Redox-regulated affinity of the third PDZ domain in the phosphotyrosine phosphatase PTP-BL for cysteine-containing target peptides.
PDZ domains are protein-protein interaction modules that are crucial for the assembly of structural and signalling complexes. They specifically bind to short C-terminal peptides and occasionally to internal sequences that structurally resemble such peptide termini. The binding of PDZ domains is dominated by the residues at the P(0) and P(-2) position within these C-terminal targets, but other residues are also important in determining specificity. In this study, we analysed the binding specificity of the third PDZ domain of protein tyrosine phosphatase BAS-like (PTP-BL) using a C-terminal combinatorial peptide phage library. Binding of PDZ3 to C-termini is preferentially governed by two cysteine residues at the P(-1) and P(-4) position and a valine residue at the P(0) position. Interestingly, we found that this binding is lost upon addition of the reducing agent dithiothrietol, indicating that the interaction is disulfide-bridge-dependent. Site-directed mutagenesis of the single cysteine residue in PDZ3 revealed that this bridge formation does not occur intermolecularly, between peptide and PDZ3 domain, but rather is intramolecular. These data point to a preference of PTP-BL PDZ3 for cyclic C-terminal targets, which may suggest a redox state-sensing role at the cell cortex.
Department of Cell Biology, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen, the Netherlands.
Lieke C J
LC
van den Berk
Elena
E
Landi
Etelka
E
Harmsen
Luciana
L
Dente
Wiljan J A J
WJ
Hendriks
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D020650:Combinatorial Chemistry Techniques; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D010446:Peptide Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases; D003545:Cysteine