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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.


UapA substrate specificity.(a) Location of the different residues involved in substrate specificity. The helical regions of the protein are shown as cylinders coloured as in Fig. 1. TM 13 from the opposite monomer is shown in light blue and indicated by the black dotted line, which traces around the surface of the TM. For clarity the majority of the loop regions of UapA have been removed. The individual residues involved in substrate specificity are shown in magenta stick model and labelled. Xanthine is shown in cyan stick model. (b) Structure of the UapA dimer as a surface representation showing the close proximity of R481 from the opposing monomer, to the substrate-binding site. TM 13 in both monomers is shown in ribbon representation, with R481 indicated in stick representation. Xanthine is shown with cyan carbon atoms.
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f3: UapA substrate specificity.(a) Location of the different residues involved in substrate specificity. The helical regions of the protein are shown as cylinders coloured as in Fig. 1. TM 13 from the opposite monomer is shown in light blue and indicated by the black dotted line, which traces around the surface of the TM. For clarity the majority of the loop regions of UapA have been removed. The individual residues involved in substrate specificity are shown in magenta stick model and labelled. Xanthine is shown in cyan stick model. (b) Structure of the UapA dimer as a surface representation showing the close proximity of R481 from the opposing monomer, to the substrate-binding site. TM 13 in both monomers is shown in ribbon representation, with R481 indicated in stick representation. Xanthine is shown with cyan carbon atoms.

Mentions: Wild-type (WT) UapA cannot bind hypoxanthine or adenine (Supplementary Fig. 6). However, mutants allowing uptake of these purines have been isolated through unbiased genetic approaches22. Mutants selected from these screens are shown in Supplementary Fig. 6. The residues affected primarily fall into one of three different regions of the protein. Phe406 (ref. 8) is located within the binding site. Val463, Ala469 (TM 12)8, Thr526 (ref. 7) and Phe528 (TM 14)22 are clustered at the interface between the gate and core domains near to the substrate-binding site (Fig. 3a). Gln113 and Ala441 (refs 7, 8) are located distant from the binding site in putative hinge regions between the two domains (Fig. 3a). In addition, specificity mutations of Arg481 arise often in the genetic screens8. These mutations do not affect transport of physiological substrates but rather modulate the specificity of the protein allowing low-affinity transport of hypoxanthine or adenine. At first glance and considering the monomer in isolation, the role of Arg481 in substrate specificity is difficult to interpret. However, the UapA dimer structure reveals that this residue lies in close proximity to the xanthine-binding site, but of the opposite monomer (Fig. 3b). MD simulations, in fact, suggest that the bound xanthine and Arg481 come in much closer proximity (2 Å) than observed in the crystal structure (12 Å) (Supplementary Figs 7 and 8).


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)

UapA substrate specificity.(a) Location of the different residues involved in substrate specificity. The helical regions of the protein are shown as cylinders coloured as in Fig. 1. TM 13 from the opposite monomer is shown in light blue and indicated by the black dotted line, which traces around the surface of the TM. For clarity the majority of the loop regions of UapA have been removed. The individual residues involved in substrate specificity are shown in magenta stick model and labelled. Xanthine is shown in cyan stick model. (b) Structure of the UapA dimer as a surface representation showing the close proximity of R481 from the opposing monomer, to the substrate-binding site. TM 13 in both monomers is shown in ribbon representation, with R481 indicated in stick representation. Xanthine is shown with cyan carbon atoms.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: UapA substrate specificity.(a) Location of the different residues involved in substrate specificity. The helical regions of the protein are shown as cylinders coloured as in Fig. 1. TM 13 from the opposite monomer is shown in light blue and indicated by the black dotted line, which traces around the surface of the TM. For clarity the majority of the loop regions of UapA have been removed. The individual residues involved in substrate specificity are shown in magenta stick model and labelled. Xanthine is shown in cyan stick model. (b) Structure of the UapA dimer as a surface representation showing the close proximity of R481 from the opposing monomer, to the substrate-binding site. TM 13 in both monomers is shown in ribbon representation, with R481 indicated in stick representation. Xanthine is shown with cyan carbon atoms.
Mentions: Wild-type (WT) UapA cannot bind hypoxanthine or adenine (Supplementary Fig. 6). However, mutants allowing uptake of these purines have been isolated through unbiased genetic approaches22. Mutants selected from these screens are shown in Supplementary Fig. 6. The residues affected primarily fall into one of three different regions of the protein. Phe406 (ref. 8) is located within the binding site. Val463, Ala469 (TM 12)8, Thr526 (ref. 7) and Phe528 (TM 14)22 are clustered at the interface between the gate and core domains near to the substrate-binding site (Fig. 3a). Gln113 and Ala441 (refs 7, 8) are located distant from the binding site in putative hinge regions between the two domains (Fig. 3a). In addition, specificity mutations of Arg481 arise often in the genetic screens8. These mutations do not affect transport of physiological substrates but rather modulate the specificity of the protein allowing low-affinity transport of hypoxanthine or adenine. At first glance and considering the monomer in isolation, the role of Arg481 in substrate specificity is difficult to interpret. However, the UapA dimer structure reveals that this residue lies in close proximity to the xanthine-binding site, but of the opposite monomer (Fig. 3b). MD simulations, in fact, suggest that the bound xanthine and Arg481 come in much closer proximity (2 Å) than observed in the crystal structure (12 Å) (Supplementary Figs 7 and 8).

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.