Unique Role For The -5′ Position In The Carboxyl Terminus Of GIRK3 Channel In Determining Binding Specificity To The PDZ Domain Of Sorting Nexin 27

Abstract

We recently established that sorting nexin 27 (SNX27) regulates trafficking of neuronal GIRK2/3 but not the closely related IRK1 channels. This regulation is achieved by the interaction of the C-terminal PDZ binding motif (-SKV) of GIRK3 with the PDZ domain of SNX27. We also found that IRK1(-SEI) binds to PSD95-PDZ2 but not to SNX27-PDZ. Due to the similarity in the PDZ binding motif between IRK1 and GIRK3, we hypothesized that amino acids in the -4′ and -5′ position are important for determining PDZ binding specificity. Using in vitro binding assays, we discovered that exchanging the these amino acids, IRK1(-ESESKV) and GIRK3(-RRESKV), reversed the binding specificity, suggesting a critical role for -5′ glutamate for binding SNX27 and -5′ arginine for binding PSD95. Further mutagenesis in IRK1 revealed that aspartate or glutamine substitution at -5′ position, IRK1(-DRESKV) and IRK1(-QRESKV), promotes binding to SNX27-PDZ. Surprisingly, substitution with the large, hydrophobic tryptophan enhanced SNX27 binding more than the wild-type glutamate; IRK1(-WRESKV) was 5±1.3 fold greater than IRK1(-ESESKV). We next investigated the consequence of altering the PDZ binding specificity using a clustering assay. CFP-tagged IRK1 or GIRK3 were co-expressed with YFP-tagged PSD95 or SNX27 in COS7 cells. Using TIRF microscopy to visualize proteins at or near the cell membrane, we observed that mutant IRK1(-ESESKV) and wild-type GIRK3(-ESESKV) partially co-localized with SNX27 but did not form clusters. By contrast, mutant GIRK3(-RRESKV) and IRK1(-RRESKV) co-clustered with PSD95. We then used X-ray crystallography to solve the crystal structure of SNX27-PDZ complexed with ESESKV and are now using the structural data to correlate with binding data. These studies provide new details into the specificity of PDZ binding among class I PDZ binding motifs