Cold environments challenge the structural and functional integrity of membrane proteins, requiring specialized adaptations to maintain activity under low thermal energy. Here, we investigate the molecular basis of cold tolerance in the peptide transporter PepT1 from the Antarctic icefish (Chionodraco hamatus, ChPepT1) using molecular dynamics simulations, binding free energy calculations (MM/GBSA), and dynamic network analysis. We compare ChPepT1 to its human ortholog (hPepT1), a non-cold-adapted variant, to reveal key features enabling psychrophilic function. Our simulations show that ChPepT1 displays enhanced global flexibility, particularly in domains adjacent to the substrate-binding site and the C-terminal domain (CTD). While hPepT1 loses substrate binding affinity as temperature increases, ChPepT1 maintains stable peptide interactions across a broad thermal range. This thermodynamic buffering results from temperature-sensitive rearrangement of hydrogen bond networks and more dynamic lipid interactions. Importantly, we identify a temperature-responsive segment (TRS, residues 660–670) within the proximal CTD that undergoes an α-helix to coil transition, modulating long-range coupling with transmembrane helices. Dynamic cross-correlation analyses further suggest that ChPepT1, unlike hPepT1, reorganizes its interdomain communication in response to temperature shifts. Our findings suggest that cold tolerance in ChPepT1 arises from a combination of structural flexibility, resilient substrate binding, and temperature-sensitive interdomain dynamics. These results provide new mechanistic insight into thermal adaptation in membrane transporters and offer a framework for engineering proteins with enhanced functionality in extreme environments.

The proton-activated chloride channel PAC/TMEM206 is broadly expressed in mammalian tissues and contributes to acid-induced cell injury in pathological settings such as ischemia, inflammation, and tumor acidosis. In addition to its established proton sensitivity, PAC is strongly modulated by temperature: heating potentiates proton-evoked currents and shifts activation toward less acidic pH yet does not open the channel at neutral pH. How proton and thermal inputs are structurally integrated remains unclear. Here, we combined site-specific incorporation of the environmentally sensitive fluorescent amino acid ANAP with automated patch-clamp electrophysiology and molecular dynamics simulations to identify structural elements underlying PAC thermal modulation. ANAP reporters introduced across the extracellular domain and vestibule–pore coupling region revealed residue-specific thermal- and proton-dependent spectral shifts, showing that heating and acidification remodel overlapping but non-identical local environments. The strongest ANAP-reported temperature-dependent changes were observed at R93, Y111, F196, R237, and F282, whereas proton-dependent changes prominently involved R93, Y111, H130, and R237. Several temperature-sensitive ANAP reporters mapped to an intersubunit region also highlighted by dynamic correlation analysis. Together, our results identify structural correlates of PAC thermal modulation consistent with a model in which protonation creates an activation-permissive landscape while heating reweights coupling across an intersubunit scaffold; this model generates testable predictions that should be addressed by targeted mutagenesis and thermodynamic characterization.