Transport domain unlocking sets the uptake rate of an aspartate transporter

Nurunisa Akyuz, Elka R. Georgieva, Zhou Zhou, Sebastian Stolzenberg, Michel A. Cuendet, George Khelashvili, Roger B. Altman, Daniel S. Terry, Jack H. Freed, Harel Weinstein, Olga Boudker () and Scott C. Blanchard ()
Additional contact information
Nurunisa Akyuz: Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA
Elka R. Georgieva: National Biomedical Center for Advanced ESR Technology, Cornell University
Zhou Zhou: Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA
Sebastian Stolzenberg: Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA
Michel A. Cuendet: Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA
George Khelashvili: Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA
Roger B. Altman: Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA
Daniel S. Terry: Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA
Jack H. Freed: National Biomedical Center for Advanced ESR Technology, Cornell University
Harel Weinstein: Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA
Olga Boudker: Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA
Scott C. Blanchard: Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA

Nature, 2015, vol. 518, issue 7537, 68-73

Abstract: 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.

Date: 2015
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DOI: 10.1038/nature14158

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