The N-lobe of human serum transferrin (hTF/2N) has been expressed in baby hamster kidney cells and crystallized in both orthorhombic (P212121) and tetragonal (P41212) space groups. Both crystal forms diffract to high resolution (1.6 and 1.8 A, respectively) and have been solved by molecular replacement. Subsequent refinement resulted in final models for the structure of hTF/2N that had crystallographic R-factors of 18.1 and 19.7% for the two crystal forms, respectively; these models represent the highest-resolution transferrin structures determined to date. The hTF/2N polypeptide has a folding pattern similar to those of other transferrins, including the presence of a deep cleft that contains the metal-binding site. In contrast to other transferrins, both crystal forms of hTF/2N display disorder at the iron-binding site; model building suggests that this disorder consists of alternative conformations of the synergistically bound carbonate anion, the side chain for Arg-124, and several solvent molecules. Subsequent refinement revealed that conformation A has an occupancy of 0.63-0. 65 and corresponds to the structure of the iron-binding site found in other transferrins. The alternative conformation B has an occupancy of 0.35-0.37; in this structure, the carbonate has rotated 30 degrees relative to the iron and the side chain for Arg-124 has moved to accommodate the new carbonate position. Several water molecules appear to stabilize the carbonate anion in the two conformations. These structures are consistent with the protonation of the carbonate and resulting partial removal of the anion from the metal; these events would occur prior to cleft opening and metal release.

No abstract

Proteins of the transferrin family, which contains serum transferrin and lactoferrin, control iron levels in higher animals through their very tight (Kapp approximately 10(20)) but reversible binding of iron. These bilobate molecules have two binding sites, one per lobe, each housing one Fe3+ and the synergistic CO3(2-) ion. Crystallographic studies of human lactoferrin and rabbit serum transferrin in their iron-bound forms have characterized their binding sites and protein structure. Physical studies show that a substantial conformational change accompanies iron binding and release. We have addressed this phenomenon through crystal structure analysis of human apolactoferrin at 2.8 A resolution. In this structure the N-lobe binding cleft is wide open, following a domain rotation of 53 degrees, mediated by the pivoting of two helices and flexing of two interdomain polypeptide strands. Remarkably, the C-lobe cleft is closed, but unliganded. These observations have implications for transferrin function and for binding proteins in general.