Experimentally Defined Structural Model of the Human Proton-Coupled Folate Transporter

Abstract

Folate cofactors, Vitamin B9, play crucial roles in more than a hundred one-carbon metabolism reactions in mammalian cells. Humans cannot synthesize folates de novo and absorption through the diet is the only source of this vitamin. The proton-coupled folate transporter (PCFT) mediates this uptake in the upper small intestine by a pH-dependent process. PCFT also transports folates into the central nervous system. Point mutations in the PCFT gene cause Hereditary Folate Malabsorption (HFM), with associated hematological and neurological defects due to impaired folate transport. Certain solid tumor cell lines express high levels of PCFT mRNA. Importantly, the overall expression of PCFT is limited to only certain tissues in humans. Therefore, PCFT is a molecular target for specific delivery of anti-folate chemotherapeutic agents to tumor cells. Unfortunately, the success rate of anti-folate agents to reach clinical use is very low due to their side-effects. A structural model of PCFT can aid the development of specific anti-folate agents for their PCFT-targeted delivery and thus minimize their side-effects. To optimize and verify an initial structural model of PCFT, we applied a wide array of experimental approaches: solvent accessibility profiling through substituted cysteine accessibility scanning, studying the helix packing, and assaying the functionality of PCFT mutants. We complemented the experimental data with theoretical approaches for structure prediction such as homology modeling/threading, ligand docking and an extensive review of other secondary transporters and folate transport proteins. With these combined approaches, we have developed a structural model of PCFT that accurately reflects our experimental observations. This model forms a basis to understand the impacts of HFM mutations, and to develop folate analogues for folate deficiency intervention. Additionally, it will aid the design of PCFT structure-based anti-folate agents for the treatment of cancer.