Heterogeneous substrate binding in a glutamate transporter homologue

Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this family, including kinetic models of transport. Recent studies have revealed transport rate heterogeneity in an archaeal glutamate transporter homologue GltPh, inconsistent with simple kinetic models. The structural and mechanistic determinants of this heterogeneity remain undefined. In a mutant form of GltPh, we demonstrate substrate binding heterogeneity in the outward-facing state, modulated by temperature and salts. We observe similar trends in wild-type GltPh that correlate with changes in the transport rate. Extensive cryo-EM analysis of the fully bound mutant GltPh provides multiple potential explanations of heterogeneous substrate binding. At equilibrium, we show subtle differences in tilts of protomers in the outward-facing state and configurations of the substrate-binding pocket. Within seconds of substrate binding, we observe perturbed helical packing of the extracellular half of the substrate-binding domain. Some or all of these may contribute to the heterogeneity observed in binding and transport. ### Competing Interest Statement The authors have declared no competing interest.