Heterogeneous substrate binding in a glutamate transporter homologue

ABSTRACT

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 Glt_Ph, inconsistent with simple kinetic models. The structural and mechanistic determinants of this heterogeneity remain undefined. In a mutant form of Glt_Ph, we demonstrate substrate binding heterogeneity in the outward-facing state, modulated by temperature and salts. We observe similar trends in wild-type Glt_Ph that correlate with changes in the transport rate. Extensive cryo-EM analysis of the fully bound mutant Glt_Ph 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.