Transport domain unlocking sets the uptake rate of an aspartate transporter

Abstract

Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signaling and preventing glutamate-mediated excitotoxicity. Crystallographic studies on an archaeal homologue, GltPh, showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold. Here, we report direct observations of these 'elevator-like' transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that GltPh bearing two “humanizing” mutations exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motions and substrate uptake. Crystallographic and computational investigations reveal that these mutations favor structurally “unlocked” states with increased solvent occupancy at the interface between the transport domain and the trimeric scaffold.

Direct visualization of glutamate transporter elevator mechanism by high-speed AFM

“…1D, Upper), consistent with crystal structures of extreme states ( Fig. S3A) (6,7), sm-FRET (8,16,17), and electron paramagnetic resonance (EPR) (19,20) studies. Kymographs converted to idealized traces of transitions between outward-and inward-facing conformations (Fig.…”

“…Notably, the outward-facing state is longer lived in the substrate-bound compared with the apo transporter. Reduced dynamics of the substrate-bound transport domain has also been observed in sm-FRET studies (8,16). At present, we do not know why the dynamics are reduced, but we note that substrate binding is associated with minor conformational changes (10) and changes in the overall charge of the transport domain.…”

“…However, the fraction of active molecules varies depending on the ligands bound to the transporter, arguing against these explanations. Alternatively, temporary inactivity could be an innate feature of the transporter; if so, it might be reminiscent of dynamic heterogeneity observed in sm-FRET studies of Glt Ph , where periods of activity were interspersed by long quiescent periods (8,16) [although not in another (17)]. Assuming that the stalled molecules observed in HS-AFM experiments reflect the reversible entry into such postulated "locked" states, we interpret the Q value (Table S1) as a fraction of unlocked active transporters.…”

“…S1). Single-molecule FRET (sm-FRET) experiments revealed Glt Ph transport dynamics in detergent and tethered vesicles (8,16,17). However, direct visualization of Glt Ph elevator transport mechanism remained elusive.…”

Protonation of Glu 135 Facilitates the Outward-to-Inward Structural Transition of Fucose Transporter

“…According to previous experiments on glutamate and aspartate transporters, the large-scale conformational changes of membrane transporters frequently fall in the timescale of seconds (47,48). Such slow molecular events therefore may escape the observing capability of all-atom equilibrium simulations that can only track the timescale of microseconds or perhaps milliseconds at most.…”