Sylvia V. Rumball

Transferrins: insights into structure and function from studies on lactoferrin

Abstract The recently determined three-dimensional structure of lactoferrin has thrown new light on aspects of transferrin structure and function, including the nature and location of the iron sites, structural models for iron binding and release, homologies within and between transferrins, and some unexpected similarities to bacterial binding proteins.

Three-Dimensional Structure of Lactoferrin in Various Functional States

The three-dimensional structures of various forms of lactoferrin, determined by high resolution crystallographic studies, have been compared in order to determine the relationship between structure and biological function. These comparisons include human apo and diferric lactoferrins, metal and anion substituted lactoferrins, the N-terminal half molecule of human lactoferrin, and bovine diferric lactoferrin. The structures themselves define the nature and location of the iron binding sites and allow anti-bacterial and putative receptor-binding regions to be mapped on to the molecular surface. The structural comparisons show that small internal adjustments can allow the accommodation of different metals and anions without altering the overall molecular structure, whereas large-scale conformational changes are associated with metal binding and release, and smaller, but significant, movements accompany species variations. The results also focus on differences in flexibility between the two lobes, and on the importance of interactions in the inter-lobe region in modulating iron release from the N-lobe and in possibly enabling binding at one site to be signalled to the other.

Structure, function and flexibility of human lactoferrin

X-ray structure analyses of four different forms of human lactoferrin (diferric, dicupric, an oxalate-substituted dicupric, and apo-lactoferrin), and of bovine diferric lactoferrin, have revealed various ways in which the protein structure adapts to different structural and functional states. Comparison of diferric and dicupric lactoferrins has shown that different metals can, through slight variations in the metal position, have different stereochemistries and anion coordination without any significant change in the protein structure. Substitution of oxalate for 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 lactoferrin also allows rigid body movements. Comparison of human and bovine lactoferrins, and of these with rabbit serum transferrin, shows that the relative orientations of the two lobes in each molecule can vary; these variations may contribute to differences in their binding properties. The structure of apo-lactoferrin 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 form being the active binding species. These structural forms are shown to be similar to those seen for bacterial periplasmic binding proteins, and lead to a common model for the various steps in the binding process.

Metal and anion binding sites in lactoferrin and related proteins

The metal and anion binding sites of the protein lactoferrin (Lf) have been defined through crystallographic analyses of FegLf and Cu2Lf. In both cases each metal ion is 6-coordinate, with four protein ligands (2 Tyr, 1 Asp, 1 His) and the synergistic C032- anion which binds as a bidentate ligand. The C032- fits into a pocket between the metal and the N-terminus of an a-helix. Binding of other metals and anions can be understood in terms of the protein structure and a suggested mechanism for binding and release. Differences between the two sites in Lf can also be explained. suggest links between bacterial and mammalian binding proteins. Striking similarities between Lf and a S042- binding protein

Purification and preliminary crystallographic studies on azurin and cytochrome c′ from Alcaligenes denitrificans and Alcaligenes sp. NCIB 11015

Purification and crystallisation procedures are reported for azurin and cytochrome c′ from Alcaligenes denitrificans and Alcaligenes sp. NCIB 11015. The azurin crystals from A. denitrificans are suitable for high-resolution X-ray structure analysis. They are orthorhombic, space group C2221 (with marked tetragonal pseudo-symmetry), cell dimensions a = 75.0 A, b = 74.1 A, c = 99.5 A, with two molecules per asymmetric unit. The cytochrome c′ crystals from both species are hexagonal, space group P6122 (or P6522), cell dimensions a = b = 54.7 A, c ~ 185 A, γ = 120 °, with one subunit (molecular weight 14,000) in the asymmetric unit.

Crystallographic data for human lactoferrin

Human lactoferrin, an iron-binding protein present in human milk, has been crystallized in a form suitable for X-ray diffraction studies. The space group is P212121, with a=155·5 A, b=97·3 A and c=55·5A. Dehydration and density measurements indicate a solvent content of about 42%, and a protein molecular weight, per asymmetric unit, of 85,000 to 87,500.

Preliminary crystallographic studies on bovine lactoferrin

The purification of bovine lactoferrin, its crystallization at low ionic strength, and preliminary X-ray crystallographic data are reported. The crystals, which grow from a two-phase system, are radiation-stable and suitable for a medium-resolution X-ray analysis. They are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 138.4 A, b = 87.1 A, c = 73.6 A, and one protein molecule in the asymmetric unit.

A Comparison of the Three-Dimensional Structures of Human Lactoferrin in its Iron Free and Iron Saturated Forms

The structures of human lactoferrin in its iron free1 (apoLf) and iron saturated2 (Fe2Lf) forms have been determined crystallographically at high resolution (2.0 and 2.2 A respectively). The root-mean-square errors in positions of the individual atoms are of the order of 0.2–0.3 A, which means that it is possible to make meaningful comparisons between the two structures with a view to understanding how iron binding and release are accomplished.

X-Ray Structural Studies of Lactoferrin

Knowledge of protein tertiary structure is fundamental to a proper understanding of function. Currently there is little detailed structural information available on human milk proteins. The structure of bovine milk β-lactoglobulin at 2.8 A has recently been determined and this has led to speculation about its function, but this protein is apparently not found in human milk. Preliminary crystallographic data on α-lactalbumin has been published but no high resolution structure has yet been forthcoming.