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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 translocation pathway and transport mechanism in UapA.(a) Surface model of the UapA dimer showing the xanthine trajectory during MD. The image is zoomed into the substrate translocation channel. The regions of the protein dimer are coloured as in Fig. 1, and R481 from the opposite monomer is labelled. The positions adopted by the xanthine during translocation (Supplementary Figs 7 and 8) are coloured from the xanthine-binding site (blue) to the intracellular side of the protein (red). (b–d) UapA is postulated to function using an elevator mechanism, involving displacement of TMs 1, 3, 8 and 10 of the core domain, which effectively carries the substrate from one side of the membrane to the other. (b) Postulated outward-facing structure of key regions of UapA, with the helices shown as cylinders. The positions of grey-coloured helices (TMs 1, 3, 8 and 10 from the core domain) are modelled based on the outward-facing structure of AE1 (PDB 4YZF). The positions of helices 5 and 12 (blue) are as in the crystal structure. The arrow indicates access to the postulated substrate-binding site shown by the grey space-filling model of xanthine. (c) Overlay of the helices 1, 3, 8 and 10 in the postulated outward-facing conformation (grey) and the inward-facing conformation from the crystal structure (red). The position of xanthine in both conformations is shown in grey (outward facing) and cyan (inward facing). The large grey–red-coloured arrow indicates the direction of movement of the core domain helices during change from the outward- to inward-facing conformations. (d) Inward-facing conformation of the helices in blue and red as seen in the crystal structure. Xanthine is shown in cyan. The large arrow indicates the substrate translocation trajectory from the binding site to the intracellular side of the membrane (as seen in a). Although this seems to be the key conformational change it is possible that other domain movements are involved, for example, TM 14 is kinked and this may need to flex for the core to move. Interestingly Thr526 and Phe528, key for specificity, are located at this kink.
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f5: Substrate translocation pathway and transport mechanism in UapA.(a) Surface model of the UapA dimer showing the xanthine trajectory during MD. The image is zoomed into the substrate translocation channel. The regions of the protein dimer are coloured as in Fig. 1, and R481 from the opposite monomer is labelled. The positions adopted by the xanthine during translocation (Supplementary Figs 7 and 8) are coloured from the xanthine-binding site (blue) to the intracellular side of the protein (red). (b–d) UapA is postulated to function using an elevator mechanism, involving displacement of TMs 1, 3, 8 and 10 of the core domain, which effectively carries the substrate from one side of the membrane to the other. (b) Postulated outward-facing structure of key regions of UapA, with the helices shown as cylinders. The positions of grey-coloured helices (TMs 1, 3, 8 and 10 from the core domain) are modelled based on the outward-facing structure of AE1 (PDB 4YZF). The positions of helices 5 and 12 (blue) are as in the crystal structure. The arrow indicates access to the postulated substrate-binding site shown by the grey space-filling model of xanthine. (c) Overlay of the helices 1, 3, 8 and 10 in the postulated outward-facing conformation (grey) and the inward-facing conformation from the crystal structure (red). The position of xanthine in both conformations is shown in grey (outward facing) and cyan (inward facing). The large grey–red-coloured arrow indicates the direction of movement of the core domain helices during change from the outward- to inward-facing conformations. (d) Inward-facing conformation of the helices in blue and red as seen in the crystal structure. Xanthine is shown in cyan. The large arrow indicates the substrate translocation trajectory from the binding site to the intracellular side of the membrane (as seen in a). Although this seems to be the key conformational change it is possible that other domain movements are involved, for example, TM 14 is kinked and this may need to flex for the core to move. Interestingly Thr526 and Phe528, key for specificity, are located at this kink.

Mentions: Molecular dynamics simulations suggest a translocation pathway from the binding site to the inside of the cell (Fig. 5a and Supplementary Figs 7 and 8). Given the resolution of the crystal structure, these data should be treated with caution; however, the results are consistent with the biochemical data. Initially, xanthine remains strongly ligated to Gln408 whilst tumbling within the binding cavity. It interacts with Leu459, Val463 and Ser466 close to the binding site while Arg481 of the opposite monomer approaches the central binding cavity. This effectively creates a specificity barrier on the pathway to the cytoplasm with the xanthine forming transient H-bond and π–π stacking interactions with Arg481 before finally moving to the cytosol with characteristic conformational changes of the Arg481 side chain and the Arg481–Gln408 interaction (Fig. 5a and Supplementary Fig. 8). During this procedure there is a gradual displacement of TM 10, which may be required for ligand exit.


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 translocation pathway and transport mechanism in UapA.(a) Surface model of the UapA dimer showing the xanthine trajectory during MD. The image is zoomed into the substrate translocation channel. The regions of the protein dimer are coloured as in Fig. 1, and R481 from the opposite monomer is labelled. The positions adopted by the xanthine during translocation (Supplementary Figs 7 and 8) are coloured from the xanthine-binding site (blue) to the intracellular side of the protein (red). (b–d) UapA is postulated to function using an elevator mechanism, involving displacement of TMs 1, 3, 8 and 10 of the core domain, which effectively carries the substrate from one side of the membrane to the other. (b) Postulated outward-facing structure of key regions of UapA, with the helices shown as cylinders. The positions of grey-coloured helices (TMs 1, 3, 8 and 10 from the core domain) are modelled based on the outward-facing structure of AE1 (PDB 4YZF). The positions of helices 5 and 12 (blue) are as in the crystal structure. The arrow indicates access to the postulated substrate-binding site shown by the grey space-filling model of xanthine. (c) Overlay of the helices 1, 3, 8 and 10 in the postulated outward-facing conformation (grey) and the inward-facing conformation from the crystal structure (red). The position of xanthine in both conformations is shown in grey (outward facing) and cyan (inward facing). The large grey–red-coloured arrow indicates the direction of movement of the core domain helices during change from the outward- to inward-facing conformations. (d) Inward-facing conformation of the helices in blue and red as seen in the crystal structure. Xanthine is shown in cyan. The large arrow indicates the substrate translocation trajectory from the binding site to the intracellular side of the membrane (as seen in a). Although this seems to be the key conformational change it is possible that other domain movements are involved, for example, TM 14 is kinked and this may need to flex for the core to move. Interestingly Thr526 and Phe528, key for specificity, are located at this kink.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Substrate translocation pathway and transport mechanism in UapA.(a) Surface model of the UapA dimer showing the xanthine trajectory during MD. The image is zoomed into the substrate translocation channel. The regions of the protein dimer are coloured as in Fig. 1, and R481 from the opposite monomer is labelled. The positions adopted by the xanthine during translocation (Supplementary Figs 7 and 8) are coloured from the xanthine-binding site (blue) to the intracellular side of the protein (red). (b–d) UapA is postulated to function using an elevator mechanism, involving displacement of TMs 1, 3, 8 and 10 of the core domain, which effectively carries the substrate from one side of the membrane to the other. (b) Postulated outward-facing structure of key regions of UapA, with the helices shown as cylinders. The positions of grey-coloured helices (TMs 1, 3, 8 and 10 from the core domain) are modelled based on the outward-facing structure of AE1 (PDB 4YZF). The positions of helices 5 and 12 (blue) are as in the crystal structure. The arrow indicates access to the postulated substrate-binding site shown by the grey space-filling model of xanthine. (c) Overlay of the helices 1, 3, 8 and 10 in the postulated outward-facing conformation (grey) and the inward-facing conformation from the crystal structure (red). The position of xanthine in both conformations is shown in grey (outward facing) and cyan (inward facing). The large grey–red-coloured arrow indicates the direction of movement of the core domain helices during change from the outward- to inward-facing conformations. (d) Inward-facing conformation of the helices in blue and red as seen in the crystal structure. Xanthine is shown in cyan. The large arrow indicates the substrate translocation trajectory from the binding site to the intracellular side of the membrane (as seen in a). Although this seems to be the key conformational change it is possible that other domain movements are involved, for example, TM 14 is kinked and this may need to flex for the core to move. Interestingly Thr526 and Phe528, key for specificity, are located at this kink.
Mentions: Molecular dynamics simulations suggest a translocation pathway from the binding site to the inside of the cell (Fig. 5a and Supplementary Figs 7 and 8). Given the resolution of the crystal structure, these data should be treated with caution; however, the results are consistent with the biochemical data. Initially, xanthine remains strongly ligated to Gln408 whilst tumbling within the binding cavity. It interacts with Leu459, Val463 and Ser466 close to the binding site while Arg481 of the opposite monomer approaches the central binding cavity. This effectively creates a specificity barrier on the pathway to the cytoplasm with the xanthine forming transient H-bond and π–π stacking interactions with Arg481 before finally moving to the cytosol with characteristic conformational changes of the Arg481 side chain and the Arg481–Gln408 interaction (Fig. 5a and Supplementary Fig. 8). During this procedure there is a gradual displacement of TM 10, which may be required for ligand exit.

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.