Interlobe Communication in ¹³C-Methionine-Labeled Human Transferrin

Abstract

[1H, 13C] NMR investigations of metal-induced conformational changes in the blood serum protein transferrin (80 kDa) are reported. These are thought to play an important role in the recognition of this protein by its cellular receptors. [1H, 13C] NMR resonance assignments are presented for all nine methionine 13CH3 groups of recombinant deglycosylated human transferrin on the basis of studies of recombinant N-lobe (40 kDa, five Met residues), NOESY-relayed [1H, 13C] HMQC spectra, and structural considerations. The first specific assignments for C-lobe resonances of transferrin are presented. Using methionine 13CH3 resonances as probes, it is shown that, with oxalate as the synergistic anion, Ga3+ binds preferentially to the C-lobe and subsequently to the N-lobe. The NMR shifts of Met464, which is in the Trp460-centered hydrophobic patch of helix 5 in the C-lobe in contact with the anion and metal binding site, show that Ga3+ binding causes movement of side chains within this helix, as is also the case in the N-lobe. The C-lobe residue Met382, which contacts the N-lobe hinge region, is perturbed when Ga3+ binds to the N-lobe, indicative of interlobe communication, a feature which may control the recognition of fully-metallated transferrin by its receptor. These results demonstrate that selective 13C labeling is a powerful method for probing the structure and dynamics of high-molecular-mass proteins.

Mutagenesis of the aspartic acid ligands in human serum transferrin: lobe–lobe interaction and conformation as revealed by antibody, receptor-binding and iron-release studies

“…In recent studies, microcalorimetry of hTF, for example, shows that iron binds preferentially to the C-lobe which raises the T m of the N-lobe 4 mC. Lobe-lobe interaction has been shown in NMR experiments of hTF in which the methionine residues were labelled with "$C [31]. Titration calorimetry has been used to measure the interaction between the isolated N-and C-lobes of hTF [17] and of oTF [32], produced by recombinant techniques and\or by proteolytic cleavage.…”

The Crystal Structure of Iron-free Human Serum Transferrin Provides Insight into Inter-lobe Communication and Receptor Binding

“…Interactions between Lobes-A cooperative effect of one lobe on the other lobe has been convincingly documented in a number of studies using a variety of techniques, which include NMR of hTF (58), absorption spectra of oTF (59), pH-dependent iron release of LTF (60), calorimetric studies of both hTF and oTF (61-63), electrospray ionization mass spectrometry studies of hTF (43), iron release from monoferric LTF (64), analysis by urea gels (22), and chemical relaxation studies (65)(66)(67). Significantly, a number of these studies specifically attribute cooperative effects between the two lobes of hTF to participation of helix 12 from the C-lobe (2,68).…”

Characterisation of Cisplatin Binding Sites in Human Serum Proteins Using Hyphenated Multidimensional Liquid Chromatography and ESI Tandem Mass Spectrometry

“…This fritless 100 mm capillary was packed with 12 cm of Eclipse XDB C 18 and a column flow rate of 0.12 mL min À1 and spray voltage of 1.8 kV were used. Only buffer solutions A and B were required for the reversed-phase chromatographic separation.…”

“…The same group has also reported an NMR spectroscopic study of cisplatin binding to human Trfe, [17] in which they observed that only the 13 C resonances that were previously assigned [18] to the d-CH 3 groups of M256 and M499 were influenced by adduct formation. In contrast, the T457 was established as a specific cisplatin binding site by Dyson et al [19,20] Cisplatin binding sites in human serum proteins have been characterised by using combined multidimensional liquid chromatography and ESI tandem mass spectrometry (MudPIT).…”