title: Induced fit substrate binding to an archeal glutamate transporter homologue.
abstract: Excitatory amino acid transporters (EAATs) are a class of glutamate transporters that terminate glutamatergic synaptic transmission in the mammalian CNS. GltPh, an archeal EAAT homolog from Pyrococcus horikoshii, is currently the only member with a known 3D structure. Here, we studied the kinetics of substrate binding of a single tryptophan mutant (L130W) GltPh in detergent micelles. At low millimolar [Na(+)], the addition of L-aspartate resulted in complex time courses of W130 fluorescence changes over tens of seconds. With increasing [Na(+)], the kinetics were dominated by a fast component [k(obs,fast); K(D) (Na(+)) = 22 ± 3 mM, n(Hill )= 1.7 ± 0.3] with values of k(obs,fast) rising in a saturable manner to ≈ 500 s(-1) (at 6 °C) with increasing [L-aspartate]. The binding kinetics of L-aspartate differed from the binding kinetics of two alternative substrates: L-cysteine sulfinic acid and d-aspartate. L-cysteine sulfinic acid bound with higher affinity than L-aspartate but involved lower saturating rates, whereas the saturating rates after D-aspartate binding were higher. Thus, after the association of two Na(+) to the empty transporter, GltPh binds amino acids by induced fit. Cross-linking and proteolysis experiments suggest that the induced fit results from the closure of helical hairpin 2. This conformational change is faster for GltPh than for most mammalian homologues, whereas the amino acid association rates are similar. Our data reveal the importance of induced fit for substrate selection in EAATs and illustrate how high-affinity binding and the efficient transport of glutamate can be accomplished simultaneously by this class of transporters.
authors.affiliation: Institut für Neurophysiologie, Medizinische Hochschule, 30625 Hannover, Germany. ewers.david@gmail.com
authors.forename: David
authors.initials: D
authors.lastname: Ewers
authors.forename: Toni
authors.initials: T
authors.lastname: Becher
authors.forename: Jan-Philipp
authors.initials: JP
authors.lastname: Machtens
authors.forename: Ingo
authors.initials: I
authors.lastname: Weyand
authors.forename: Christoph
authors.initials: C
authors.lastname: Fahlke
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000737:chemistry / Q000378:metabolism*; D001105:Archaea / Q000378:metabolism*; D007536:Isomerism; D008958:Models, Molecular; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D027322:Amino Acid Transport System X-AG
keywords: sodium-coupled transport; stopped flow; uptake