Inward-facing conformation of glutamate transporters as revealed by their inverted-topology structural repeats.
Glutamate transporters regulate synaptic concentrations of this neurotransmitter by coupling its flux to that of sodium and other cations. Available crystal structures of an archeal homologue of these transporters, GltPh, resemble an extracellular-facing state, in which the bound substrate is occluded only by a small helical hairpin segment called HP2. However, a pathway to the cytoplasmic side of the membrane is not clearly apparent. We previously modeled an alternate state of a transporter from the neurotransmitter:sodium symporter family, which has an entirely different fold, solely on the presence of inverted-topology structural repeats. In GltPh, we identified two distinct sets of inverted-topology repeats and used these repeats to model an inward-facing conformation of the protein. To test this model, we introduced pairs of cysteines into the neuronal glutamate transporter EAAC1, at positions that are >27 A apart in the crystal structures of GltPh, but approximately = 10 A apart in the inward-facing model. Transport by these mutants was activated by pretreatment with the reducing agent dithithreitol. Subsequent treatment with the oxidizing agent copper(II)(1,10-phenantroline)(3) abolished this activation. The inhibition of transport was potentiated under conditions thought to promote the inward-facing conformation of the transporter. By contrast, the inhibition was reduced in the presence of the nontransportable substrate analogue D,L-threo-beta-benzyloxyaspartate, which favors the outward-facing conformation. Other conformation-sensitive accessibility measurements are also accommodated by our inward-facing model. These results suggest that the inclusion of inverted-topology repeats in transporters may provide a general solution to the requirement for two symmetry-related states in a single protein.
Crisman
Thomas J
TJ
Computational Structural Biology Group, The Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt am Main, Germany.
Qu
Shaogang
S
Kanner
Baruch I
BI
Forrest
Lucy R
LR
D027322:Amino Acid Transport System X-AG / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D019843:Archaeal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000187:drug effects; D004229:Dithiothreitol / Q000494:pharmacology; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D043902:Pyrococcus horikoshii / Q000187:drug effects / Q000378:metabolism*; D011817:Rabbits; D020449:Repetitive Sequences, Amino Acid; D013379:Substrate Specificity / Q000187:drug effects; D014981:Xenopus
D027322:Amino Acid Transport System X-AG; D019843:Archaeal Proteins; D003432:Cross-Linking Reagents; D050505:Mutant Proteins; D010618:Phenanthrolines; D003545:Cysteine; D004229:Dithiothreitol