Transport mechanism of a bacterial homologue of glutamate transporters

Abstract

Glutamate transporters are integral membrane proteins that catalyze a thermodynamically uphill uptake of the neurotransmitter glutamate from the synaptic cleft into the cytoplasm of glial and neuronal cells by harnessing the energy of pre-existing electrochemical gradients of ions. The linchpin of the reaction is the conformational transition of the transporters between outward and inward facing states, in which the substrate binding sites are accessible from the extracellular space and the cytoplasm respectively. Here we describe a crystal structure of a double cysteine mutant of a bacterial homologue of glutamate transporters, GltPh, which is trapped in the inward facing state by cysteine cross-linking. Together with the previously determined crystal structure of GltPh in the outward facing state, the structure of the cross-linked mutant allows us to propose a molecular mechanism, by which GltPh and, by analogy, mammalian glutamate transporters, mediate sodium-coupled substrate uptake.

Structural and mechanistic insights into prokaryotic energy-coupling factor transporters

“…A scaffold domain is needed to facilitate the elevator-type movement of the transport domain. The Na + -coupled aspartate transporter Glt Ph is the prototypical example of a transporter that uses this mechanism 72 . d | Hypothetical model for ATPdriven transport by ECF transporters using the toppling mechanism.…”

Crystal structure of a substrate-free aspartate transporter

“…Structural information is available for the glutamate transporter homolog Glt Ph from the archaeon Pyrococcus horikoshii, which is selective for aspartate rather than glutamate and which couples the uptake of substrate to the symport of three sodium ions [3][4][5][6][7][8] . The protein is a homotrimer, with each protomer consisting of two domains.…”

“…The trimerization domain mediates the subunit contacts, and the transport domain carries the binding sites for aspartate and sodium ions. Different crystal structures of substrate-bound Glt Ph have revealed that the trimerization domains form a stable scaffold 6,9 . The transport domains have been captured in three states: outward facing, intermediate and inward facing [5][6][7] .…”

“…Different crystal structures of substrate-bound Glt Ph have revealed that the trimerization domains form a stable scaffold 6,9 . The transport domains have been captured in three states: outward facing, intermediate and inward facing [5][6][7] . The structures suggest that the transport domains behave as rigid bodies and move across the membrane like an elevator to shuttle the substrate and coupled ions from one side to the other.…”

Asymmetric perturbations of signalling oligomers

“…This is well exemplified by a recent XRC analysis of a functional double mutant of Glt Ph , a glutamate transporter from Pyrococcus horikoshii (Verdon and Boudker, 2012). While previous structure determinations captured Glt Ph (Yernool et al, 2004;Boudker et al 2007) and its analogue (Reyes et al, 2009) as symmetric trimers with all their protomers facing either outward or inward, the structure of the Glt Ph -V198CA380C Hg double mutant is an asymmetric trimer with two protomers in IFS and one in an intermediate outward-facing state (iOFS) (Fig. 4A).…”