The high-energy transition state of a membrane transporter

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 a model glutamate transporter homologue suggested that the slow conformational transition from the outward-to the inwardfacing state, when the bound substrate is translocated from the extracellular to the cytoplasmic side of the membrane, is rate-limiting to transport. Here, we aim to gain insight into the structure of the high-energy transition state that limits the rate of this critical isomerization reaction. Using bioinformatics, we identify gain-of-function mutants of the transporter and apply 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 for allosteric modulation of these transporters.

Large domain movements through lipid bilayer mediate substrate release and inhibition of glutamate transporters

“…Single-molecules studies of the OFS to IFS translocation dynamics showed that the ratelimiting high-energy transition state most likely resembles the IFS structurally and that the transport domain might make multiple attempts to achieve a stable observable IFS (Huysmans et al, 2020). These studies suggest that multiple IFS conformations exist and are separated by significant energetic barriers.…”

“…One remarkable aspect of the observed structural transitions is the conformational plasticity of HP2 and the interface between the transport domain and scaffold, which differ in each functional intermediate of the transporter. Our recent studies suggest that both translocation of the transport domain and substrate release into the cytoplasm are the slow steps of the GltPh functional cycle (Oh e Boudker, 2018;Huysmans et al, 2020). Most strikingly, subtle packing mutations in HP2 at sites distant from the substrate-binding site decrease substrate affinity in the OFS and IFS, and increase the dynamics of the OFS to IFS transitions (Huysmans et al, 2020).…”

Large domain movements through the lipid bilayer mediate substrate release and inhibition of glutamate transporters

“…Also, in the inward-facing neutral amino acid transporter ASCT2 (Garaeva et al, 2018, Garaeva et al, 2019 Boudker, 2018, Huysmans et al, 2020). Most strikingly, subtle packing mutations in HP2 at sites distant from the substrate-binding site decrease affinity in the OFS and IFS and increase the elevator transitions frequency (Huysmans et al, 2020).…”

“…These movements rely on the remarkable conformational plasticity of HP2 and the interface between the transport and scaffold domains, which differ in each functional intermediate of the transporter. Our recent studies suggest that both translocation of the transport domain and substrate release into the cytoplasm are slow processes ( Oh and Boudker, 2018 ; Huysmans et al, 2020 ). Most strikingly, subtle packing mutations in HP2 at sites distant from the substrate-binding site decrease affinity in the OFS and IFS and increase the elevator transitions frequency ( Huysmans et al, 2020 ).…”

“…Single-molecule studies of the elevator dynamics showed that the rate-limiting high-energy transition state most likely structurally resembles the IFS, and the transport domain might make multiple attempts to achieve a stable observable IFS ( Huysmans et al, 2020 ). These studies suggest that multiple IFS conformations exist and are separated by significant energetic barriers.…”