Proton-coupled alternating access in a versatile mycobacterial Spns drug transporter

Spns transporters are a mechanistically distinct branch of the major facilitator superfamily that regulate lipid transport, lysosomal homeostasis, immunity and disease. How the conserved Spns fold integrates protonation, substrate binding and alternating access to support chemically and directionally diverse transport remains unresolved. Here we combine DEER spectroscopy in lipid nanodiscs with DEER- and AlphaFold-guided modeling and protonation-mimetic mutagenesis to define the conformational landscape of the Mycobacterium smegmatis homolog MsSpns. Protonation shifts MsSpns toward an inward-facing state, whereas deprotonation favors a broader outward-facing ensemble through remodeling of intracellular and extracellular gates. Protonation-mimetic substitutions identify Glu126 as a switch that stabilizes an inward-facing, substrate-entry-competent conformation, while Asp38 and Asp57 favor outward-facing states and tune the extracellular proton-sensing network. The substrate-binding cavity displays distinct proton sensitivity and weaker cooperativity than gating networks. Hydrophilic cationic substrates stabilize the outward-facing state, consistent with efflux antiport, whereas lipophilic compounds 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.