Gies, S.; Jagessar, K. L.; Wu, T.; Miller, I.; Dastvan, K.; Dastvan, R.

Spns transporters are major facilitator superfamily proteins that regulate lipid transport, lysosomal homeostasis, immunity and disease, yet how protonation enables their chemically diverse transport functions remains unclear. Here, we combine double electron-electron resonance spectroscopy in lipid nanodiscs with DEER- and AlphaFold-guided modeling to define the conformational landscape of the Mycobacterium smegmatis Spns homolog MsSpns. Protonation shifts MsSpns toward an inward-facing state, whereas deprotonation favors a broader outward-facing ensemble through coordinated rearrangements of the intracellular and extracellular gates. These transitions are governed by membrane-embedded protonation switches and proton-sensing networks on both sides of the membrane, while the substrate-binding cavity exhibits distinct proton sensitivity and weaker cooperativity. Hydrophilic cationic substrates, including capreomycin and ethidium bromide, stabilize the outward-facing state, consistent with efflux antiport, whereas lipophilic compounds, including rifampicin, epicholesterol and certain phospholipids, favor the inward-facing state, suggesting uptake or allosteric stabilization. Thus, conserved proton-coupling elements can drive substrate transport in opposite directions, revealing the mechanistic versatility of the Spns fold with therapeutic potential.