TY - JOUR

T1 - Molecular determinants for the complex binding specificity of the PDZ domain in PICK1

AU - Madsen, Kenneth L

AU - Beuming, Thijs

AU - Niv, Masha Y

AU - Chang, Chiun-Wen

AU - Dev, Kumlesh K

AU - Weinstein, Harel

AU - Gether, Ulrik

N1 - Keywords: Animals; Binding Sites; Carrier Proteins; Computer Simulation; Dopamine Plasma Membrane Transport Proteins; Fluorescence Polarization; Glutathione Transferase; Humans; Lysine; Membrane Glycoproteins; Membrane Transport Proteins; Models, Molecular; Mutagenesis; Nerve Tissue Proteins; Nuclear Proteins; Protein Binding; Protein Kinase C; Protein Kinase C-alpha; Rats; Receptors, Adrenergic, beta-2; Recombinant Fusion Proteins; Structure-Activity Relationship

PY - 2005

Y1 - 2005

N2 - 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.

AB - 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.

U2 - 10.1074/jbc.M500577200

DO - 10.1074/jbc.M500577200

M3 - Journal article

C2 - 15774468

VL - 280

SP - 20539

EP - 20548

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 21

ER -