Structure and Molecular Mechanism of a Nucleobase–Cation–Symport-1 Family Transporter

GAT-1 is a sodium-and chloride-coupled ␥-aminobutyric acid (GABA) transporter, which fulfills an essential role in the synaptic transmission by this neurotransmitter. Cysteine-399 is the major site of inhibition of GAT-1 by membrane-permeant sulfhydryl reagents. This cysteine residue was previously thought to reside on a cytoplasmic loop connecting transmembrane domains (TMs) 8 and 9. However, the crystal structure of LeuT, a bacterial homologue of the mammalian neurotransmitter:sodium symporters, revealed that the residue corresponding to Cys-399 is in fact located in the middle of TM 8. This residue is located to the cytoplasmic side of Asp-395 and Ser-396, whose side chains are thought to ligand one of the two cotransported sodium ions. To determine how the sulfhydryl reagents approach cysteine-399, a cysteine scan of all 35 residues of TM 8 was performed. Sulfhydryl reagents inhibited transport when a cysteine residue was present at either of the positions 399, 402, 406, and 410. SKF-89976A and other non-transportable analogues, which are expected to lock the transporter in a conformation facing the extracellular medium, protected against the sulfhydryl modification at positions 399, 402, and 406. Such a protection was not seen by GABA itself, which actually modestly potentiated the modification at positions 399 and 402. Our results point to an ␣-helical stripe on TM8 lining an aqueous access pathway from the cytoplasm into the binding pocket, which gets occluded in the conformation of the transporter where the binding pocket is exposed to the extracellular medium.

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Transmembrane Domain 8 of the γ-Aminobutyric Acid Transporter GAT-1 Lines a Cytoplasmic Accessibility Pathway into Its Binding Pocket

Ben-Yona

Kanner

2009

“…Transporters alternate between two conformations to expose their binding sites to the cytoplasmic and extracellular side (15)(16)(17)(18)(19)(20)(21)(22). However, prior to conformational changes substrates have to be recognized and bound.…”

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confidence: 99%

Substrate Binding Tunes Conformational Flexibility and Kinetic Stability of an Amino Acid Antiporter

Bippes

Zeltina

Casagrande

et al. 2009

We used single molecule dynamic force spectroscopy to unfold individual serine/threonine antiporters SteT from Bacillus subtilis. The unfolding force patterns revealed interactions and energy barriers that stabilized structural segments of SteT. Substrate binding did not establish strong localized interactions but appeared to be facilitated by the formation of weak interactions with several structural segments. Upon substrate binding, all energy barriers of the antiporter changed thereby describing the transition from brittle mechanical properties of SteT in the unbound state to structurally flexible conformations in the substrate-bound state. The lifetime of the unbound state was much shorter than that of the substrate-bound state. This leads to the conclusion that the unbound state of SteT shows a reduced conformational flexibility to facilitate specific substrate binding and a reduced kinetic stability to enable rapid switching to the bound state. In contrast, the bound state of SteT showed an increased conformational flexibility and kinetic stability such as required to enable transport of substrate across the cell membrane. This result supports the working model of antiporters in which alternate substrate access from one to the other membrane surface occurs in the substrate-bound state.

“…Such residues are expected to face other hydrophilic parts of the protein and/or the solvent-accessible environment of the binding pocket and often play crucial roles in substrate binding and the mechanism of energy coupling in active transport (11)(12)(13)(14). Employing systematic site-directed mutagenesis of a set of 14 putatively charged and 7 highly polar residues predicted to lie in TMs ( Fig.…”

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confidence: 99%

Role of Intramembrane Polar Residues in the YgfO Xanthine Permease

Karena

Frillingos

2009

Using the YgfO xanthine permease of Escherichia coli as a bacterial model for the study of the evolutionarily ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family, we performed a systematic Cys-scanning and site-directed mutagenesis of 14 putatively charged (Asp, Glu, His, Lys, or Arg) and 7 highly polar (Gln or Asn) residues that are predicted to lie in transmembrane helices (TMs). Of 21 single-Cys mutants engineered in the background of a functional YgfO devoid of Cys residues (C-less), only four are inactive or have marginal activity (H31C, N93C, E272C, D304C). The 4 residues are conserved throughout the family in TM1 (His-31), TM3 (Asn-93/Ser/Thr), TM8 (Glu-272), and putative TM9a (Asp-304/Asn/Glu). Extensive site-directed mutagenesis in wild-type background showed that H31N and H31Q have high activity and affinity for xanthine but H31Q recognizes novel purine bases and analogues, whereas H31C and H31L have impaired affinity for xanthine and analogues, and H31K or H31R impairs expression in the membrane. N93S and N93A are highly active but more promiscuous for recognition of analogues at the imidazole moiety of substrate, N93D has low activity, N93T has low affinity for xanthine or analogues, and N93Q or N93C is inactive. All mutants replacing Glu-272 or Asp-304, including E272D, E272Q, D304E, and D304N, are inactive, although expressed to high levels in the membrane. Finally, one of the 17 assayable single-Cys mutants, Q258C, was sensitive to inactivation by N-ethylmaleimide. The findings suggest that polar residues important for the function of YgfO cluster in TMs 1, 3, 8 and 9a.The nucleobase-ascorbate transporter (NAT) 2 or nucleobase-cation symporter-2 (NCS2) family is an evolutionarily ubiquitous family of purine, pyrimidine, and L-ascorbate transporters, with members specific for cellular uptake of uracil, xanthine, or uric acid (microbial and plant genomes) or vitamin C (mammalian genomes) (1, 2). Despite their importance for the recognition and uptake of several frontline purine-related drugs, NAT/NCS2 members have not been studied systematically at the molecular level, and high resolution structures or mechanistic models are missing. More than 1000 sequence entries are known, but few have been functionally characterized to date. The best studied eukaryotic member is UapA, a high affinity uric acid/xanthine:H ϩ symporter from the ascomycote Aspergillus nidulans (3-7). Studies with chimeric transporter constructs (3), site-directed mutagenesis, second-site suppressors, and kinetic inhibition analysis of ligand specificity have shown that a conserved NAT/NCS2 motif region between putative transmembrane helices 8 and 9 of UapA includes determinants of substrate recognition and selectivity, with at least one residue (Gln-408) implicated in binding with the imidazole moiety of purine (4), whereas a conserved QH motif at the middle of TM1 is important for activity and/or correct targeting to the plasma membrane (5), and an aromatic residue at the middle of TM12 (Phe-528) may act as a purine subst...