Insight into the mechanism of H + -coupled nucleobase transport

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TL;DR

This study presents the 2.80 Å crystal structure of PurT~Cp~, a bacterial H⁺-coupled purine transporter from the NAT family, revealing an occluded conformation with a single purine binding site at the crossover of transmembrane helices 3 and 10. Functional assays demonstrate that transport is strictly dependent on an inwardly directed H⁺ gradient and that conserved Asp276 is essential for pH-dependent substrate binding and H⁺/purine symport. These findings provide mechanistic insight into ion-coupled nucleobase transport, with implications for understanding NAT family transporters and designing therapeutic nucleobase analogs.

Full summary

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**Background:** The nucleobase/ascorbate transporter (NAT) family mediates uptake of nucleobases across all kingdoms of life and also transports vitamin C in vertebrates. Nucleobase analogs are used therapeutically for cancers, viral infections, and inflammatory diseases. While mammalian NAT members are Na⁺-coupled, bacterial, fungal, and plant NATs are thought to be H⁺-coupled. Previous crystal structures of UraA and UapA provided static snapshots, but the mechanism of H⁺-coupled transport remained unclear. This study aimed to elucidate the structural and functional basis of H⁺/nucleobase symport using a bacterial NAT homolog, PurT~Cp~ from *Colwellia psychrerythraea*.

**Methods:** PurT~Cp~ was cloned, expressed in *E. coli*, and purified. Its structure was determined by X-ray crystallography at 2.80 Å resolution (Rwork/Rfree = 21/25%). Functional characterization included scintillation proximity assays (SPA) for binding of radiolabeled purines and pyrimidines, competition binding assays, and transport assays using proteoliposomes with defined H⁺ gradients. Solid-supported membrane (SSM) electrophysiology measured charge movement. Mutants D276A and D276N were generated to test the role of conserved Asp276.

**Key Results:** The structure shows PurT~Cp~ as a homodimer, each protomer with 14 transmembrane segments (TMs) forming a transport domain (TMs 1-4, 8-11) and a scaffold domain (TMs 5-7, 12-14). A bound guanine molecule is occluded at the crossover of TM3 and TM10, coordinated by residues including Asp276. Binding assays revealed that PurT~Cp~ binds purines (guanine, xanthine, adenine, hypoxanthine) with Kd ~2 µM for xanthine and ~3 µM for guanine, but not pyrimidines or ascorbic acid. Binding was pH-dependent: maximal at pH ≥7.5, with half-maximal at ~pH 6.8. Mutants D276A and D276N showed pH-independent binding, with similar activity at pH 5.5 and 8.0, whereas wild-type binding was nearly abolished at pH 5.5. Transport of ³H-xanthine in proteoliposomes required an inwardly directed H⁺ gradient (pHin 8.5, pHout 5.5) and was abolished by the protonophore CCCP. Transport kinetics gave Km ~1.8 µM and Vmax ~100 nmol·mg⁻¹·min⁻¹ (kcat ~0.1 s⁻¹). SSM electrophysiology showed a transient inward current upon xanthine addition to PurT~Cp~-containing proteoliposomes, ~6-fold larger than controls, consistent with H⁺ influx. D276A and D276N mutants were impaired in transport.

**Clinical Implications:** This study provides a structural and mechanistic framework for H⁺-coupled nucleobase transport by NAT family members. The identification of Asp276 as a critical residue for pH sensing and H⁺ coupling may inform the design of nucleobase analog drugs (e.g., allopurinol, 5-fluorouracil, acyclovir) by predicting how they interact with transporters. Understanding the transport mechanism could also aid in developing strategies to modulate drug uptake in therapeutic contexts. However, as this is a basic science study on a bacterial homolog, direct clinical applications require further translational research.