This is the supplementary data for our paper "The mechanism of mammalian proton-coupled peptide transporters". Abstract: Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuoussubstrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of thePOT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the protonelectrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into differentconformational states of two mammalian POTs, SLC15A1 and SLC15A2. Nevertheless, these studies leave openimportant questions regarding the mechanism of proton and substrate coupling, while simultaneously providing aunique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employextensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and itsthermodynamic driving forces. By computing conformational free energy landscapes in different protonation states andin the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps thatdrive inward-directed alternating access. These simulations identify key differences in the extracellular gate betweenmammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results fromconstant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between protonbinding and peptide recognition, revealing key details underpining secondary active transport in POTs. This studyprovides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave theway to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organbioavailability. This project was funded by the Wellcome Trust (Grant ID: 218514/Z/19/Z). Compute resources were also provided bythe EPSRC ARCHER2, Jade 2 and N8 CIR BEDE facilities, granted via the High-End Computing Consortium for BiomolecularSimulation (HECBioSim), supported by EPSRC (EP/X035603/1).