Specific interdomain synergy in the UapA transporter determines its unique specificity for uric acid among NAT carriers.
UapA, a uric acid-xanthine permease of Aspergillus nidulans, has been used as a prototype to study structure-function relationships in the ubiquitous nucleobase-ascorbate transporter (NAT) family. Using novel genetic screens, rational mutational design, chimeric NAT molecules, and extensive transport kinetic analyses, we show that dynamic synergy between three distinct domains, transmembrane segment (TMS)1, the TMS8-9 loop, and TMS12, defines the function and specificity of UapA. The TMS8-9 loop includes four residues absolutely essential for substrate binding and transport (Glu356, Asp388, Gln408, and Asn409), whereas TMS1 and TMS12 seem to control, through steric hindrance or electrostatic repulsion, the differential access of purines to the TMS8-9 domain. Thus, UapA specificity is determined directly by the specific interactions of a given substrate with the TMS8-9 loop and indirectly by interactions of this loop with TMS1 and TMS12. We finally show that intramolecular synergy among UapA domains is highly specific and propose that it forms the basis for the evolution of the unique specificity of UapA for uric acid, a property not present in other NAT members.
Papageorgiou
Ioannis
I
Faculty of Biology, University of Athens, Panepistimioupolis 15781, Athens, Greece.
Gournas
Christos
C
Vlanti
Anna
A
Amillis
Sotiris
S
Pantazopoulou
Areti
A
Diallinas
George
G
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001233:Aspergillus nidulans / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D033702:Nucleobase Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014527:Uric Acid / Q000378:metabolism*
D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D033702:Nucleobase Transport Proteins; C083852:UAPA protein, Aspergillus nidulans; D014527:Uric Acid