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. Recent studies revealed transport rate heterogeneity in an archaeal homologue GltPh, inconsistent with simple kinetic models, but its structural and mechanistic determinants remain undefined. In a mutant GltPh, which exclusively populates the outward-facing state, we demonstrate the co-existence of at least two sub-states in slow equilibrium binding the substrate with different apparent affinities. Wild-type GltPh shows similar binding properties, and modulation of the sub-state equilibrium correlates with transport rates. Following binding, the low-affinity sub-state of the mutant is transient. Consistently, cryo-EM on samples frozen within seconds after substrate addition reveals the presence of structural classes with perturbed helical packing of the extracellular half of the transport domain in regions adjacent to the binding site. In contrast, an equilibrated structure does not show such classes. The structure at 2.2 [A] resolution details a pattern of waters in the intracellular half of the domain and resolves classes with subtle differences in the substrate-binding site. We hypothesize that the rigid cytoplasmic half of the domain mediates substrate and ion recognition and coupling, while the extracellular labile half sets the affinity and dynamic properties.