Unsynchronised subunit motion in single trimeric sodium-coupled aspartate transporters

Abstract

Excitatory amino acid transporters (EAATs) are secondary transport proteins that mediate the uptake of glutamate and other amino acids. EAATs fulfil an important role in neuronal signal transmission by clearing the excitatory neurotransmitters from the synaptic cleft after depolarization of the postsynaptic neuron. An intensively studied model system for understanding the transport mechanism of EAATs is the archaeal aspartate transporter GltPh. Each subunit in the homotrimeric GltPh supports the coupled translocation of one aspartate molecule and three Na(+) ions as well as an uncoupled flux of Cl(-) ions. Recent crystal structures of GltPh revealed three possible conformations for the subunits, but it is unclear whether the motions of individual subunits are coordinated to support transport. Here, we report the direct observation of conformational dynamics in individual GltPh trimers embedded in the membrane by applying single-molecule fluorescence resonance energy transfer (FRET). By analysing the transporters in a lipid bilayer instead of commonly used detergent micelles, we achieve conditions that approximate the physiologically relevant ones. From the kinetics of FRET level transitions we conclude that the three GltPh subunits undergo conformational changes stochastically and independently of each other.

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.…”

“…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.…”

Structural and mechanistic insights into prokaryotic energy-coupling factor transporters

“…Spectroscopic techniques, such as electron paramagnetic resonance (EPR) 39,54 and Förster resonance energy transfer (FRET) 55 , can be used to assay the dynamics of proteins during catalysis and seem to be suitable for studying the proposed toppling of the S component during the translocation cycle. In addition, crystal structures of ECF complexes in the presence of nucleotides and transported substrates, are likely to provide further mechanistic insight, and may reveal, for example, whether the proposed toppling event is specifically coupled to ATP binding or hydrolysis.…”

Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh

“…The conformational change reported by F273W is significantly slower than the initial substrate binding (k on ), which is manifested by the difference between the K d values calculated from the rate constants by stopped-flow fluorescence measurements and thermodynamics using ITC measurements (18 M versus 2.7 nM, respectively). The conformation change could for example be associated with an opening of the inward-facing lid HP1 (37,38) or a reorientation of the whole transport domain after binding of both Na ϩ and aspartate (32). However, for the F273W mutant the k obs values of aspartate binding did not saturate at high aspartate concentration, in contrast to L130W (28).…”