A conserved asparagine residue stabilizes iron binding in Manduca sexta transferrin-1

Abstract

Transferrin 1 (Tsf1) is an insect-specific iron-binding protein that is abundant in hemolymph and other extracellular fluids. It binds iron tightly at neutral pH and releases iron under acidic conditions. Tsf1 influences the distribution of iron in the body and protects against infection. Elucidating the mechanisms by which Tsf1 achieves these functions will require an understanding of how Tsf1 binds and releases iron. Previously, crystallized Tsf1 from Manduca sexta was shown to have a novel type of iron coordination that involves four iron-binding ligands: two tyrosine residues (Tyr90 and Tyr204), a buried carbonate anion, and a solvent-exposed carbonate anion. The solvent-exposed carbonate anion was bound by a single amino acid residue, a highly conserved asparagine at position 121 (Asn121); thus, we predicted that Asn121 would be essential for high-affinity iron binding. To test this hypothesis, we analyzed the iron-binding and -release properties of five forms of recombinant Tsf1: wild-type, a Y90F/Y204F double mutant (negative control), and three Asn121 mutants (N121A, N121D and N121S). Each of the Asn121 mutants exhibited altered spectral properties, confirming that Asn121 contributes to iron coordination. The N121D and N121S mutations resulted in slightly lower affinity for iron, especially at acidic pH, while iron binding and release by the N121A mutant was indistinguishable from that of the wild-type protein. The surprisingly minor consequences of mutating Asn121, despite its high degree of conservation in diverse insect species, suggest that Asn121 may play a role that is essential in vivo but non-essential for high affinity iron binding in vitro.

Keywords: Carbonate; Hemolymph; Insect; Iron; Mutant; Transferrin.

Figure 1.. Asn121 forms hydrogen bonds with the solvent-exposed carbonate anion and Tyr338.

Hydrogen bonds are shown as dashed lines, and distances are in angstroms. The two iron-ligating tyrosines, buried carbonate anion, and Fe³⁺ are also shown. Both carbonate anions function as bidentate iron ligands. The Tyr338-Asn121 bond is an N1-N2 intralobal interaction.

Figure 2.. Iron-binding region of models of Asn121 mutants superimposed on the crystal structure of holo-MsTsf1.

The PyMOL Molecular Graphics System was used to superimpose the top-scoring AlphaFold2 model of each Asn121 mutant on the crystal structure of holo-MsTsf1. The conformations adopted by the position 121 side chains (alanine, aspartate and serine) closely match the conformation of the Asn121 side chain in the crystal structure, and no significant changes in the predicted structures of the iron-binding region of the mutants were observed.

Figure 3.. Purified WT and mutant forms of MsTsf1.

Purified WT and mutant forms of MsTsf1 were analyzed by reducing SDS-PAGE followed by Coomassie staining. The molecular mass standards (in kDa) are shown to the left. The expected mass of MsTsf1 is 73 kDa. Few contaminating protein bands were visible.

Figure 4.. Far-UV CD spectra of the apo- and holo-forms of WT and mutant forms of MsTsf1.

A) The CD spectra of the apo-proteins. B) The CD spectra of the holo-proteins. WT MsTsf1 is shown in black, N121A in red, N121D in blue, N121S in green, and Y90F/Y204F in orange. Proteins were in a buffer containing 10 mM HEPES, 20 mM sodium bicarbonate, pH 7.4, and a buffer blank was subtracted from each protein spectrum. All spectra were normalized to the protein concentration by converting the units to molar ellipticity (mdeg•cm²/dmol). Only the holo-form of the N121S mutant had a CD spectrum appreciably different from the WT protein.

Figure 5.. Difference spectra of the LMCT peak for WT and mutant forms of MsTsf1.

WT MsTsf1 is shown in black, N121A in red, N121D in blue, N121S in green, and Y90F/Y204F in orange. Proteins were at approximately 2 mg/mL in 10 mM HEPES, 20 mM sodium bicarbonate, pH 7.4. To obtain each difference spectrum, the apo-protein spectrum was subtracted from the holo-protein spectrum. Only the Y90F/Y204F mutant lacked an LMCT peak, but the LMCT λ_max of each Asn121 mutant differed from that of the WT protein.

Figure 6.. The effect of pH on iron binding.

WT MsTsf1 is shown in black, N121A in red, N121D in blue, and N121S in green. Holo-proteins at 5 mg/mL were dialyzed against various buffers with a pH range of 4 to 8. The percent iron saturation was calculated by measuring the absorbance change in the LMCT λ_max before and after dialysis. A sigmoidal curve was fitted to the data using GraphPad Prism software. The effect of pH was identical for the WT protein and the N121A mutant, whereas the pH₅₀ for the N121D and N121S mutants were higher. (Iron release by WT MsTsf1 and the N121A mutant could not be measured at pH 4 because they precipitated at this pH.)