Choline is an essential dietary nutrient required for phosphatidylcholine synthesis— the most abundant lipid in cell membranes. Despite its metabolic importance, the mechanisms governing choline trafficking remain poorly understood. Recent studies identified FLVCR2 (Feline Leukemia Virus Subgroup C Receptor 2) as a high-affinity choline transporter at the blood–brain barrier1. While structural data have resolved its 12 transmembrane helices and transport mechanism, the ~100-residue N-terminal domain (NTD) remains unresolved due to its intrinsically disordered nature. This domain contains a conserved 5x His-Pro motif implicated in thermogenesis through heme binding2, yet the mechanism and functional consequences of this interaction are unknown. Further, zinc has also been shown to bind to His-Pro repeats, and recent literature further highlighted the first known zinc-heme mirror binding sites in cyanobacteria’s DRI (Domain Related to Iron)3.
To investigate heme and zinc binding to the FLVCR2 NTD, we first expressed and purified the isolated NTDs of WT and a HA mutant (in which all five histidine in the His–Pro motif are substituted with alanine) for detailed biophysical characterisation. Heme and zinc bound specifically to the WT NTD in a penta-coordinated, enthalpy-driven manner, while no binding was observed for the HA mutant. Circular dichroism spectroscopy further indicated that heme and zinc binding induces subtle compaction, shifting the WT NTD toward a pre-molten-globule–like state while remaining largely disordered. In parallel, we generated full-length WT-, HA-, and ΔNTD-FLVCR2 constructs for future experiments investigating if heme binding allosterically regulated FLVCR2 function. WT and HA variants were successfully expressed at the plasma membrane and purified to homogeneity, whereas the ΔNTD-FLVCR2 was mislocalised and aggregated.
These findings establish that the conserved HP motif within the FLVCR2 NTD mediates heme and zinc binding, laying the foundation and providing tools for future studies into how this interaction may allosterically regulate choline transport.