Two High-Resolution Crystal Structures of the Recombinant N-Lobe of Human Transferrin Reveal a Structural Change Implicated in Iron Release^,

Abstract

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.

Transferrins, the mechanism of iron release by ovotransferrin

“…Nevertheless, in both proteins, proton-assisted carbonate loss seems to be the indispensable trigger for iron release [13]. This is confirmed by an X±ray diffraction study on the N-site of serum-transferrin [14]. With serum-transferrin, the release of the metal from the N-site involves a single slow proton-transfer reaction which we ascribed to an acid-base reaction occurring on the Hist ligand (Eqn 2) and probably controlled by the change of conformation from close to open occurring upon iron loss [12,16].…”

“…is too fast to be analysed and, the rates and amplitudes of all the other observed kinetic processes are independent of the CO 2 partial pressures. It was, therefore, assumed that this fast phenomenon may describe protonassisted decarbonation of the protein in acidic media, because this decarbonation is a prerequisite for iron release [13,14]. The reciprocal relaxation time equations associated with Eqn (10), the second kinetic process of Fig.…”

Anion-independent Iron Coordination by the Campylobacter jejuni Ferric Binding Protein

“…Carbonate is synergistic for iron binding to transferrin (33), and a low pH crystal structure suggests a role for bound carbonate in the initiation of iron release (34). In Class I FbpAs (hFbpA and nFbpA), phosphate and the two conserved Tyr residues form an iron half-site that is proposed to bind iron before forming a closed holo structure (32).…”

The low pKa value of iron‐binding ligand Tyr188 and its implication in iron release and anion binding of human transferrin

“…Iron in the ferric form is bound to hTF in plasma at neutral pH and transported into cells where it is then released to an unknown chelator in the acidic environment of the endosome. A large conformational change between the apo-and iron-bound forms of the N-lobe of hTF (hTF/2N) has been demonstrated by X-ray crystallographic analysis [4,5]. When iron is released, the two domains of this lobe rotate 63°around a central hinge leading to an 'open' conformation.…”