Refinement of the Central Steps of Substrate Transport by the Aspartate Transporter GltPh: Elucidating the Role of the Na2 Sodium Binding Site.
Glutamate homeostasis in the brain is maintained by glutamate transporter mediated accumulation. Impaired transport is associated with several neurological disorders, including stroke and amyotrophic lateral sclerosis. Crystal structures of the homolog transporter GltPh from Pyrococcus horikoshii revealed large structural changes. Substrate uptake at the atomic level and the mechanism of ion gradient conversion into directional transport remained enigmatic. We observed in repeated simulations that two local structural changes regulated transport. The first change led to formation of the transient Na2 sodium binding site, triggered by side chain rotation of T308. The second change destabilized cytoplasmic ionic interactions. We found that sodium binding to the transiently formed Na2 site energized substrate uptake through reshaping of the energy hypersurface. Uptake experiments in reconstituted proteoliposomes confirmed the proposed mechanism. We reproduced the results in the human glutamate transporter EAAT3 indicating a conserved mechanics from archaea to humans.
Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.
SanthoshKannan
S
Venkatesan
Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.
Kusumika
K
Saha
Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.
Azmat
A
Sohail
Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.
Walter
W
Sandtner
Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.
Michael
M
Freissmuth
Division of Drug Design & Medicinal Chemistry, Department of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria.
Gerhard F
GF
Ecker
Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.
Harald H
HH
Sitte
Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.
Thomas
T
Stockner
D026905:Amino Acid Transport Systems / Q000737:chemistry* / Q000648:ultrastructure*; D001224:Aspartic Acid / Q000737:chemistry*; D001665:Binding Sites; D004058:Diffusion; D008956:Models, Chemical; D062105:Molecular Docking Simulation; D011485:Protein Binding; D011487:Protein Conformation; D012964:Sodium / Q000737:chemistry*
D026905:Amino Acid Transport Systems; D001224:Aspartic Acid; D012964:Sodium