Energetic determinants of internal motif recognition by PDZ domains

PDZ domains are protein-protein interaction modules that organize intracell ular signaling complexes. Most PDZ domains recognize specific peptide motif s followed by a required COOH-terminus. However, several PDZ domains have b een found which recognize specific internal peptide motifs. The best charac terized example is the syntrophin PDZ domain which, in addition to binding peptide ligands with the consensus sequence -E-S/T-X-V-COOH, also binds the neuronal nitric oxide synthase (nNOS) PDZ domain in a manner that does not depend on its precise COOH-terminal sequence. In the structure of the synt rophin-nNOS PDZ heterodimer complex, the two PDZ domains interact in a head -to-tail fashion, with an internal sequence from the nNOS PDZ domain bindin g precisely at the peptide binding groove of the syntrophin PDZ domain. To understand the energetic basis of this alternative mode of PDZ recognition, we have undertaken an extensive mutagenic and biophysical analysis of the nNOS PDZ domain and its interaction with the syntrophin PDZ domain. Our dat a indicate that the presentation of the nNOS internal motif within the cont ext of a rigid beta -hairpin conformation is absolutely essential to bindin g; amino acids crucial to the structural integrity of the hairpin are as im portant or more important than residues that make direct contacts. The resu lts reveal the general rules of PDZ recognition of diverse ligand types.