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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

Importance of dimer formation for UapA transport function.(a), In vivo inverted fluorescence microscopy of growing hyphal cells of isogenic strains expressing GFP-tagged WT UapA or mutant versions Q408P, Q408E, N409D or G411V, or co-expressing two copies of UapA (UapA/UapA) or a copy of UapA with each one of the four mutants (Q408P/UapA, Q408E/UapA, N409D/UapA or G411V/UapA). In all cases UapA-GFP fluorescence is associated with the plasma membrane and the septa of growing hyphal cells, a picture typical of properly folded and correctly localized UapA78. Minor cytoplasmic GFP labelling reflects the normal vacuolar turnover of UapA56. Scale bar shown in the UapA-GFP panel, 5 μm. (b) Relevant growth tests of the same strains on either ammonium (NH4+) or xanthine (Xa) as sole nitrogen sources. Notice that full growth on Xa occurs in UapA or UapA/UapA strains, whereas co-expression of the selected loss-of-function mutants with WT UapA (last four subpanels) reduces growth. (c) Initial uptake rates of [3H]-xanthine in strains expressing WT or mutant versions of UapA. Rates of WT UapA are taken as 100%. Measurable Km values for xanthine are also indicated. Error bars represent s.d., n=9. (d) The effect of hypoxanthine on [3H]-xanthine uptake on strains expressing WT UapA (blue line) or co-expressing Q408E/UapA (red line) was assessed. Rates of xanthine uptake in the absence of hypoxanthine for both strains are taken as 100%. Ki values are also indicated. Error bars represent s.d., n=9. Note that solubility issues mean it is not possible to use higher concentrations of hypoxanthine.
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f4: Importance of dimer formation for UapA transport function.(a), In vivo inverted fluorescence microscopy of growing hyphal cells of isogenic strains expressing GFP-tagged WT UapA or mutant versions Q408P, Q408E, N409D or G411V, or co-expressing two copies of UapA (UapA/UapA) or a copy of UapA with each one of the four mutants (Q408P/UapA, Q408E/UapA, N409D/UapA or G411V/UapA). In all cases UapA-GFP fluorescence is associated with the plasma membrane and the septa of growing hyphal cells, a picture typical of properly folded and correctly localized UapA78. Minor cytoplasmic GFP labelling reflects the normal vacuolar turnover of UapA56. Scale bar shown in the UapA-GFP panel, 5 μm. (b) Relevant growth tests of the same strains on either ammonium (NH4+) or xanthine (Xa) as sole nitrogen sources. Notice that full growth on Xa occurs in UapA or UapA/UapA strains, whereas co-expression of the selected loss-of-function mutants with WT UapA (last four subpanels) reduces growth. (c) Initial uptake rates of [3H]-xanthine in strains expressing WT or mutant versions of UapA. Rates of WT UapA are taken as 100%. Measurable Km values for xanthine are also indicated. Error bars represent s.d., n=9. (d) The effect of hypoxanthine on [3H]-xanthine uptake on strains expressing WT UapA (blue line) or co-expressing Q408E/UapA (red line) was assessed. Rates of xanthine uptake in the absence of hypoxanthine for both strains are taken as 100%. Ki values are also indicated. Error bars represent s.d., n=9. Note that solubility issues mean it is not possible to use higher concentrations of hypoxanthine.

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)

Importance of dimer formation for UapA transport function.(a), In vivo inverted fluorescence microscopy of growing hyphal cells of isogenic strains expressing GFP-tagged WT UapA or mutant versions Q408P, Q408E, N409D or G411V, or co-expressing two copies of UapA (UapA/UapA) or a copy of UapA with each one of the four mutants (Q408P/UapA, Q408E/UapA, N409D/UapA or G411V/UapA). In all cases UapA-GFP fluorescence is associated with the plasma membrane and the septa of growing hyphal cells, a picture typical of properly folded and correctly localized UapA78. Minor cytoplasmic GFP labelling reflects the normal vacuolar turnover of UapA56. Scale bar shown in the UapA-GFP panel, 5 μm. (b) Relevant growth tests of the same strains on either ammonium (NH4+) or xanthine (Xa) as sole nitrogen sources. Notice that full growth on Xa occurs in UapA or UapA/UapA strains, whereas co-expression of the selected loss-of-function mutants with WT UapA (last four subpanels) reduces growth. (c) Initial uptake rates of [3H]-xanthine in strains expressing WT or mutant versions of UapA. Rates of WT UapA are taken as 100%. Measurable Km values for xanthine are also indicated. Error bars represent s.d., n=9. (d) The effect of hypoxanthine on [3H]-xanthine uptake on strains expressing WT UapA (blue line) or co-expressing Q408E/UapA (red line) was assessed. Rates of xanthine uptake in the absence of hypoxanthine for both strains are taken as 100%. Ki values are also indicated. Error bars represent s.d., n=9. Note that solubility issues mean it is not possible to use higher concentrations of hypoxanthine.
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f4: Importance of dimer formation for UapA transport function.(a), In vivo inverted fluorescence microscopy of growing hyphal cells of isogenic strains expressing GFP-tagged WT UapA or mutant versions Q408P, Q408E, N409D or G411V, or co-expressing two copies of UapA (UapA/UapA) or a copy of UapA with each one of the four mutants (Q408P/UapA, Q408E/UapA, N409D/UapA or G411V/UapA). In all cases UapA-GFP fluorescence is associated with the plasma membrane and the septa of growing hyphal cells, a picture typical of properly folded and correctly localized UapA78. Minor cytoplasmic GFP labelling reflects the normal vacuolar turnover of UapA56. Scale bar shown in the UapA-GFP panel, 5 μm. (b) Relevant growth tests of the same strains on either ammonium (NH4+) or xanthine (Xa) as sole nitrogen sources. Notice that full growth on Xa occurs in UapA or UapA/UapA strains, whereas co-expression of the selected loss-of-function mutants with WT UapA (last four subpanels) reduces growth. (c) Initial uptake rates of [3H]-xanthine in strains expressing WT or mutant versions of UapA. Rates of WT UapA are taken as 100%. Measurable Km values for xanthine are also indicated. Error bars represent s.d., n=9. (d) The effect of hypoxanthine on [3H]-xanthine uptake on strains expressing WT UapA (blue line) or co-expressing Q408E/UapA (red line) was assessed. Rates of xanthine uptake in the absence of hypoxanthine for both strains are taken as 100%. Ki values are also indicated. Error bars represent s.d., n=9. Note that solubility issues mean it is not possible to use higher concentrations of hypoxanthine.
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.


Related in: MedlinePlus