Oxalate- and Ga(3+)-induced structural changes in human serum transferrin and its recombinant N-lobe. 1H NMR detection of preferential C-lobe Ga3+ binding.

(1) The binding of the synergistic anion oxalate and Ga3+ to human serum transferrin (HTF, 80 kDa) and its recombinant N-lobe (HTF/2N, 40 kDa) has been studied by one- and two-dimensional 1H NMR spectroscopy, at 310 K, pH*7.25. (2) Specific protein resonances are sensitive to oxalate binding (fast exchange on the NMR time scale) and allowed determination of the apparent binding constant for oxalate binding to the N-lobe (log K 4.04). (3) Slow exchange between apo-HTF and Ga-loaded HTF or HTF/2N was observed. Binding of Ga3+ appeared to be accompanied by small changes in the orientations of residues in hydrophobic pockets in the interdomain hinge region close to the metal binding site. (4) Under the conditions used, preferential binding of Ga3+ (added as Ga(NTA)2) to the C-lobe of HTF was observed. Binding to the C-lobe markedly perturbed resonances in the glycan N-acetyl region of the spectrum, suggesting that metal binding is communicated to the surface of the protein. This could be important in receptor recognition of metallotransferrins. (5) The displacement of Ga3+ from Ga-ox-HTF with Fe3+ was studied, and the paramagnetic broadening effects allowed identification of resonances from groups close to Fe3+. The passage of Fe3+ from the exterior to the interior of the protein was followed by 1H NMR spectroscopy, and the half-life for Ga(3+)-Fe3+ exchange was determined to be 4.3 h (310 K).