Abstract
The Escherichia coli uracil:proton symporter UraA is a prototypical member of the nucleobase/ascorbate transporter (NAT) or nucleobase/cation symporter 2 (NCS2) family, which corresponds to the human solute carrier family SLC23. UraA consists of 14 transmembrane segments (TMs) that are organized into two distinct domains, the core domain and the gate domain, a structural fold that is also shared by the SLC4 and SLC26 transporters. Here we present the crystal structure of UraA bound to uracil in an occluded state at 2.5 Å resolution. Structural comparison with the previously reported inward-open UraA reveals pronounced relative motions between the core domain and the gate domain as well as intra-domain rearrangement of the gate domain. The occluded UraA forms a dimer in the structure wherein the gate domains are sandwiched by two core domains. In vitro and in vivo biochemical characterizations show that UraA is at equilibrium between dimer and monomer in all tested detergent micelles, while dimer formation is necessary for the transport activity. Structural comparison between the dimeric UraA and the recently reported inward-facing dimeric UapA provides important insight into the transport mechanism of SLC23 transporters.
Highlights
The nucleobase-ascorbate transporter (NAT) family, known as the nucleobase-cation symporter-2 (NCS2) family, mediates the uptake of nucleobases in bacteria, fungi, plants, and animals, and L-ascorbic acid in mammals [1, 2]
An evident difference of the two structures exists at the connecting segment between transmembrane segments (TMs) 13 and 14, which forms a flexible loop in the previous UraAIO structure, but a more rigid β-hairpin in the new structure (Figure 1A)
After refinement of the protein structure, an omit electron density map corresponding to uracil was unambiguously recognized between the core domain (TMs 1-4 and 8-11) and the gate domain (TMs 5-7 and 12-14) (Supplementary information, Figure S1C)
Summary
The nucleobase-ascorbate transporter (NAT) family, known as the nucleobase-cation symporter-2 (NCS2) family, mediates the uptake of nucleobases in bacteria, fungi, plants, and animals, and L-ascorbic acid (vitamin C) in mammals [1, 2]. The mammalian NAT family members include SVCT1 and SVCT2 (the sodium-dependent vitamin C transporter, encoded by SLC23A1 and SLC23A2 genes) [3,4,5,6,7]. The architecture of the NAT family (or SLC23 in human) was originally elucidated from the crystal structure of UraA, which was captured in an inward-open conformation with substrate bound [16]. UraA comprises fourteen transmembrane segments (TMs) that are organized into a pair of “7 + 7” inverted repeats. The substrate uracil is sandwiched by the two domains and coordinated predominantly by residues in the core domain. An unusual feature of UraA is the presence of short anti-parallel β-strands formed by the middle segments of TM3 and TM10, which provide the primary accommodation for uracil [16]
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