Supplementary data for: The mechanism of mammalian proton-coupled peptide transporters

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 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.

This project was funded by the Wellcome Trust (Grant ID: 218514/Z/19/Z). Compute resources were also provided by
the EPSRC ARCHER2, Jade 2 and N8 CIR BEDE facilities, granted via the High-End Computing Consortium for Biomolecular
Simulation (HECBioSim), supported by EPSRC (EP/X035603/1).