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.
Serum transferrin binds ferric ions in the bloodstream and transports them to cells, where they are released in a process involving receptor-mediated endocytosis. Iron release is believed to be pH dependent and is coupled with a large conformational change. To help define the steps in iron release, we have determined the three-dimensional structure of the iron-free (apo) form of the recombinant N-lobe half-molecule of human serum transferrin (ApoTfN) by X-ray crystallography. Two crystal forms were obtained, form 1 with four molecules in the asymmetric unit and form 2 with two molecules in the asymmetric unit. The structures of both forms were determined by molecular replacement and were refined at 2.2 and 3.2 A resolution, respectively. Final R-factors were 0.203 (free R = 0. 292) for form 1 and 0.217 (free R = 0.312) for form 2. All six copies of the ApoTfN structure are essentially identical. Comparison with the holo form (FeTfN) shows that a large rigid-body domain movement of 63 degrees has occurred in ApoTfN, to give an open binding cleft. The extent of domain opening is the same as in the N-lobe of human lactoferrin, showing that it depends on internal constraints that are conserved in both proteins, and that it is unaffected by the presence or absence of the C-lobe. Although the conformational change is primarily a rigid-body motion, several local adjustments occur. In particular, two iron ligands, Asp 63 and His 249, change conformation to form salt bridges, with Lys 296 and Glu 83, respectively, in the binding cleft of the apo protein. Both salt bridges would have to break for iron coordination to occur. Most importantly, the structure, determined at a pH (5.3) that is close to the pH of physiological iron release, indicates that protonation of His 249 is a key step in iron release.
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