Competition between Al3+ and Fe3+ binding to human transferrin and toxicological implications: structural investigations using ultra-high resolution ESI MS and CD spectroscopy

Abstract

Human serum transferrin (hTF) is an iron binding protein with the primary task of ensuring well-controlled transport of Fe³⁺-ions in the bloodstream. Furthermore, hTF has been identified as a key component in the trafficking of Al³⁺-ions from the serum to cells. It is clear that binding alone does not guarantee cellular uptake via the transferrin receptor, since this is determined by the structural properties of the metal–protein complex. The conformation of the metallated hTF is critically important for delivery of Fe³⁺ or any other metal into the cell. The combination of ultra-high resolution ESI mass spectrometry and CD spectroscopy together provide accurate species distribution of the Fe³⁺ during stepwise addition to apo-hTF and an indirect indication of the tertiary structure of the metallated protein. These two methods together are extremely fine probes of structural changes as a function of precise metal binding status at micromolar concentrations. Simulation of the precise domain distribution could be determined during the stepwise metallation from 0 to 2 Fe³⁺ added. Analysis of the ESI-MS data for the stepwise metallation of apo-hTF and Al_1 or 2-hTF with Fe³⁺ was carried out and used to simulate the experimental speciation based on the reported K_F values. There are six main conclusions: (1) Fe³⁺ binds predominantly, initially to the C-lobe. (2) The CD spectral properties indicate that the C-lobe metallation dominates the structural properties of both binding sites; N-lobe metallation modifies the C-lobe structure. (3) Fe³⁺ metallation of the mixed Al_1–2-hTF results in the dominant form of Fe₁Al₁-hTF. (4) The first Fe³⁺ bound to Al₁-hTF binds predominantly in the C-lobe domain. (5) The CD spectral properties when Fe³⁺ binds to Al_1–2-hTF indicates that Al–N-lobe occupation mirrors the structural effects of N-lobe occupation by Fe³⁺. (6) With respect to how Al³⁺ might enter the cell, the formation of a hybrid form Al₁Fe₁-hTF might enable the Al³⁺ to enter the cell via receptor-mediated endocytosis due to the binding of Fe³⁺ in the C-lobe of the protein which is primarily responsible for the structure of the metal–protein complex.