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Structure of eukaryotic purine/H(+) symporter UapA suggests a role for homodimerization in transport activity.

Alguel Y, Amillis S, Leung J, Lambrinidis G, Capaldi S, Scull NJ, Craven G, Iwata S, Armstrong A, Mikros E, Diallinas G, Cameron AD, Byrne B - Nat Commun (2016)

Bottom Line: The structure shows UapA in an inward-facing conformation with xanthine bound to residues in the core domain.Analysis of dominant negative mutants is consistent with dimerization playing a key role in transport.We postulate that UapA uses an elevator transport mechanism likely to be shared with other structurally homologous transporters including anion exchangers and prestin.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College London, London SW7 2AZ, UK.

ABSTRACT
The uric acid/xanthine H(+) symporter, UapA, is a high-affinity purine transporter from the filamentous fungus Aspergillus nidulans. Here we present the crystal structure of a genetically stabilized version of UapA (UapA-G411VΔ1-11) in complex with xanthine. UapA is formed from two domains, a core domain and a gate domain, similar to the previously solved uracil transporter UraA, which belongs to the same family. The structure shows UapA in an inward-facing conformation with xanthine bound to residues in the core domain. Unlike UraA, which was observed to be a monomer, UapA forms a dimer in the crystals with dimer interactions formed exclusively through the gate domain. Analysis of dominant negative mutants is consistent with dimerization playing a key role in transport. We postulate that UapA uses an elevator transport mechanism likely to be shared with other structurally homologous transporters including anion exchangers and prestin.

No MeSH data available.


Related in: MedlinePlus

Structure of UapA.(a) Topology diagram of UapA. α-helices are represented by cylinders and β-strands by arrows. The substrate-binding site between the amino termini of TMs 3 and 10 is indicated by the schematic of xanthine (cyan). The protein is arranged into two domains, a gate domain consisting of TMs 5, 6, 7, 12, 13 and 14 (blue), and a core domain consisting of TMs 1, 2, 3, 4, 8, 9, 10 and 11 (red). The half helices and short β-strand regions of TMs 3 and 10 (both part of the core domain) are coloured yellow and green, respectively. (b) Ribbon representation of the UapA monomer. The gate domain is shown in blue while most of the core domain is shown in red. The amphipathic helices that link the core and gate domains are shown in grey. Xanthine is shown in cyan as a space-filling model and the disulphide bond is shown in orange sticks. Only the N-terminal (residues 12–65) and C-terminal (residues 546–574) ends are missing from the structure. (c) UapA dimer from the cytoplasmic side. One monomer is shown as in b, with the helices numbered, and the other is shown in surface representation with the same colouring as in a. The xanthine in the surface representation is labelled. (d) Surface representation of the dimer looking through the membrane. The pale blue lines indicate the likely location of the membrane.
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f1: Structure of UapA.(a) Topology diagram of UapA. α-helices are represented by cylinders and β-strands by arrows. The substrate-binding site between the amino termini of TMs 3 and 10 is indicated by the schematic of xanthine (cyan). The protein is arranged into two domains, a gate domain consisting of TMs 5, 6, 7, 12, 13 and 14 (blue), and a core domain consisting of TMs 1, 2, 3, 4, 8, 9, 10 and 11 (red). The half helices and short β-strand regions of TMs 3 and 10 (both part of the core domain) are coloured yellow and green, respectively. (b) Ribbon representation of the UapA monomer. The gate domain is shown in blue while most of the core domain is shown in red. The amphipathic helices that link the core and gate domains are shown in grey. Xanthine is shown in cyan as a space-filling model and the disulphide bond is shown in orange sticks. Only the N-terminal (residues 12–65) and C-terminal (residues 546–574) ends are missing from the structure. (c) UapA dimer from the cytoplasmic side. One monomer is shown as in b, with the helices numbered, and the other is shown in surface representation with the same colouring as in a. The xanthine in the surface representation is labelled. (d) Surface representation of the dimer looking through the membrane. The pale blue lines indicate the likely location of the membrane.

Mentions: The overall structure of UapA contains 14 transmembrane domains (TMs) organized into a 7+7 TM fold divided into a core (TMs 1–4 and 8–11) and a gate (TMs 5–7 and 12–14) domain, similar to UraA10 (Fig. 1a,b and Supplementary Fig. 1). A key feature is that TMs 3 and 10 only extend halfway through the protein, being followed by short β-strands and random coils that crossover in the centre of the protein (Fig. 1a,b). Compared with the bacterial protein UraA, UapA contains substantially longer loop regions (Supplementary Fig. 2). Interestingly, the extracellular loop between TMs 3 and 4 contains a disulphide bond between Cys174 and Cys185, residues highly conserved in fungi (Fig. 1b and Supplementary Fig. 3). Mutating either residue to serine results in both marked reduction in heterologous expression and almost complete abolition of sorting to the membrane (Supplementary Fig. 3) indicating that the formation of this disulphide bond is important for correct intracellular folding and localization of UapA. This is also the case for other eukaryotic membrane transport proteins2021. UapA forms a dimer with an extensive interface and a buried surface area of 6,000 Å2, involving TMs 12, 13 and 14 of the gate domain. TM 13 is particularly closely associated with dimer formation, fitting into a cleft formed by the opposite monomer (Fig. 1c,d).


Structure of eukaryotic purine/H(+) symporter UapA suggests a role for homodimerization in transport activity.

Alguel Y, Amillis S, Leung J, Lambrinidis G, Capaldi S, Scull NJ, Craven G, Iwata S, Armstrong A, Mikros E, Diallinas G, Cameron AD, Byrne B - Nat Commun (2016)

Structure of UapA.(a) Topology diagram of UapA. α-helices are represented by cylinders and β-strands by arrows. The substrate-binding site between the amino termini of TMs 3 and 10 is indicated by the schematic of xanthine (cyan). The protein is arranged into two domains, a gate domain consisting of TMs 5, 6, 7, 12, 13 and 14 (blue), and a core domain consisting of TMs 1, 2, 3, 4, 8, 9, 10 and 11 (red). The half helices and short β-strand regions of TMs 3 and 10 (both part of the core domain) are coloured yellow and green, respectively. (b) Ribbon representation of the UapA monomer. The gate domain is shown in blue while most of the core domain is shown in red. The amphipathic helices that link the core and gate domains are shown in grey. Xanthine is shown in cyan as a space-filling model and the disulphide bond is shown in orange sticks. Only the N-terminal (residues 12–65) and C-terminal (residues 546–574) ends are missing from the structure. (c) UapA dimer from the cytoplasmic side. One monomer is shown as in b, with the helices numbered, and the other is shown in surface representation with the same colouring as in a. The xanthine in the surface representation is labelled. (d) Surface representation of the dimer looking through the membrane. The pale blue lines indicate the likely location of the membrane.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837479&req=5

f1: Structure of UapA.(a) Topology diagram of UapA. α-helices are represented by cylinders and β-strands by arrows. The substrate-binding site between the amino termini of TMs 3 and 10 is indicated by the schematic of xanthine (cyan). The protein is arranged into two domains, a gate domain consisting of TMs 5, 6, 7, 12, 13 and 14 (blue), and a core domain consisting of TMs 1, 2, 3, 4, 8, 9, 10 and 11 (red). The half helices and short β-strand regions of TMs 3 and 10 (both part of the core domain) are coloured yellow and green, respectively. (b) Ribbon representation of the UapA monomer. The gate domain is shown in blue while most of the core domain is shown in red. The amphipathic helices that link the core and gate domains are shown in grey. Xanthine is shown in cyan as a space-filling model and the disulphide bond is shown in orange sticks. Only the N-terminal (residues 12–65) and C-terminal (residues 546–574) ends are missing from the structure. (c) UapA dimer from the cytoplasmic side. One monomer is shown as in b, with the helices numbered, and the other is shown in surface representation with the same colouring as in a. The xanthine in the surface representation is labelled. (d) Surface representation of the dimer looking through the membrane. The pale blue lines indicate the likely location of the membrane.
Mentions: The overall structure of UapA contains 14 transmembrane domains (TMs) organized into a 7+7 TM fold divided into a core (TMs 1–4 and 8–11) and a gate (TMs 5–7 and 12–14) domain, similar to UraA10 (Fig. 1a,b and Supplementary Fig. 1). A key feature is that TMs 3 and 10 only extend halfway through the protein, being followed by short β-strands and random coils that crossover in the centre of the protein (Fig. 1a,b). Compared with the bacterial protein UraA, UapA contains substantially longer loop regions (Supplementary Fig. 2). Interestingly, the extracellular loop between TMs 3 and 4 contains a disulphide bond between Cys174 and Cys185, residues highly conserved in fungi (Fig. 1b and Supplementary Fig. 3). Mutating either residue to serine results in both marked reduction in heterologous expression and almost complete abolition of sorting to the membrane (Supplementary Fig. 3) indicating that the formation of this disulphide bond is important for correct intracellular folding and localization of UapA. This is also the case for other eukaryotic membrane transport proteins2021. UapA forms a dimer with an extensive interface and a buried surface area of 6,000 Å2, involving TMs 12, 13 and 14 of the gate domain. TM 13 is particularly closely associated with dimer formation, fitting into a cleft formed by the opposite monomer (Fig. 1c,d).

Bottom Line: The structure shows UapA in an inward-facing conformation with xanthine bound to residues in the core domain.Analysis of dominant negative mutants is consistent with dimerization playing a key role in transport.We postulate that UapA uses an elevator transport mechanism likely to be shared with other structurally homologous transporters including anion exchangers and prestin.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College London, London SW7 2AZ, UK.

ABSTRACT
The uric acid/xanthine H(+) symporter, UapA, is a high-affinity purine transporter from the filamentous fungus Aspergillus nidulans. Here we present the crystal structure of a genetically stabilized version of UapA (UapA-G411VΔ1-11) in complex with xanthine. UapA is formed from two domains, a core domain and a gate domain, similar to the previously solved uracil transporter UraA, which belongs to the same family. The structure shows UapA in an inward-facing conformation with xanthine bound to residues in the core domain. Unlike UraA, which was observed to be a monomer, UapA forms a dimer in the crystals with dimer interactions formed exclusively through the gate domain. Analysis of dominant negative mutants is consistent with dimerization playing a key role in transport. We postulate that UapA uses an elevator transport mechanism likely to be shared with other structurally homologous transporters including anion exchangers and prestin.

No MeSH data available.


Related in: MedlinePlus