Dataset Information

Iron release from recombinant N-lobe and single point Asp63 mutants of human transferrin by EDTA.

ABSTRACT: Transferrins bind ferric ion and deliver the iron to cells. The mechanism of the iron release has been studied kinetically, in vitro, with the aid of single point mutants in which the iron-binding ligand, Asp63 (aspartic acid-63, D63), has been changed to Ser, Asn, Glu and Ala. Iron release from the unmutated N-lobe of human serum transferrin (hTF/2N) by EDTA is influenced by a variety of factors. The rate-determining conformational-change mechanism may be a major pathway for iron release from hTF/2N's having a 'closed' conformation, which leads to a saturation kinetic mode with respect to ligand concentration. The effect of chloride depends on the protein conformation, showing a negative action in the case of tight binding and a positive action when the protein has an 'open' or 'loose' conformation. The negative effect of chloride could originate from the binding competition between chloride and the chelate to the active site for iron release, and the positive effect could derive from the synergistic participation of chloride in iron removal. The 'open' conformation may be induced by decreasing pH: the transitional point appears to be at about pH 6.3 for the wild-type hTF/2N; the 'loose' conformation may be facilitated by mutations at D63, which result in the loss of a key linking component in interdomain interactions of the protein. In the latter case, structural factors dominate over other potential negative effects because the weak interdomain contacts derived from the mutation of D63 cause the binding site to open easily, even at pH 7.4. Therefore chloride exhibits an accelerating action on iron release by EDTA from all the D63 mutants.

SUBMITTER: He QY

PROVIDER: S-EPMC1218939 | biostudies-other | 1997 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:The major function of human transferrin is to deliver iron from the bloodstream to actively dividing cells. Upon iron release, the protein changes its conformation from 'closed' to 'open'. Extensive studies in vitro indicate that iron release from transferrin is very complex and involves many factors, including pH, the chelator used, an anion effect, temperature, receptor binding and intra-lobe interactions. Our earlier work [He, Mason and Woodworth (1997) Biochem. J. 328, 439-445] using the isolated transferrin N-lobe (recombinant N-lobe of human transferrin comprising residues 1-337; hTF/2N) has shown that anions and pH modulate iron release from hTF/2N in an interdependent manner: chloride retards iron release at neutral pH, but accelerates the reaction at acidic pH. The present study supports this idea and further details the nature of the dual effect of chloride: the anion effect on iron release is closely related to the strength of anion binding to the apoprotein. The negative effect seems to originate from competition between chloride and the chelator for an anion-binding site(s) near the metal centre. With decreasing pH, the strength of anion binding to hTF/2N increases linearly, decreasing the contribution of competition with the chelator. In the meantime, the 'open' or 'loose' conformation of hTF/2N, induced by the protonation of critical residues such as the Lys-206/Lys-296 pair at low pH, enables chloride to enter the cleft and bind to exposed side chains, thereby promoting cleft opening and synergistically allowing removal of iron by the chelator, leading to a positive anion effect. Disabling one or more of the primary anion-binding residues, namely Arg-124, Lys-206 and Lys-296, substantially decreases the anion-binding ability of the resulting mutant proteins. In these cases, the competition for the remaining binding residue(s) is increased, leading to a negative chloride effect or, at most, a very small positive effect, even at low pH.

Dataset Information

Iron release from recombinant N-lobe and single point Asp63 mutants of human transferrin by EDTA.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets