Molecular Determinants for the Complex Binding Specificity of the PDZ Domain in PICK1

Kenneth L Madsen,Thijs Beuming,Masha Y Niv,Chiun-Wen Chang,Kumlesh K Dev,Harel Weinstein,Ulrik Gether

doi:10.1074/jbc.m500577200

Abstract

PICK1 (protein interacting with C kinase 1) contains a single PDZ domain known to mediate interaction with the C termini of several receptors, transporters, ion channels, and kinases. In contrast to most PDZ domains, the PICK1 PDZ domain interacts with binding sequences classifiable as type I (terminating in (S/T)XPhi; X, any residue) as well as type II (PhiXPhi; Phi, any hydrophobic residue). To enable direct assessment of the affinity of the PICK1 PDZ domain for its binding partners we developed a purification scheme for PICK1 and a novel quantitative binding assay based on fluorescence polarization. Our results showed that the PICK1 PDZ domain binds the type II sequence presented by the human dopamine transporter (-WLKV) with an almost 15-fold and >100-fold higher affinity than the type I sequences presented by protein kinase Calpha (-QSAV) and the beta(2)-adrenergic receptor (-DSLL), respectively. Mutational analysis of Lys(83) in the alphaB1 position of the PDZ domain suggested that this residue mimics the function of hydrophobic residues present in this position in regular type II PDZ domains. The PICK1 PDZ domain was moreover found to prefer small hydrophobic residues in the C-terminal P(0) position of the ligand. Molecular modeling predicted a rank order of (Val > Ile > Leu) that was verified experimentally with up to a approximately 16-fold difference in binding affinity between a valine and a leucine in P(0). The results define the structural basis for the unusual binding pattern of the PICK1 PDZ domain by substantiating the critical role of the alphaB1 position (Lys(83)) and of discrete side chain differences in position P(0) of the ligands.

Highlights

With over 540 domains in more than 300 different proteins, PDZ (PSD-95/Disc-large/ZO-1 homology)1 domains are among the most common protein domains in the human genome [1,2,3]
Our results showed that the PICK1 PDZ domain binds the type II sequence presented by the human dopamine transporter (-WLKV) with an almost 15-fold and >100-fold higher affinity than the type I sequences presented by protein kinase C␣ (-QSAV) and the ␤2-adrenergic receptor (-DSLL), respectively
We have aimed to elucidate the determinants for the unusual binding pattern of the PICK1 PDZ domain, in the structural context of a molecular model of the protein. To this end we have developed a purification scheme for PICK1 and a quantitative binding assay based on fluorescence polarization (FP) that enables direct assessment of the affinity of the PICK1 PDZ domain for binding partners

Summary

Introduction

With over 540 domains in more than 300 different proteins, PDZ (PSD-95/Disc-large/ZO-1 homology) domains are among the most common protein domains in the human genome [1,2,3]. It was recently shown that a specific mutation in the carboxylate-binding loop of the PICK1 PDZ domain results in loss of interaction with the type II binding sequence of the L-␣-amino-3-hydroxy-5-methyl-4isoxazole propionic acid (AMPA) receptor subunit GluR2, whereas binding of the type I ligand PKC␣ appears unaffected [25]. This suggests different binding modes of type I versus type II ligands, but does not explain their structural basis

Methods

Results

Conclusion