Molecular determinants of selective and high-affinity binding of the scaffold protein PDZK1 to the transporter URAT1

The renal solute carrier URAT1 (SLC22A12) is essential for urate homeostasis, with loss-of-function linked to renal hypouricemia, nephrolithiasis and lower gout risk. URAT1 function depends on binding the multi-PDZ domain scaffold protein PDZK1 (NHERF3), with a similar role suggested for the related NHERF1. The molecular basis of these interactions remains poorly understood. Using fluorescence anisotropy, we show that full-length human PDZK1 binds the C-terminal peptide of URAT1 with high affinity ( K _D 170 nM), unlike NHERF1 ( K _D >70 µM). The PDZ1 domain of PDZK1 alone is sufficient for high-affinity binding ( K _D 160 nM), while PDZ4 provides a secondary site ( K _D 1.35 µM), with both interactions characterized by rapid kinetics. Gel filtration shows that PDZK1 can bind two URAT1 peptides. X-ray structures of individual PDZ domains from PDZK1 and NHERF1 complexed with the URAT1 peptide reveal the molecular determinants for PDZK1’s higher affinity and selectivity. Murine Pdzk1 and Nherf1 bind Urat1 with high affinity indicating species-specific interactions. These data provide insights into URAT1 regulation by PDZ scaffold proteins with relevance for understanding urate homeostasis regulation and related disorders.