Transport domain unlocking sets the uptake rate of an aspartate transporter.
Nature
; 518(7537): 68-73, 2015 Feb 05.
Article
em En
| MEDLINE
| ID: mdl-25652997
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.
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Ácido Aspártico
/
Sistemas de Transporte de Aminoácidos Acídicos
/
Pyrococcus horikoshii
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Nature
Ano de publicação:
2015
Tipo de documento:
Article
País de afiliação:
Estados Unidos