Insights into the function and three-dimensional structure of the human heme exporter and viral receptor FLVCR1a

Heme is a fundamental cofactor for a number of hemoproteins. Nevertheless, heme is also highly cytotoxic. As so, its homeostasis must be stringently regulated in order to permit optimal cell functionality. While it has been believed for a long time that heme regulation is mainly based on its biosynthesis and degradation, in recent years it was unraveled that heme transport plays a crucial role as well. The Feline Leukemia Virus sub-group C Receptor 1 isoform a (FLVCR1a) is a heme exporter localized on the plasma membrane of a wide number of cell types. As the name suggests, FLVCR1a plays a role also as a viral receptor for the Feline Leukemia Virus sub-group C, which has been found to cause pure red blood cell anemia in viremic cats. Despite its numerous roles both physiologically and as a viral receptor, little is known about FLVCR1a. Following the clues given by the symptoms of FeLV-infected cats, given by heme overload in erythroblasts, the role of FLVCR1a in heme export has been discovered. Additionally, FLVCR1a displays affinity also for heme precursors, adding further complexity layers to the cellular function of this exporter. Given its insolubility in aqueous solution, it is possible that cytoplasmic carriers are needed to deliver heme to the transporter. Extracellular carriers have been identified, hemopexin and albumin both necessary for heme export activity via FLVCR1a. Based on its sequence, FLVCR1a has been classified as a member of the Major Facilitator Superfamily of transporter trafficking small solutes across cell membranes. Nevertheless, further information with regards to its structure and mode of transport is lacking. Finally, a characterization of the interaction between FLVCR1a and the surface protein (SU) of FeLV is still absent as well. In this thesis, the focus was directed towards all these aspects. First, FLVCR1a was recombinantly expressed, purified, and investigated through cryo-electron microscopy in an effort to characterize its 3D structure. Moreover, its topology and conformational transitions have been investigated through Hydrogen Deuterium Exchange. Second, attempting to better characterize the interactions of FLVCR1 with heme and coproporphyrin, as well as with the viral SU, Microscale Thermophoresis was employed, together with the evaluation of the thermal stability of such complexes. Although further investigations still need to be conducted, interesting insights in FLVCR1a export function and 3D structure were reached, hopefully contributing to unravel the delicate, yet complex mechanisms of cellular heme trafficking and viral infection.