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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.


Substrate-binding site and specificity regions of UapA.(a) A monomer of UapA shown in grey with the key substrate-binding regions of the protein coloured as in Fig. 1. The blue triangle indicates the solvent-filled translocation channel from the substrate-binding site to the cytoplasm. Xanthine is shown in cyan space-filling model. (b) Zoomed-in version of the xanthine-binding site coloured as in Fig. 1 and with xanthine shown in stick model. Residues involved in substrate binding are shown in stick representation and labelled. Side chain and main chain groups of Glu356, Gln408 and Ala407 and main chain groups of Phe155, Val153 are all within H-bonding distance of the xanthine. Essentially the main contacts are between the amide N of Phe155 and N9 of xanthine, Glu356 and N7–H, Gln408 and N1–H and C2=O, and the amide N of Ala407 and C6=O. The side chain groups of Phe155 and Phe406 also pack around the xanthine. Other residues from TMs 1, 3 and 8 contribute to the architecture of the binding site as indicated.
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f2: Substrate-binding site and specificity regions of UapA.(a) A monomer of UapA shown in grey with the key substrate-binding regions of the protein coloured as in Fig. 1. The blue triangle indicates the solvent-filled translocation channel from the substrate-binding site to the cytoplasm. Xanthine is shown in cyan space-filling model. (b) Zoomed-in version of the xanthine-binding site coloured as in Fig. 1 and with xanthine shown in stick model. Residues involved in substrate binding are shown in stick representation and labelled. Side chain and main chain groups of Glu356, Gln408 and Ala407 and main chain groups of Phe155, Val153 are all within H-bonding distance of the xanthine. Essentially the main contacts are between the amide N of Phe155 and N9 of xanthine, Glu356 and N7–H, Gln408 and N1–H and C2=O, and the amide N of Ala407 and C6=O. The side chain groups of Phe155 and Phe406 also pack around the xanthine. Other residues from TMs 1, 3 and 8 contribute to the architecture of the binding site as indicated.

Mentions: Density consistent with xanthine is visible in a clearly defined binding site approximately halfway across the membrane in a cleft formed by the half helices of TMs 3 and 10 (Fig. 2a; and extended data, Fig. 4), similar to the uracil-binding site in UraA. A number of residues were predicted to be involved in xanthine binding by previous mutational analysis7819. The current structure confirms many of these earlier predictions, revealing xanthine is within hydrogen-bonding distance of the side chains of Gln408 (TM 10) and Glu356 (TM 8), the main chain nitrogen of Ala407 at the N terminus of TM 10 and the carbonyl oxygen of Val153 preceding TM 3, as well as the main chain nitrogen of Phe155 of TM 3. The phenyl rings of both Phe155 and Phe406 pack onto the xanthine (Fig. 2b). The orientation of the substrate (Fig. 2b) is corroborated by the position of the electron density peak in an anomalous difference map obtained for a complex of UapA crystallized with 8-bromoxanthine (Supplementary Fig. 4), and with structure activity relationships of xanthine analogues indicating Q408 interacts with N1–H and C2=O of xanthine while E356 interacts with N7–H (ref. 9). With the current structure we cannot completely rule out the substrate adopting the alternative conformation with E356 interacting with N9–H, however in light of all the current evidence the most likely orientation of xanthine is the one presented in Fig. 2b. The residues directly interacting with the xanthine are all from the core domain and in the structure a detergent molecule packs over the xanthine separating it from TM 12 of the gate domain (Supplementary Fig. 5). The xanthine is fully accessible to the inward-facing side of the protein through this detergent-filled cavity.


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)

Substrate-binding site and specificity regions of UapA.(a) A monomer of UapA shown in grey with the key substrate-binding regions of the protein coloured as in Fig. 1. The blue triangle indicates the solvent-filled translocation channel from the substrate-binding site to the cytoplasm. Xanthine is shown in cyan space-filling model. (b) Zoomed-in version of the xanthine-binding site coloured as in Fig. 1 and with xanthine shown in stick model. Residues involved in substrate binding are shown in stick representation and labelled. Side chain and main chain groups of Glu356, Gln408 and Ala407 and main chain groups of Phe155, Val153 are all within H-bonding distance of the xanthine. Essentially the main contacts are between the amide N of Phe155 and N9 of xanthine, Glu356 and N7–H, Gln408 and N1–H and C2=O, and the amide N of Ala407 and C6=O. The side chain groups of Phe155 and Phe406 also pack around the xanthine. Other residues from TMs 1, 3 and 8 contribute to the architecture of the binding site as indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Substrate-binding site and specificity regions of UapA.(a) A monomer of UapA shown in grey with the key substrate-binding regions of the protein coloured as in Fig. 1. The blue triangle indicates the solvent-filled translocation channel from the substrate-binding site to the cytoplasm. Xanthine is shown in cyan space-filling model. (b) Zoomed-in version of the xanthine-binding site coloured as in Fig. 1 and with xanthine shown in stick model. Residues involved in substrate binding are shown in stick representation and labelled. Side chain and main chain groups of Glu356, Gln408 and Ala407 and main chain groups of Phe155, Val153 are all within H-bonding distance of the xanthine. Essentially the main contacts are between the amide N of Phe155 and N9 of xanthine, Glu356 and N7–H, Gln408 and N1–H and C2=O, and the amide N of Ala407 and C6=O. The side chain groups of Phe155 and Phe406 also pack around the xanthine. Other residues from TMs 1, 3 and 8 contribute to the architecture of the binding site as indicated.
Mentions: Density consistent with xanthine is visible in a clearly defined binding site approximately halfway across the membrane in a cleft formed by the half helices of TMs 3 and 10 (Fig. 2a; and extended data, Fig. 4), similar to the uracil-binding site in UraA. A number of residues were predicted to be involved in xanthine binding by previous mutational analysis7819. The current structure confirms many of these earlier predictions, revealing xanthine is within hydrogen-bonding distance of the side chains of Gln408 (TM 10) and Glu356 (TM 8), the main chain nitrogen of Ala407 at the N terminus of TM 10 and the carbonyl oxygen of Val153 preceding TM 3, as well as the main chain nitrogen of Phe155 of TM 3. The phenyl rings of both Phe155 and Phe406 pack onto the xanthine (Fig. 2b). The orientation of the substrate (Fig. 2b) is corroborated by the position of the electron density peak in an anomalous difference map obtained for a complex of UapA crystallized with 8-bromoxanthine (Supplementary Fig. 4), and with structure activity relationships of xanthine analogues indicating Q408 interacts with N1–H and C2=O of xanthine while E356 interacts with N7–H (ref. 9). With the current structure we cannot completely rule out the substrate adopting the alternative conformation with E356 interacting with N9–H, however in light of all the current evidence the most likely orientation of xanthine is the one presented in Fig. 2b. The residues directly interacting with the xanthine are all from the core domain and in the structure a detergent molecule packs over the xanthine separating it from TM 12 of the gate domain (Supplementary Fig. 5). The xanthine is fully accessible to the inward-facing side of the protein through this detergent-filled cavity.

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.