3 (2-) ions. The C-terminal half (residues, Tyr342 - Arg689, C-lobe) of bovine lactoferrin (residues Ala1 - Arg689) was prepared by limited proteolysis using trypsin. Both C-lobe and intact bovine lactoferrin were saturated to 100%. Both of them retained up to nearly 85% of iron at pH 6.5. At pH 5.0, C-lobe retained 75% of iron whereas intact protein could retain only slightly more than 60%. At pH 4.0 both contained 25% iron and at pH 2.0 they were left with iron concentration of only 10%. The structure of iron saturated C-lobe was determined at 2.79 Å resolution and refined to Rcryst and Rfree factors of 0.205 and 0.273 respectively. The structure contains two crystallographically independent molecules, A and B. They were found to have identical structures with an r.m.s. shift of 0.5 Å for their C(α) atoms. A high solvent content of 66% was observed in the crystals. The average value...
Structure of Human Lactoferrin at 3.2- angstrom Resolution
Proceedings of The National Academy of Sciences, 1987
The three-dimensional structure of human milk lactoferrin, a member of the transferrin family, has been determined crystallographically at 3.2- angstrom resolution. The molecule has two-fold internal homology. The N- and C-terminal halves form two separate globular lobes, connected by a short α -helix, and carry one iron-binding site each. Each lobe has the same folding, based on two domains of similar supersecondary structure, with the iron site at the domain interface. Each iron atom is coordinated by four protein ligands: two tyrosines, one histidine, and one aspartate. A probable CO32- (or HCO3-) ion is suggested by the electron density, bound to iron and adjacent to an arginine side chain and a helix N terminus. The protein folding and location of the binding sites show marked similarities with those of other binding proteins, notably the sulfate-binding protein from Salmonella typhimurium.
Structure, function and flexibility of human lactoferrin
International journal of …, 1991
X-ray structure analyses of/bur different jbrms of human lactoferrin ( diferric, dieupric, an oxalate-substituted dicupric, and apo-lactoJerrin ), and o[" bovine di/erric lactoferrin, have revealed various ways in which the protein structure adapts to different structural and Junctional states. Comparison of diferric and dicupric lactoferrins has shown that different metals can, through slight variations in the metal position, have difJerent stereochemistries and anion coordination without an)' significant change in the protein structure. Substitution of oxalate Jor carbonate, as seen in the structure of a hybrid dicupric complex with oxalate in one site and carbonate in the other, shows that larger anions can be accommodated by small side-chain movements in the binding site. The multidomain nature of lactoJerrin also allows rigid body movements. Comparison of human and bovine lactoJerrins, and of these with rabbit serum transJerrin, shows that the relative orientations 0[ the two lobes in each molecule can var.v ; these variations may contribute to differences in their binding properties. The structure of apo-lactojerrin demonstrates the importance of large-scale domain movements .for metal binding and release and suggests that in solution an equilibrium exists between open and closed forms, with the open.[brm being the active binding species. These structural jorms are shown to be similar to those seen for bacterial periplasmie binding proteins, and lead to a common model Jbr the various steps in the binding process.
FEBS Journal, 2014
Bovine lactoferrin, a 76-kDa glycoprotein (Ala1-Arg689) consists of two similar N-and C-terminal molecular halves with the ability to bind two Fe 3+ ions. The N-terminal half, designated as the N-lobe (Ala1-Arg341) and the C-terminal half designated as the C-lobe (Tyr342-Arg689) have similar iron-binding properties, but the resistant C-lobe prolongs the physiological role of bovine lactoferrin in the digestive tract. Here, we report the crystal structure of true C-lobe, which was produced by limited proteolysis of bovine lactoferrin using trypsin. In the first proteolysis step, two fragments of 21 kDa (Glu86-Lys282) and 45 kDa (Ser283-Arg689) were generated because two lysine residues, Lys85 and Lys282, in the structure of iron-saturated bovine lactoferrin were fully exposed. The 45-kDa fragment was further digested at the newly exposed side chain of Arg341, generating a 38-kDa perfect C-lobe (Tyr342-Arg689). By contrast, the apo-lactoferrin was cut by trypsin only at Arg341, which was exposed in the structure of apo-lactoferrin, whereas the other two sites with Lys85 and Lys282 are inaccessible. The purified iron-saturated C-lobe was crystallized at pH 4.0. The structure was determined by the molecular replacement method using coordinates of the C-terminal half (Arg342-Arg689) of intact camel apolactoferrin. The structure determination revealed that the iron atom was absent and the iron-binding cleft was found in a wide-open conformation, whereas in the previously determined structure of iron-saturated C-lobe of bovine lactoferrin, the iron atom was present and the iron-binding site was in the closed confirmation.
Three-dimensional structure of diferric bovine lactoferrin at 2.8 Å resolution
Journal of Molecular Biology, 1997
The three-dimensional structure of diferric bovine lactoferrin (bLf) has been determined by X-ray crystallography in order to investigate the factors that in¯uence iron binding and release by transferrins. The structure was solved by molecular replacement, using the coordinates of diferric human lactoferrin (hLf) as a search model, and was re®ned with data to 2.8 A Ê resolution by simulated annealing (X-PLOR) and restrained least squares (TNT). The ®nal model comprises 5310 protein atoms (residues 5 to 689), 124 carbohydrate atoms (from ten monosaccharide units, in three glycan chains), 2 Fe 3 , 2 CO 3 2À and 50 water molecules. This model gives an R-factor of 0.232 for 21440 re¯ections in the resolution range 30.0 to 2.8 A Ê . The folding of the bLf molecule is essentially the same as that of hLf, but bLf differs in the extent of closure of the two domains of each lobe, and in the relative orientations of the two lobes. Differences in domain closure are attributed to amino acid changes in the interface, and differences in lobe orientations to slightly altered packing of two hydrophobic patches between the lobes. Changed interdomain interactions may explain the lesser iron af®nity of bLf, compared with hLf, and two lysine residues behind the N-lobe iron site of bLf offer new insights into thè`d ilysine trigger'' mechanism proposed for iron release by transferrins. The bLf structure is also notable for several well-de®ned oligosaccharide units which demonstrate the structural factors that stabilise carbohydrate structure. One glycan chain, attached to Asn545, appears to contribute to interdomain interactions and may modulate iron release from the C-lobe.
AN INVESTIGATION OF THE PROTONATION STATES OF HUMAN LACTOFERRIN IRON-BINDING PROTEIN
In this study, the protonation states of ionizable groups of human lactoferrin in various conformations were investigated theoretically, at physiological pH (7.365). These calculations show that the transition of the protein from a conformation to another one is accompanied by changes in the protonation state of specific amino acid residues. Analysis of the pK a calculatons underlined the importance of participation of two arginines and one lysine in the opening / closing of the protein. In addition, it was found that the mechanism of iron release depends on the protonation state of TYR-192. Protonated state of this residue in the closed form of lactoferrin will trigger the opening of protein and release of iron ions.