Structural and functional characterization of scaffold protein Par-3

Cell polarity is one of the most fundamental processes throughout multicellular organisms. The establishment and maintenance of cell polarity rely heavily on a set of cellular signaling cascades tightly controlling trafficking and segregation of bioactive macromolecules to specific cellular destinations at particular time periods. The evolutionarily conserved multi-PDZ domain containing Par protein complex, Par-3/Par-6/aPKC, is a central participant in the regulation of cell polarity. Asymmetric positioning of the Par complex is absolutely required for both establishment and maintenance of cell polarity, presumably by assembling and targeting cell polarity protein complexes and other determinants to specific cellular localizations. However, the biochemical and structural basis of such Par complex-mediated cell polarity regulation is poorly understood prior to this thesis study. The central theme of this thesis work has been focusing on the elucidation of the molecular and functional mechanisms of Par-3 in the regulation of cell polarity.;Chapter 1 and 2 provide an overall introduction of the backgrounds of the thesis work. In Chapter 3, I comprehensively characterized the structure and function of the N-terminal oligomerization domain (NTD) of Par-3. The 3D solution structure of the NTD monomer adopts a ubiquitin-like fold similar to the PB1 domain. The structural characterization and biochemical analysis revealed that the NTD domains form a front-to-back homomultimer with a helical fiber-like structure. I also demonstrated that Par-3 NTD plays an essential role for its correct cellular localization and cell polarity regulation in an epithelial cell model.;In Chapter 4, I discovered that PDZ2 domain from Par-3 serves as a lipid-membrane binding module and the molecular basis for such PDZ-lipid interaction is deciphered in detail for the first time by combinations of multiple structural and biochemical approaches. Both a conserved positively charged surface and a specific binding pocket for the head group of phosphoinositides are required for PDZ2-lipid interaction. A flexible hydrophobic loop of Par-3 PDZ2 inserts into membrane bilayers upon membrane binding. Interestingly, the canonical peptide ligand binding groove of the PDZ domain partially overlaps with the lipid head group binding pocket, suggesting a potential functional switch of Par-3 PDZ2. We further demonstrated that binding to lipid membrane is a general property of PDZ domain. We assayed the lipid binding capacities of around 100 different mammalian PDZ domains and found that a subset of PDZ domains (15 PDZs) are positive lipid binders. One of these lipid-binding PDZ domains is PICK1 PDZ (elaborated in Chapter 5), which contains a distinct binding mode from that of Par-3 PDZ2. We conclude that binding to lipid membrane is likely to be the second most prevalent function of PDZs known to data. The biological implications of PDZ-lipid membrane interactions are also discussed in both Chapter 4 and 5.