2 HIGH-RESOLUTION CRYSTAL-STRUCTURES OF THE RECOMBINANT N-LOBE OF HUMAN TRANSFERRIN REVEAL A STRUCTURAL-CHANGE IMPLICATED IN IRON RELEASE

The N-lobe of human serum transferrin (hTF/2N) has been expressed in b aby hamster kidney cells and crystallized in both orthorhombic (P2(1)2 (1)2(1)) and tetragonal (P4(1)2(1)2) space groups. Both crystal forms diffract to high resolution (1.6 and 1.8 Angstrom, respectively) and h ave been solved by molecular replacement. Subsequent refinement result ed in final models for the structure of hTF/2N that had crystallograph ic 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 simi lar to those of other transferrins, including the presence of a deep c left that contains the metal-binding site. In contrast to other transf errins, both crystal forms of hTF/2N display disorder at the iron-bind ing site; model building suggests that this disorder consists of alter native conformations of the synergistically bound carbonate anior., th e side chain for Arg-124, and several solvent molecules. Subsequent re finement revealed that conformation A has an occupancy of 0.63-0.65 an d 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 relativ e to the iron and the side chain for Arg-124 has moved to accommodate the new carbonate position. Several water molecules appear to stabiliz e the carbonate anion in the two conformations. These structures are c onsistent 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.