Interactions outside the boundaries of the canonical binding groove of a PDZ domain influence ligand binding.

The PDZ domain is a protein-protein interaction module with a shallow binding groove where protein ligands bind. However, interactions that are not part of this canonical binding groove are likely to modulate peptide binding. We have investigated such interactions beyond the binding groove for PDZ3 from PSD-95 and a peptide derived from the C-terminus of the natural ligand CRIPT. We found by NMR experiments that up to eight residues of the peptide ligand interact with the PDZ domain, showing that the interaction surface extends far outside of the binding groove as defined by the crystal structure. PDZ3 contains an extra structural element, a C-terminal helix (α3), which is known to affect affinity. Deletion of this helix resulted in loss of several inter-molecular NOEs from peptide residues outside of the binding pocket, suggesting that α3 forms part of the extra binding surface in wild type PDZ3. Site-directed mutagenesis, isothermal titration calorimetry and fluorescence intensity experiments confirmed the importance of both α3 and the N-terminal part of the peptide for the affinity. Our data suggest a general mechanism where different binding surfaces outside of the PDZ binding groove could provide sites for specific interactions.