Cryo-EM structure determination of the human reduced folate carrier SLC19A1 in complex with methotrexate.

Folates are important nutrients that play critical roles in cellular metabolism. Owing to the fact mammals lack systems for folate biogenesis, cells rely on folate transporters and receptors for membrane translocation of these organic anions. The human reduced folate carrier (hRFC), member of the solute carrier 19 family (SLC19), is the main transporter of dietary and reduced folates in humans. Further, hRFC is an anion exchanger that is also responsible for cellular entry of antifolate chemotherapeutics such as methotrexate and pemetrexed. This is exemplified by the fact genetic variants in hRFC or the expression level of hRFC have been implicated in methotrexate resistant cancers. Despite the importance to human metabolism and pharmacology, little was known about the molecular basis of antifolate drug recognition by hRFC. As hRFC is a small solute carrier that was refractory to crystallization, we turned to cryo-electron microscopy for structure determination. An unanticipated limitation encountered was absence of stable methotrexate binding to detergent-purified transporter. Utilizing an engineered fiducial, a methotrexate analog that covalently modifies the substrate binding site, and ad hoc image processing strategies, we were successful in solving a cryo-EM structure of hRFC trapped in a methotrexate bound state. In combination with transport assays and molecular dynamics simulations, our studies uncover the molecular basis of methotrexate recognition and other key features of anion selectivity by hRFC. These findings are discussed in depth in our companion poster [“Methotrexate recognition by the human reduced folate carrier SLC19A1”].