Structure of human lactoferrin at 3.2-A resolution.

Abstract

The three-dimensional structure of human milk lactoferrin, a member of the transferrin family, has been determined crystallographically at 3.2-resolution. The molecule has two-fold internal homology. The N-and C-terminal halves form two separate globular lobes, connected by a short a-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 CO2-(or HCO5) 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 sulfatebinding protein from Salmonella typhimurium.Lactoferrin [also known as lactotransferrin (1)] is a member of the family of iron-binding proteins that also includes transferrin and ovotransferrin (2,3). These proteins are widely distributed in the physiological fluids of vertebrates. Although they have been the subject of intensive investigation over many years no definitive three-dimensional structural information has hitherto been available. All are monomeric glycoproteins with =700 amino acid residues and molecular weight -80,000. Each binds reversibly two iron atoms (as Fe3"), concomitantly with two C02- (or HCO ) ions. Notable features of their binding properties are (i) the synergistic relationship between cation and anion binding (4), (ii) the extremely tight binding of iron (binding constant 1020 for lactoferrin), and (iii) the fact that this tightly bound iron is nevertheless available in vivo, apparently through binding at specific receptors (5, 6).The known or proposed biological functions ofthe transferrins depend on their iron-binding properties. Thus serum transferrin, the iron transport protein in plasma, provides an iron source for hemoglobin synthesis and other metabolic requirements. Lactoferrin, widely distributed through many exocrine secretions, notably milk, and an important component of leukocytes, has strong bacteriostatic properties (7). These result from its avidity for iron, depriving bacteria of iron essential for growth. It may also protect cells from free radical damage by binding potentially catalytic free iron (8).All three proteins have bilobal structures. This is indicated by (i) fragmentation studies (ref. 9 and references therein), which demonstrate that the polypeptide chain can be cleaved into two halves, each carrying one iron site, and (ii) lowresolution x-ray studies (10). Amino acid sequence alignments (11) show that, in addition to the extensive homology between different transferring, each also shows strong twofold internal homology, indicative of gene duplication from a one-iron 40,000 molecular weight precursor molecule. For lactoferrin, there is =40% sequence identity between its Nand C-terminal halves.Here we report the resu...

The N‐terminal domain I of human lactotransferrin binds specifically to phytohemagglutinin‐stimulated peripheral blood human lymphocyte receptors

“…These observations point out the importance of the protein conformation to lymphocyte receptor binding. Moreover, the lactotransferrin recognition site seems to be located precisely in the N-terminal domain I (residues 4-90 and 258-281) described by Anderson et al [7] because the N2-glycopeptide (residues 91-257), corresponding to the N-terminal domain II, was not bound to the lymphocyte receptor. Up to now, the Nterminal domain I has not been isolated because of its degradation during tryptic hydrolysis.…”

“…Lactotransferrin, also called lactoferrin, serotransferrin and ovotransferrin are bilobed ironbinding glycoproteins of the transferrin class whose primary structure [1-3] and threedimensional conformation, as revealed by partial proteolytic hydrolysis studies [4-61 and X-ray diffraction data [7,8], are very similar. However, these glycoproteins differ in the number, the structure and the location of their glycans [9][10][11][12].…”

“…Both 30 kDa N-and 50 kDa C-tryptic fragments were re-associated into a non-covalent N,C-tryptic complex [18]. In addition; an iron-binding 20 kDa glycopeptide (residues 91-257) called N2-glycopeptide, which corresponds to the N-terminal domain II of human lactotransferrin described by Anderson et al [7], was prepared from tryptic hydrolysate of the 30 kDa N-tryptic fragment [6].…”

Mutagenesis of the aspartic acid ligands in human serum transferrin: lobe–lobe interaction and conformation as revealed by antibody, receptor-binding and iron-release studies

“…The cleft is formed by two domains, N-I and N-II in the N-lobe and C-I and C-II in the Clobe. In human lactoferrin (hLF), human serum transferrin (hTF), rabbit serum transferrin (rTF) and chicken ovotransferrin (oTF), crystallographic data show that each ferric ion is directly coordinated to the side chains of two tyrosine residues, one histidine residue, one aspartic acid residue and two oxygens from the synergistic carbonate anion [3][4][5].…”

Human melanotransferrin (p97) has only one functional iron‐binding site

“…It is membrane-bound, and it differs in certain key amino acid residues which in other transferrins are involved in iron binding. Examination of the MTf sequence in relation td the three-dimenqional structures of human lactoferrin [7,83 and rabbit serum transferrin [9] has Ied to the proposal that it has an intact transferrin-type iron binding site in its N-terminal half but a possibly defective site in its C-terminal half [lo]. We have tested this proposal by investigation of the iron-binding properties of MTf, and discuss the results in the light of its presumed three-dimensional structure.…”