SummaryPDZ protein interaction domains are typically selective for C-terminal ligands, however, non-Cterminal, "internal" ligands have also been identified. The PDZ domain from the cell polarity protein Par-6 binds C-terminal ligands and an internal sequence from the protein Pals1/Stardust. The structure of the Pals1-Par-6 PDZ complex reveals that the PDZ ligand-binding site is deformed to allow for internal binding. While binding of the Rho GTPase Cdc42 to a CRIB domain adjacent to the Par-6 PDZ regulates binding of C-terminal ligands, the conformational change that occurs upon binding of Pals1 renders its binding independent of Cdc42. These results suggest a mechanism by which the requirement for a C-terminus can be readily bypassed by PDZ ligands and reveal a complex set of cooperative and competitive interactions in Par-6 that are likely to be important for cell polarity regulation.Protein interaction domains form the backbone of cellular information flow 1 . The PDZ protein interaction domain participates in a wide variety of signaling pathways and is one of the most common in metazoan genomes 2 . Because they often occur in multiple instances in the same polypeptide, PDZ domains are thought to serve an organizational role in signal transduction pathways 3 . Given the large number of PDZ domains, several modes of ligand recognition exist whose mechanisms are still being elucidated.PDZ domains bind to short sequences of five to seven residues in their target proteins 4,5 . Although these recognition sequences have a low information content, specificity is typically enhanced by the requirement that the sequence occurs at the C-terminus. C-terminal recognition is found to fall into several different classes, depending on the identity of critical binding residues 3 . For example, class I PDZ ligands have a consensus sequence of -S-X-V-COOH.The requirement for a C-terminus results from a steric rather than electrostatic mechanism. The peptide-binding pocket is constructed such that residues that extend past the C-terminus clash with a conserved PDZ segment known as the carboxylate-binding loop 3,4 . Although Cterminal ligands possess a negatively charged carboxylate, studies of salt effects on the binding reaction suggest that electrostatic contributions are negligible 6 . Because of the additional specificity provided by the C-terminus, enforcement of C-terminal binding is an important component of PDZ -ligand recognition.While recognition of C-terminal motifs appears to be the dominant mode of PDZ-ligand interaction, non-C-terminal (also known as "internal"), PDZ ligands also exist. Compared to our understanding of C-terminal PDZ ligands, however, the mechanism of internal ligand recognition is much less clear. The best-characterized internal PDZ interactions involve ligands that adopt a specific conformation that adheres to the steric requirements of the PDZ binding Correspondence should be addressed to K.E.P. (prehoda@molbio.uoregon.edu) . pocket. For example, in the hetero-oligomerization of the nNOS and sy...

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