title: Interactions outside the boundaries of the canonical binding groove of a PDZ domain influence ligand binding.
abstract: The postsynaptic density protein-95/discs large/zonula occludens-1 (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 via nuclear magnetic resonance 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 the loss of several intermolecular nuclear Overhauser enhancements 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 in which different binding surfaces outside of the PDZ binding groove could provide sites for specific interactions.
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123 Uppsala, Sweden.
authors.forename: Celestine N
authors.initials: CN
authors.lastname: Chi
authors.forename: S Raza
authors.initials: SR
authors.lastname: Haq
authors.forename: Serena
authors.initials: S
authors.lastname: Rinaldo
authors.forename: Jakob
authors.initials: J
authors.lastname: Dogan
authors.forename: Francesca
authors.initials: F
authors.lastname: Cutruzzolà
authors.forename: Åke
authors.initials: Å
authors.lastname: Engström
authors.forename: Stefano
authors.initials: S
authors.lastname: Gianni
authors.forename: Patrik
authors.initials: P
authors.lastname: Lundström
authors.forename: Per
authors.initials: P
authors.lastname: Jemth
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002151:Calorimetry; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D019277:Entropy; D008024:Ligands; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000378:metabolism; D054731:PDZ Domains / Q000187:drug effects*; D011485:Protein Binding
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C112203:CRIPT protein, human; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D008024:Ligands; D009842:Oligopeptides; C118970:USH1C protein, human