Mechanistic insights into inactivating mutations in the proton-coupled folate transporter (SLC46A1), and compensatory mutations that restore function.

Abstract

Hereditary folate malabsorption (HFM) is a rare, autosomal recessive disorder characterized by impaired intestinal absorption and impaired transport of folates across the choroid plexus into cerebral spinal fluid due to inactivating mutations in the hPCFT gene, which encodes the proton-coupled folate transporter (PCFT-SLC46A1). Understanding the structural impact of these mutations is crucial for elucidating the mechanistic basis for PCFT function and the pathophysiology of HFM. Recently, the cryo-electron microscopic structural characterization of the Gallus gallus PCFT (gPCFT) was obtained, which shares significant sequence identity with hPCFT. We conducted molecular dynamics (MD) simulations of human PCFT (hPCFT) based on this structure, to explore structural changes induced by functionally defective disease-causing and other mutant proteins and mutations that restore function. Simulations revealed that the mutually mechanistic basis for the loss of function is partial loss of structural integrity of hPCFT primarily manifested in an enlarged and distorted pore accompanied by loss of long-range contacts, less stable, fluctuating inner helices with reduced solvent accessibility and a marked loss of ordered secondary structures. These changes are reversed by the introduction of compensatory mutations. These findings provide novel insights into the structural and functional consequences of PCFT mutations associated with HFM and provide correlations with kinetic and biochemical properties of the mutant proteins.