Crystal Structure of an Asymmetric Trimer of a Bacterial Glutamate Transporter Homologue

Abstract

Excitatory amino acid transporters (EAATs), or glutamate transporters, are trimeric electrochemically driven pumps. In the brain, EAATs reside in membranes of glial cells and neurons, and couple the uptake of the neurotransmitter glutamate to the co-transport of three sodium ions and a proton, and to the counter-transport of a potassium ion. Also, they mediate a bidirectional chloride flux, which is gated by glutamate and sodium binding but is not required for transport. This channel-like activity may play an important role in regulating synaptic transmission, particularly in retina. Insights into EAATs transport mechanism came from crystal structures of an archaeal homolog, GltPh, which pictured symmetrical trimers with protomers in either outward or inward facing conformations. These structures show that each protomer harbors a distinct transport domain, which coordinates substrate and coupled ions, and traverses ∼15 Å of the lipid bilayer to move its cargo between extracellular and intracellular solutions. Here, we report an asymmetric structure of GltPh with two protomers in the inward facing conformation and one protomer in a novel state, which may represent an early intermediate between outward and inward facing states. This structure illustrates the autonomous nature of the transport domain, consistent with the functional independence of the protomers. It further suggests that transitions between outward and inward facing states may proceed via relatively low-energy intermediates, and provides a glimpse of their unusual structural properties. Specifically, the intermediate state shows compromised packing between protein domains that results in the opening of a lipid-exposed crevice and the formation of a cavity, potentially accessible to the solvent. Based on this structure and published electrophysiology data, we hypothesize that transport cycle intermediates may serve as conduits for uncoupled permeation by polar molecules and ions. Further experiments will be presented.