Mutagenesis of the Histidine Ligand in Human Lactoferrin:  Iron Binding Properties and Crystal Structure of the Histidine-253 → Methionine Mutant^,

Abstract

The contribution of the conserved His ligand to iron binding in transferrins has been addressed by site-directed mutagenesis and X-ray crystallographic analysis. His 253 in the N-terminal half-molecule of human lactoferrin, LfN (residues 1-333), has been changed to Gly, Ala, Pro, Thr, Leu, Phe, Met, Tyr, Glu, Gln, and Cys by oligonucleotide-directed mutagenesis. The proteins have been expressed in baby hamster kidney cells, at high levels, and purified. The results show that the His ligand is essential for the stability of the iron binding site. All of the substitutions destabilized iron binding irrespective of whether the replacements were potential iron ligands or not. Iron was lost below pH approximately 6 for the Cys, Glu, and Tyr mutants and below pH 7 or higher for the others, compared with pH 5.0 for LfN. The destabilization is attributed to both steric and electronic effects. The importance of electronic effects has been shown by the crystal structure of the H253M mutant, which has been determined at an effective resolution of 2.5 A and refined to a final R factor of 0.173. The iron atom is changed from six-coordinate to five-coordinate; the Met 253 side chain is not bound to iron even though there appears to be no steric barrier. This is attributed to the poorer affinity of the thioether ligand for Fe(III) compared with imidazole nitrogen. The decreased stability of the iron binding is attributed solely to the loss of the His ligand as the protein conformation and interdomain interactions are unchanged.

Lactoferrin

“…It comprises four protein ligands (2 Tyr, 1 Asp, 1 His) that provide three negative charges to balance the 3+ charge of Fe 3+ , together with a helix N-terminus and Arg sidechain whose positive charge balances the negative charge on the CO 3 2-anion. Mutagenesis experiments on the Asp, His and Tyr ligands [35][36][37] show that any change is strongly deleterious to iron binding, as is mutation of the Arg residue that helps bind the CO 3 2-anion [38]. That shown here for the N-lobe of human Lf.…”

Circulating Retinol-Binding Protein-4 Concentration Might Reflect Insulin Resistance–Associated Iron Overload

“…In addition to increased urinary excretion of RBP4, fenretide also leads to decreased total hemoglobin and anemia (13), hinting at iron-induced depletion as another potential mechanism through which fenretide could improve insulin sensitivity. Furthermore, lactoferrin, a well-known iron transport protein and iron donor (24), led to significant RBP4 release in a dosedependent manner and to increased RBP4 expression in adipose tissue explants. Our findings imply that iron supplementation leads to enhanced RBP4 release from cells due to enhanced transcription.…”

Iron‐Binding Properties of TTf, a Salt‐Induced Transferrin from the AlgaDunaliella salina

“…Specifically, Asp-63 is replaced by Gly in all three lobes, and His-249 is replaced by Asn and Gln in a1, a2, and in the b lobe, respectively. While Asn and Gln can serve as weak iron ligands, [18,19] Gly cannot fulfill this function. The importance of the conservation of Fe liganding residues was demonstrated in the Neissria Fbp iron binding protein which has little sequence similarity to Tfs but shares with them a similar set of iron binding residues.…”