Single-amino acid substitutions alter the specificity and affinity of PDZ domains for their ligands

PDZ domains are modular protein-protein interaction domains that bind to sp ecific C-terminal sequences of membrane proteins and/or to other PDZ domain s. Certain PDZ domains in PSD-95 and syntrophins interact with C-terminal p eptide ligands and heterodimerize with the extended nNOS PDZ domain. The ca pacity to interact with nNOS correlates with the presence of a Lys residue in the carboxylate-binding loop of these PDZ domains. Here, we report that substitution of an Arg for Lys-165 in PSD-95 PDZ2 disrupted its interaction with nNOS, but not with the C terminus of the Shaker-type K+ channel Kv1.4 . The same mutation affected nNOS binding to alpha1- and beta1-syntrophin P DZ domains to a lesser extent, due in part to the stabilizing effect of ter tiary interactions with the canonical nNOS PDZ domain. PDZ domains with an Arg in the carboxylate-binding loop do not bind nNOS; however, substitution with Lys or Ala was able to confer nNOS binding. Our results indicate that the carboxylate-binding loop Lys or Arg is a critical determinant of nNOS binding and that the identity of this residue can profoundly alter one mode of PDZ recognition without affecting another. We also analyzed the effects of mutating Asp-143, a residue in the alphaB helix of alpha1-syntrophin th at forms a tertiary contact with the nNOS PDZ domain. This residue is impor tant for both nNOS and C-terminal peptide binding and confers a preference for peptides with a positively charged residue at position -4. On this basi s, we have identified the C terminus of the Kir2.1 channel as a possible bi nding partner for syntrophin PDZ domains. Together, our results demonstrate that single-amino acid substitutions alter the specificity and affinity of PDZ domains for their ligands.