Integrated analysis of Shank1 PDZ interactions with C-terminal and internal binding motifs.

PDZ domains constitute a large family of modular domains that are well-known for binding C-terminal motifs of target proteins. Some of them also bind to internal PDZ binding motifs (PDZbms), but this aspect of the PDZ interactome is poorly studied. Here we explored internal PDZbm-mediated interactions using the PDZ domain of Shank1 as a model. We identified a series of human Shank1 ligands with C-terminal or internal PDZbms using proteomic peptide-phage display, and established that while the consensus sequence of C-terminal ligands is x-T-x-(L/F)-COOH, the consensus of internal PDZbm is exclusively x-T-x-F-x, where x is any amino acid. We found that the affinities of PDZbm interactions are in the low micromolar range. The crystal structure of the complex between Shank1 PDZ and an internal PDZbm revealed that the binding mode of internal PDZbms was similar to that of C-terminal ligands. Pull-down experiments confirmed that both C-terminal and internal PDZbm interactions can occur in the context of full-length proteins. Our study expands the interactome of Shank1 and hints at a largely unexplored interaction space of PDZ domains.

Identification of PDZ Interactions by Proteomic Peptide Phage Display.

PSD95-Disc large-Zonula occludens (PDZ) domains are among the most abundant modular domains in the human proteome. They typically bind short carboxy-terminal sequence motifs of their ligand proteins, which may be transmembrane proteins such as ion channels and GPCRs, as well as soluble proteins. The identity of the endogenous ligands of many PDZ domains remains unclear despite more than two decades of PDZ research. Combinatorial peptide phage display and bioinformatics predictions have contributed to shed light on PDZ-mediated interactions. However, the efficiency of these methods for the identification of interactions of potential biological relevance is hampered by different biases. Proteomic peptide-phage display (ProP-PD) was developed to overcome these limitations. Here we describe a ProP-PD protocol for the identification of C-terminal PDZ domain ligands. The method efficiently identifies peptide ligands within a proteome of interest, and pinpoint targets of potential biological relevance.

Deciphering the Unexpected Binding Capacity of the Third PDZ Domain of Whirlin to Various Cochlear Hair Cell Partners.

Hearing is a mechanical and neurochemical process, which occurs in the hair cells of inner ear that converts the sound vibrations into electrical signals transmitted to the brain. The multi-PDZ scaffolding protein whirlin plays a critical role in the formation and function of stereocilia exposed at the surface of hair cells. In this article, we reported seven stereociliary proteins that encode PDZ binding motifs (PBM) and interact with whirlin PDZ3, where four of them are first reported. We solved the atomic resolution structures of complexes between whirlin PDZ3 and the PBMs of myosin 15a, CASK, harmonin a1 and taperin. Interestingly, the PBM of CASK and taperin are rare non-canonical PBM, which are not localized at the extreme C terminus. This large capacity to accommodate various partners could be related to the distinct functions of whirlin at different stages of the hair cell development.

PDZ Domains as Drug Targets.

Protein-protein interactions within protein networks shape the human interactome, which often is promoted by specialized protein interaction modules, such as the postsynaptic density-95 (PSD-95), discs-large, zona occludens 1 (ZO-1) (PDZ) domains. PDZ domains play a role in several cellular functions, from cell-cell communication and polarization, to regulation of protein transport and protein metabolism. PDZ domain proteins are also crucial in the formation and stability of protein complexes, establishing an important bridge between extracellular stimuli detected by transmembrane receptors and intracellular responses. PDZ domains have been suggested as promising drug targets in several diseases, ranging from neurological and oncological disorders to viral infections. In this review, the authors describe structural and genetic aspects of PDZ-containing proteins and discuss the current status of the development of small-molecule and peptide modulators of PDZ domains. An overview of potential new therapeutic interventions in PDZ-mediated protein networks is also provided.