title: Cobalt and the iron acquisition pathway: competition towards interaction with receptor 1.
abstract: During iron acquisition by the cell, complete homodimeric transferrin receptor 1 in an unknown state (R1) binds iron-loaded human serum apotransferrin in an unknown state (T) and allows its internalization in the cytoplasm. T also forms complexes with metals other than iron. Are these metals incorporated by the iron acquisition pathway and how can other proteins interact with R1? We report here a four-step mechanism for cobalt(III) transfer from CoNtaCO(3)(2-) to T and analyze the interaction of cobalt-loaded transferrin with R1. The first step in cobalt uptake by T is a fast transfer of Co(3+) and CO(3)(2-) from CoNtaCO(3)(2-) to the metal-binding site in the C-lobe of T: direct rate constant, k(1)=(1.1+/-0.1) x 10(6) M(-1) s(-1); reverse rate constant, k(-1)=(1.9+/-0.6) x 10(6) M(-1) s(-1); and equilibrium constant, K=1.7+/-0.7. This step is followed by a proton-assisted conformational change of the C-lobe: direct rate constant, k(2)=(3+/-0.3) x 10(6) M(-1) s(-1); reverse rate constant, k(-2)=(1.6+/-0.3) x 10(-2) s(-1); and equilibrium constant, K(2a)=5.3+/-1.5 nM. The two final steps are slow changes in the conformation of the protein (0.5 h and 72 h), which allow it to achieve its final thermodynamic state and also to acquire second cobalt. The cobalt-saturated transferrin in an unknown state (TCo(2)) interacts with R1 in two different steps. The first is an ultra-fast interaction of the C-lobe of TCo(2) with the helical domain of R1: direct rate constant, k(3)=(4.4+/-0.6)x10(10) M(-1) s(-1); reverse rate constant, k(-3)=(3.6+/-0.6) x 10(4) s(-1); and dissociation constant, K(1d)=0.82+/-0.25 muM. The second is a very slow interaction of the N-lobe of TCo(2) with the protease-like domain of R1. This increases the stability of the protein-protein adduct by 30-fold with an average overall dissociation constant K(d)=25+/-10 nM. The main trigger in the R1-mediated iron acquisition is the ultra-fast interaction of the metal-loaded C-lobe of T with R1. This step is much faster than endocytosis, which in turn is much faster than the interaction of the N-lobe of T with the protease-like domain. This can explain why other metal-loaded transferrins or a protein such as HFE-with a lower affinity for R1 than iron-saturated transferrin but with, however, similar or higher affinities for the helical domain than the C-lobe-competes with iron-saturated transferrin in an unknown state towards interaction with R1.
authors.affiliation: Laboratoire ITODYS: Interfaces, Traitements et Organisation des Systèmes, Université Paris-Diderot, CNRS UMR 7086, 1 rue Guy de la Brosse, 75005 Paris, France.
authors.forename: Zohra
authors.initials: Z
authors.lastname: Chikh
authors.forename: Miryana
authors.initials: M
authors.lastname: Hémadi
authors.forename: Geneviève
authors.initials: G
authors.lastname: Miquel
authors.forename: Nguyêt-Thanh
authors.initials: NT
authors.lastname: Ha-Duong
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: El Hage Chahine
mesh_terms: D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001059:Apoproteins / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D003035:Cobalt / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D005260:Female; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010920:Placenta / Q000737:chemistry; D011247:Pregnancy; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011522:Protons; D011990:Receptors, Transferrin / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014168:Transferrin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D015703:Antigens, CD; D001059:Apoproteins; C068126:CD71 antigen; D004220:Disulfides; C103673:HFE protein, human; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins; D011522:Protons; D011990:Receptors, Transferrin; D014168:Transferrin; C028284:apotransferrin; D003035:Cobalt; D007501:Iron