Abstract
<title>Abstract</title> <p> Spns transporters are a mechanistically distinct branch of the major facilitator superfamily that regulate lipid transport, lysosomal homeostasis, immunity and disease, yet how the conserved Spns fold integrates protonation, substrate binding and alternating access to support chemically and directionally diverse transport activities remains unresolved. Here, we combine DEER spectroscopy in lipid nanodiscs with DEER- and AlphaFold-guided modeling to define the conformational landscape of the <italic>Mycobacterium smegmatis</italic> homolog <italic>Ms</italic> Spns. Protonation shifts <italic>Ms</italic> Spns toward an inward-facing state, whereas deprotonation favors a broader outward-facing ensemble through coordinated remodeling of intracellular and extracellular gates. These transitions are governed by membrane-embedded protonation switches and proton-sensing networks, while the substrate-binding cavity shows distinct proton sensitivity and weaker cooperativity. Hydrophilic cationic substrates, capreomycin and ethidium bromide, stabilize the outward-facing state, consistent with efflux antiport, whereas lipophilic compounds, including rifampicin, epicholesterol and selected phospholipids, favor the inward-facing state, suggesting uptake or allosteric stabilization. Thus, conserved proton-coupling elements can power opposing transport modes, revealing the mechanistic versatility of the Spns fold and its therapeutic potential. </p>