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) × 10 6 M − 1 s − 1 ; reverse rate constant, k − 1 = (1.9 ± 0.6) × 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) × 10 6 M − 1 s − 1 ; reverse rate constant, k − 2 = (1.6 ± 0.3) × 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) × 10 10 M − 1 s − 1 ; reverse rate constant, k − 3 = (3.6 ± 0.6) × 10 4 s − 1 ; and dissociation constant, K 1d = 0.82 ± 0.25 μM. 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.