The proton‐coupled folate transporter (PCFT, SLC46A1) transports folic acid across the plasma membrane, together with an excess of protons such that the net charge translocation is positive. We developed 3D structural models of PCFT threaded onto the X‐ray structures of major facilitator superfamily (MFS) members that were identified as close structural homologues. The model of PCFT threaded onto the glycerol‐3‐phosphate transporter (GlpT) structure is consistent with detailed accessibility studies in the absence of extracellular substrate and at pH 7.4 presented here, and additionally with a multitude of other mutagenesis and functional studies. Characteristic MFS structural features are preserved in this PCFT model, such as 12 transmembrane helices divided into two pseudosymmetric bundles, and a high density of positive charges on the periphery of the cytoplasmic site that allow interactions with negatively charged lipid head‐groups. Under the experimental conditions, PCFT predominantly samples the resting state, which in this case is inward‐open. Several positions lining the substrate cavity have been identified. Motif A, a helix‐turn‐helix motif that is a hallmark of MFS transporters between transmembrane segments II and III is oriented appropriately to interact with residues from transmembrane segments IV as well as XI upon conformational transition to the outward‐open state. A charge‐relay system between three charged residues as well as apposing glycines in two α‐helices, both contributed to by motif A, become engaged when PCFT is modeled on the outward‐open state of a putative proton‐driven transporter (YajR).

symporter (vSGLT), a close structural homologue exhibiting the same topological fold. Based on the topological identity we also used the x-ray structure of Aquifex aeolicus leucine transporter (LeuT) to further improve the model of our system. Sequence alignment was performed to identify important residues and potential substrate/ion binding sites. Radioactive labeled biomarkers were used as potential substrates/inhibitors, in line with the clinical applications. Aside from the radio-labeled iodide, we considered tetrafluoroborate, perchlorate, bromide, and thiocyanate. Firstly, the binding sites were identified through a docking process. Following which in order to enhance I-and inhibitor transport, necessary for improved imaging, we had to expand our system beyond the wild-type protein. The mutants we considered are all essential for transport and they are Q72N, I147C, E368A, and M68A. By inducing different rates of transport we can deduce how the protein responds to specific mutants. We have successfully identified a favorable response to our mutations and we are hopeful our approach to the engineered NIS protein will further aid in the treatment and imaging of thyroidal and extrathyroidal cancers. 1545-Pos Board B496Effect of Mutations on Transport by the Sodium/Iodide Symporter (NIS)