The structural and functional characterizations of some novel PDZ domains as well as the USH1 protein complex assembly with its implications in human Usher Syndrome disease is the main theme of this dissertation. Since elucidation of the three dimensional structures is one of the best way to reveal the molecular mechanism governing the cellular functions of proteins, I use NMR spectroscopy in conjunction with X-ray crystallography to determine the atomic 3D structures of particular proteins. As one of the most abundant protein-protein interaction modules in the eukaryotic genomes, PDZ domain participates in a variety of cellular activities including neuronal signal transduction, protein trafficking, epithelial cell-cell adhesion and hair cell stereociliar development and maintain. Based...[ Read more ]

The structural and functional characterizations of some novel PDZ domains as well as the USH1 protein complex assembly with its implications in human Usher Syndrome disease is the main theme of this dissertation. Since elucidation of the three dimensional structures is one of the best way to reveal the molecular mechanism governing the cellular functions of proteins, I use NMR spectroscopy in conjunction with X-ray crystallography to determine the atomic 3D structures of particular proteins. As one of the most abundant protein-protein interaction modules in the eukaryotic genomes, PDZ domain participates in a variety of cellular activities including neuronal signal transduction, protein trafficking, epithelial cell-cell adhesion and hair cell stereociliar development and maintain. Based on the unique abilities of PDZ domains, PDZ-containing scaffold proteins play critical role in the spatio-temporal control of macromolecular complex assembly and targeting within cells for different cellular events. To better understand how PDZ domains function, the first part of this dissertation focus on two previously un-clarified PDZ domains including the PDZ domain of PICK1 and the second PDZ domain of ZO-1. The structures together with other biochemical, biophysical and molecular cell biology results provide mechanistic insights into the novel lipid and ligand binding properties of PDZ domains. In particular, the structural and functional characterizations from PICK1 PDZ demonstrate that PICK1 PDZ domain can directly bind to membrane lipids and the PDZ-lipid interaction is relevant for PICK1’s cellular function; The PDZ2 domain of ZO-1 can form domain-swapped dimer to generate specific and regulatory target binding sites for connexin43. The study of ZO-1 PDZ2 present a new paradigm for understanding how some PDZ domain proteins specifically bind to and regulate the functions of their target proteins.

The second part of this dissertation describes the biochemical and structural study of USH1 protein complex assembly with its implications in Usher Syndrome. I first focus on the interaction between USH1C harmonin and USH1D cadherin23. In addition to the second PDZ domain, we have discovered a novel N-terminal domain of harmonin, which can function as a protein-protein interaction module to specifically interact with the cytoplasmic tail of cadherin23. The detailed structural and functional characterizations of interactions between harmonin and cadherin23 reveal a multi-dentate binding mode between these two USH1 proteins, which is critical for the harmonin-mediated assembly of stable tip link complex in the auditory hair cells. After characterization of the interaction between harmonin and cadherin23, we tend to uncover the interaction mode between USH1C harmonin and USH1G Sans. The solved complex structure between harmonin NPDZ1 and Sans SAM-PBM reveals that the N-terminal domain and the first PDZ domain of harmonin are tethered by a small domain C-terminal to PDZ1 to form a structural and functional supramodule responsible for binding to Sans, and the SAM domain of Sans specifically binds to the PDZ domain of harmonin, revealing a previously unknown interaction modes for both PDZ and SAM domains. I further demonstrate that the synergistic PDZ1/SAM and PDZ1/carboxyl PDZ binding-motif interactions between harmonin and Sans lock the two scaffold proteins into a highly stable complex. Mutations in harmonin and Sans found in USH1 patients are shown to destabilize the complex formation of the two proteins. In summary, the work described in this dissertation also contributes significant scientific knowledge to the biological academic field, in particular the results obtained from the Usher Syndrome project, since we are the first and the only group in the world to characterize this important human disease from the structural angle.