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.

Heterogeneous substrate binding in a glutamate transporter homologue

“…Therefore, it is possible that the heterogeneity in the extracellular substrate-binding contributes to the heterogeneous uptake rates or reflects different substrate-binding properties of fast and slow transporter sub-populations. Consistently, R276S/M395R, A345V, and V366A mutations lead to increased elevator dynamics and altered substrate binding (Huysmans et al, 2021), showing that structural perturbations can affect substrate binding and translocation in a concerted fashion.…”

“…To test where S1 and S2 may correspond to OFS and IFS, we measured single-molecule FRET (smFRET) signals using TIRF microscopy from fluorescently labeled P-Glt Ph solubilized in DDM. We introduced a single cysteine mutation into a cysteine-free background (P-Glt Ph C321A/N378C), labeled with donor and acceptor fluorophores, and analyzed by smFRET as in earlier studies (Akyuz et al, 2013; Akyuz et al, 2015; Huysmans et al, 2021). Conformations of protomer pairs within individual Glt Ph trimers can be distinguished by FRET efficiency ( E FRET ) as either both in OFS (~0.4), a mixture of OFS and IFS (~0.6), or both in IFS (~0.8).…”

“…Switching between the modes is rare, occurring on a time scale of hundreds of seconds and resulting in the so-called static disorder of the transport kinetics. These transport modes were attributed to sub-populations that show different elevator dynamics and intracellular substrate-release rates (Huysmans et al, 2021; Matin et al, 2020). Here, we observed a correlation, where nitrate changes the ratio of different substrate-binding states and increases the fraction of slow WT transporters ( Figure 1f ).…”

“…Recently, high-speed atomic force microscopy, single-molecule Förster resonance energy transfer (smFRET) total internal reflection fluorescence (TIRF) microscopy, and 19 F-NMR revealed a more complex picture of Glt Ph transport and dynamics (Akyuz, Altman, Blanchard, & Boudker, 2013; Akyuz et al, 2015; Ciftci et al, 2020; Erkens et al, 2013; Huang et al, 2020; Huysmans, Ciftci, Wang, Blanchard, & Boudker, 2021; Matin, Heath, Huysmans, Boudker, & Scheuring, 2020). These studies established the existence of additional conformational states in OFS and IFS, of which some translocate and transport at different rates.…”

Structural understanding of the transport cycle of glutamate transporters

“…Singlemolecule transport assay has recently shown that individual Glt Ph transporters can function at rates varying by more than two orders of magnitude that persist for multiple turnovers (Ciftci et al, 2020). Mutations with enhanced Asp uptake rates show a shift of population to faster OFS/IFS dynamics and substrate-releasing rates (Huysmans et al, 2020). Therefore, uncharacterized conformational states might be responsible for distinguishing the subset of transporters with fast dynamics from those with slow dynamics.…”