Phenylalanine Versus Tyrosine (Pos. 367/332 in MCT1/MCT4) in the Substrate Binding Site Defines Affinity and Preferred Directionality of Human Monocarboxylate Transporters 1-4

Human monocarboxylate transporters 1-4, MCT, are key for the lactate/H⁺ exchange between glycolytic and oxidative cancer cells, white and red muscle fibers, or in the astrocyte-neuron shuttle. The common MCT transport mechanism involves three conserved residues, that is, a substrate-attracting Lys and a conformation-locking Asp/Arg salt bridge (positions 38 and 309/313 in MCT1). Yet, it remained unclear which sites define isoform-specific substrate affinity and preferred transport directionality.

Here, we analyzed structural differences in the binding sites of MCT1-4 and determined their impact on the biophysical transport properties and inhibitor binding using radiolabeled transport assays in yeast.

We found differences in amino acid positions with sidechain hydroxyl groups. While the higher affinity MCT1 and MCT2 carry Phe/Ser-OH (pos. 367/371 in MCT1), the lower affinity MCT3 and MCT4 have Tyr-OH/Gly. Mutation of Phe/Ser-OH in MCT1 to Tyr-OH/Gly markedly decreased the affinity for lactate and pyruvate, while it did not change the affinity for propionate. The maximal transport velocity increased with decreasing affinity, and the preferred transport directionality shifted toward export. Likewise, replacing Met151 by Ala shifted transport bias possibly by eliminating conformation-stabilizing sulfur-aromatic interactions. Moreover, these mutations lowered the activity of the clinical candidate MCT1 inhibitor AZD3965 by three orders of magnitude providing insight into the molecular drug binding mode and explaining the strong preference for MCT1 over MCT4.

Together, subtle changes in the arrangement of sidechains in the MCT binding site determine basic monocarboxylate/H⁺ transport properties that impact lactate-related physiology, namely cellular metabolism, reprogramming, and signaling.