Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1, and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability.

The proteasome controls levels of most cellular proteins, and its activity is regulated under stress, quiescence, and inflammation. However, factors determining the proteasomal degradation rate remain poorly understood. Proteasome substrates are conjugated with small proteins (tags) like ubiquitin and Fat10 to target them to the proteasome. It is unclear if the structural plasticity of proteasome-targeting tags can influence substrate degradation. Fat10 is upregulated during inflammation, and its substrates undergo rapid proteasomal degradation. We report that the degradation rate of Fat10 substrates critically depends on the structural plasticity of Fat10. While the ubiquitin tag is recycled at the proteasome, Fat10 is degraded with the substrate. Our results suggest significantly lower thermodynamic stability and faster mechanical unfolding in Fat10 compared to ubiquitin. Long-range salt bridges are absent in the Fat10 structure, creating a plastic protein with partially unstructured regions suitable for proteasome engagement. Fat10 plasticity destabilizes substrates significantly and creates partially unstructured regions in the substrate to enhance degradation. NMR-relaxation-derived order parameters and temperature dependence of chemical shifts identify the Fat10-induced partially unstructured regions in the substrate, which correlated excellently to Fat10-substrate contacts, suggesting that the tag-substrate collision destabilizes the substrate. These results highlight a strong dependence of proteasomal degradation on the structural plasticity and thermodynamic properties of the proteasome-targeting tags.