Transport domain unlocking sets the uptake rate of an aspartate transporter

• Published in: Nature (London) Vol. 518; no. 7537; pp. 68 - 73
• Main Authors: Akyuz, Nurunisa, Georgieva, Elka R., Zhou, Zhou, Stolzenberg, Sebastian, Cuendet, Michel A., Khelashvili, George, Altman, Roger B., Terry, Daniel S., Freed, Jack H., Weinstein, Harel, Boudker, Olga, Blanchard, Scott C.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 05.02.2015; Nature Publishing Group
• Subjects: 631/45/612/1237; 631/535/1266; 631/57/2265; 82; Amino Acid Sequence; Amino Acid Transport Systems, Acidic - chemistry; Amino Acid Transport Systems, Acidic - genetics; Amino Acid Transport Systems, Acidic - metabolism; Aspartic Acid - metabolism; Biological Transport; Crystallography, X-Ray; Detergents; Energy transfer; Experiments; Fluorescence Resonance Energy Transfer; Humanities and Social Sciences; Humans; Kinetics; Ligands; Lipids; Models, Molecular; Molecular Dynamics Simulation; Molecular Sequence Data; Movement; multidisciplinary; Mutant Proteins - chemistry; Mutant Proteins - genetics; Mutant Proteins - metabolism; Mutation; Mutation - genetics; Physiology; Protein Stability; Protein Structure, Tertiary; Proteins; Proteolipids - metabolism; Pyrococcus horikoshii - chemistry; Science; Sodium - metabolism; Solvents; Thermodynamics
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature14158

Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii , Glt Ph , 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 Glt Ph bearing two mutations introduced to impart characteristics of the human transporter exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motion and substrate uptake. Crystallographic and computational investigations corroborated these findings by revealing that the ‘humanizing’ mutations favour structurally ‘unlocked’ intermediate states in the transport cycle exhibiting increased solvent occupancy at the interface between the transport domain and the trimeric scaffold. An analysis of a bacterial homologue of the human glutamate transporter using single-molecule FRET and X-ray crystallography reveals that opening of the interface between its distinct transport and scaffold domains is rate determining for the transport cycle. Glutamate transporter mechanisms In this manuscript, the authors use single-molecule fluorescence resonance energy transfer imaging, X-ray crystallography and molecular dynamics simulations to probe the dynamics of Glt Ph , an aspartate transporter from the archaeon Pyrococcus horikoshii , and of a double mutant form of the enzyme that has a lower affinity for the substrate, but an increased rate of substrate transport. Glt Ph is a homologue of the glutamate transporters that play an important role in neural transmission by maintaining low concentrations of neurotransmitter within synapses in the brain, and the two mutations bring the enzyme closer in action to the human equivalent. The structures reveal that opening of the interface between Glt Ph 's distinct transport and scaffold domains — linked to the transition between between outward- and inward-facing states — are directly correlated with the rate of substrate transport.