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Structural ensemble of a glutamate transporter homologue in lipid nanodisc environment.

ABSTRACT: Glutamate transporters are cation-coupled secondary active membrane transporters that clear the neurotransmitter L-glutamate from the synaptic cleft. These transporters are homotrimers, with each protomer functioning independently by an elevator-type mechanism, in which a mobile transport domain alternates between inward- and outward-oriented states. Using single-particle cryo-EM we have determined five structures of the glutamate transporter homologue GltTk, a Na+- L-aspartate symporter, embedded in lipid nanodiscs. Dependent on the substrate concentrations used, the protomers of the trimer adopt a variety of asymmetrical conformations, consistent with the independent movement. Six of the 15 resolved protomers are in a hitherto elusive state of the transport cycle in which the inward-facing transporters are loaded with Na+ ions. These structures explain how substrate-leakage is prevented - a strict requirement for coupled transport. The belt protein of the lipid nanodiscs bends around the inward oriented protomers, suggesting that membrane deformations occur during transport.

SUBMITTER: Arkhipova V

PROVIDER: S-EPMC7035293 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Structural ensemble of a glutamate transporter homologue in lipid nanodisc environment.

Arkhipova Valentina V Guskov Albert A Slotboom Dirk J DJ

Nature communications 20200221 1

Glutamate transporters are cation-coupled secondary active membrane transporters that clear the neurotransmitter L-glutamate from the synaptic cleft. These transporters are homotrimers, with each protomer functioning independently by an elevator-type mechanism, in which a mobile transport domain alternates between inward- and outward-oriented states. Using single-particle cryo-EM we have determined five structures of the glutamate transporter homologue Glt<sub>Tk</sub>, a Na<sup>+</sup>- L-aspar ...[more]

PMID: 32081874

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Project description:Excitatory amino acid transporters (EAATs) are membrane proteins responsible for reuptake of glutamate from the synaptic cleft to terminate neurotransmission and help prevent neurotoxically high, extracellular glutamate concentrations. Important structural information about these proteins emerged from crystal structures of GltPh, a bacterial homologue of EAATs, in conformations facing outward and inward. These remarkably different conformations are considered to be end points of the substrate translocation path (STP), suggesting that the transport mechanism involves major conformational rearrangements that remain uncharted. To investigate possible steps in the structural transitions of the STP between the two end-point conformations, we applied a combination of computational modeling methods (motion planning, molecular dynamics simulations, and mixed elastic network models). We found that the conformational changes in the transition involve mainly the repositioning the "transport domain" and the "trimerization domain" identified previously in the crystal structures. The two domains move in opposite directions along the membrane normal, and the transport domain also tilts by ?17° with respect to this axis. Moreover, the TM3-4 loop undergoes a flexible, "restraining bar"-like conformational change with respect to the transport domain. As a consequence of these conformational rearrangements along the transition path we calculated a significant decrease of nearly 20% in the area of the transport-to-trimerization domain interface (TTDI). Water penetrates parts of the TTDI in the modeled intermediates but very much less in the end-point conformations. We show that these characteristics of the modeled intermediate states agree with experimental results from residue-accessibility studies in individual monomers and identify specific residues that can be used to test the proposed STP. Moreover, MD simulations of complete GltPh trimers constructed from initially identical monomer intermediates suggest that asymmetry can appear in the trimer, consonant with available experimental data showing independent transport kinetics by individual monomers in the trimers.

Dataset Information

Structural ensemble of a glutamate transporter homologue in lipid nanodisc environment.

Publications

Structural ensemble of a glutamate transporter homologue in lipid nanodisc environment.

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