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TL;DR
The findings support a model where protonation of a conserved aspartate residue triggers conformational changes for substrate translocation.
Proceedings of the National Academy of Sciences of the United States of America · 8 authors, 4 centres
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The findings support a model where protonation of a conserved aspartate residue triggers conformational changes for substrate translocation.
The structure was captured in an occluded conformation. Functional experiments using proteoliposomes and electrophysiology confirmed that PurT mediates H+/nucleobase symport, a process dependent on a transmembrane proton gradient and dissipated by the protonophore CCCP. Site-directed mutagenesis revealed that the conserved Asp276 residue is essential for transport; mutating it to Ala or Asn impaired H+-coupled transport but made nucleobase binding pH-independent. Wild-type PurT showed decreased nucleobase binding at lower pH, suggesting Asp276 protonation may trigger the release of substrate and proton to the cytoplasm. The work provides structural and mechanistic insight into the H+-dependent members of the NAT transporter family.