Transport domain unlocking sets the uptake rate of an aspartate transporter. Academic Article uri icon

Overview

MeSH

  • Amino Acid Sequence
  • Biological Transport
  • Crystallography, X-Ray
  • Detergents
  • Fluorescence Resonance Energy Transfer
  • Humans
  • Kinetics
  • Ligands
  • Models, Molecular
  • Molecular Dynamics Simulation
  • Molecular Sequence Data
  • Movement
  • Mutant Proteins
  • Mutation
  • Protein Stability
  • Protein Structure, Tertiary
  • Proteolipids
  • Sodium
  • Solvents
  • Thermodynamics

MeSH Major

  • Amino Acid Transport Systems, Acidic
  • Aspartic Acid
  • Pyrococcus horikoshii

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.

publication date

  • February 5, 2015

has subject area

  • Amino Acid Sequence
  • Amino Acid Transport Systems, Acidic
  • Aspartic Acid
  • Biological Transport
  • Crystallography, X-Ray
  • Detergents
  • Fluorescence Resonance Energy Transfer
  • Humans
  • Kinetics
  • Ligands
  • Models, Molecular
  • Molecular Dynamics Simulation
  • Molecular Sequence Data
  • Movement
  • Mutant Proteins
  • Mutation
  • Protein Stability
  • Protein Structure, Tertiary
  • Proteolipids
  • Pyrococcus horikoshii
  • Sodium
  • Solvents
  • Thermodynamics

Research

keywords

  • Journal Article

Identity

Language

  • eng

PubMed Central ID

  • PMC4351760

Digital Object Identifier (DOI)

  • 10.1038/nature14158

PubMed ID

  • 25652997

Additional Document Info

start page

  • 68

end page

  • 73

volume

  • 518

number

  • 7537