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The high-energy transition state of the glutamate transporter homologue GltPh.

ABSTRACT: Membrane transporters mediate cellular uptake of nutrients, signaling molecules, and drugs. Their overall mechanisms are often well understood, but the structural features setting their rates are mostly unknown. Earlier single-molecule fluorescence imaging of the archaeal model glutamate transporter homologue Glt_Ph from Pyrococcus horikoshii suggested that the slow conformational transition from the outward- to the inward-facing state, when the bound substrate is translocated from the extracellular to the cytoplasmic side of the membrane, is rate limiting to transport. Here, we provide insight into the structure of the high-energy transition state of Glt_Ph that limits the rate of the substrate translocation process. Using bioinformatics, we identified Glt_Ph gain-of-function mutations in the flexible helical hairpin domain HP2 and applied linear free energy relationship analysis to infer that the transition state structurally resembles the inward-facing conformation. Based on these analyses, we propose an approach to search for allosteric modulators for transporters.

SUBMITTER: Huysmans GHM

PROVIDER: S-EPMC7780239 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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The high-energy transition state of the glutamate transporter homologue GltPh.

Huysmans Gerard H M GHM Ciftci Didar D Wang Xiaoyu X Blanchard Scott C SC Boudker Olga O

The EMBO journal 20201113 1

Membrane transporters mediate cellular uptake of nutrients, signaling molecules, and drugs. Their overall mechanisms are often well understood, but the structural features setting their rates are mostly unknown. Earlier single-molecule fluorescence imaging of the archaeal model glutamate transporter homologue Glt<sub>Ph</sub> from Pyrococcus horikoshii suggested that the slow conformational transition from the outward- to the inward-facing state, when the bound substrate is translocated from the ...[more]

PMID: 33185289

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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

The high-energy transition state of the glutamate transporter homologue GltPh.

Publications

The high-energy transition state of the glutamate transporter homologue GltPh.

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