A Biochemical and Structural Look into the Functional Role of Transferrin in D. melanogaster

Abstract

Transferrins (TFs) are a family of vertebrate and invertebrate proteins that have high affinity for ferric iron. The extensively studied mammalian TFs are typically bilobal in structure and have iron binding sites in both their lobes—critical to their essential role in proper iron sequestration and transport in blood. These sites coordinate iron binding through four conserved residues—aspartate, tyrosine, tyrosine and histidine—and a carbonate ion cofactor. Aside from their function of transporting iron to cells, their ability to sequester and tightly bind iron is a crucial defense against invading pathogens. Many insect TFs show less sequence conservation and have only one iron binding site. They have not been adequately studied for their roles in iron metabolism and immunity. Our research aims to determine the function(s) of insect TF via biochemical and structural analysis using Drosophila melanogaster transferrin (DmTsf1) as a model. We purified DmTsf1 from a baculovirus expression system using ammonium sulfate precipitation, and ion exchange and gel filtration chromatography, and generated apo‐ and holo‐forms. Apo‐DmTsf1 but not holo‐DmTsf1 inhibited Escherichia coli growth, suggesting an immune function that relies on iron sequestration. A homology model of DmTsf1 showed a similar bilobal structure but iron binding in only one lobe. The putative binding residues are glutamate, tyrosine, and tyrosine. If the model is correct, a loss of the histidine residue in DmTsf1 should result in a lower binding affinity for iron. We are using crystallography to identify the actual iron binding residues. Initial protein‐ligand binding kinetics using equilibrium dialysis have shown Apo‐DmTsf1 to indeed having one binding site for ferric iron and a lower Kd of 10−18. Further biochemical pursuits into the role of insect TFs will include examining DmTsf1's ability to bind other metals.Support or Funding InformationNIH NGMS R37 GM041247, NSF IOS 1656388This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.