title: 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 signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii, 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 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.
authors: Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA.
authors: Nurunisa
authors: N
authors: Akyuz
authors: 1] National Biomedical Center for Advanced ESR Technology, Cornell University, Ithaca, New York 14853, USA [2] Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA.
authors: Elka R
authors: ER
authors: Georgieva
authors: Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA.
authors: Zhou
authors: Z
authors: Zhou
authors: Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA.
authors: Sebastian
authors: S
authors: Stolzenberg
authors: 1] Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA [2] Swiss Institute of Bioinformatics, Quartier Sorge - Batiment Genopode, 1015 Lausanne, Switzerland.
authors: Michel A
authors: MA
authors: Cuendet
authors: Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA.
authors: George
authors: G
authors: Khelashvili
authors: Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA.
authors: Roger B
authors: RB
authors: Altman
authors: Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA.
authors: Daniel S
authors: DS
authors: Terry
authors: 1] National Biomedical Center for Advanced ESR Technology, Cornell University, Ithaca, New York 14853, USA [2] Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA.
authors: Jack H
authors: JH
authors: Freed
authors: 1] Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA [2] HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College, Cornell University, 1305 York Avenue, New York, New York 10065, USA.
authors: Harel
authors: H
authors: Weinstein
authors: Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA.
authors: Olga
authors: O
authors: Boudker
authors: 1] Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA [2] Tri-Institutional Training Program in Chemical Biology, 445 East 69th Street, New York, New York 10065, USA.
authors: Scott C
authors: SC
authors: Blanchard
mesh_terms: D000595:Amino Acid Sequence; D026922:Amino Acid Transport Systems, Acidic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism*; D001692:Biological Transport; D018360:Crystallography, X-Ray; D003902:Detergents; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009068:Movement; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000378:metabolism; D043902:Pyrococcus horikoshii / Q000737:chemistry*; D012964:Sodium / Q000378:metabolism; D012997:Solvents; D013816:Thermodynamics
chemical_list: D026922:Amino Acid Transport Systems, Acidic; D003902:Detergents; D008024:Ligands; D050505:Mutant Proteins; D011510:Proteolipids; D012997:Solvents; C020485:proteoliposomes; D001224:Aspartic Acid; D012964:Sodium
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