Solution Structure And Functional Study Of The Second PDZ Domain Of Human ZO2

This PhD thesis focuses on the cloning, expression, purification and structural and functional studies of the second PDZ domains of human proteins ZO1 and ZO2. We cloned, expressed and purified the second PDZ domains of ZO1 and ZO2. The structure of the second PDZ domains of ZO2 was determined by NMR. The results revealed a novel dimerization mode for PDZ domains via three-dimensional (3D) domain swapping. The results of fast-protein liquid chromatography size-exclusion chromatography and analytical ultracentrifugation confirmed that ZO1-PDZ2 and ZO2-PDZ2 both exist as dimers and the two dimers are very tight, since no monomeric fraction was detected by sedimentation velocity analysis. Furthermore, GST pull-down experiments and immunoprecipitation studies demonstrated that interactions between ZO1-PDZ2 and ZO2-PDZ2 and their self-associations indeed exist both in vitro and in vivo. Chemical cross-linking and dynamic laser light scattering experiments revealed that both ZO1-PDZ2 and ZO2-PDZ2 can form oligomers in solution. This PDZ domain-mediated oligomerization of ZOs may provide a structural basis for the polymerization of claudins namely the formation of TJs.Chapter I, It is the review of the structures and physiological functions of tight junction and ZO proteins. Epithelial and endothelial cells can form selective barriers between tissues and different body compartments. They polarize and adhere to each other through intercellular complexes including tight junctions (TJs), adherens junctions and desmosomes. TJs, the most apical component of the junction complex, separate the apical from the lateral plasma membrane through forming a continuous belt-like attachment at the outer end of the intercellular space between cells. TJs play important roles in regulating the passage of ions and molecule through paracellular pathway. TJs are also crucial for correct function of blood-brain barrier (BBB). Disruption of the TJs in BBB is a hallmark of many central nervous system pathologies.TJs consist of different types of transmembrane proteins including occludin, claudin and JAMs. These transmembrane protein components connect to the cytoskeleton via adaptor/scaffold proteins such as zonula occludens (ZOs). ZO1, ZO2 and ZO3 belong to a membrane associated guanylate kinase protein family (MAGUKs). These proteins contain several protein-protein interaction domains, including three PDZ (for PSD-95, DlgA, ZO1 homology) domains, one SH3 domain, and one GK domain. They are mainly located at the submembranous region of the junction complex. They cross-link multiple integral membrane proteins at the cytoplasmic surface and create specialized membrane domains.Recent studies have provided accumulated evidences to unveil domains that are responsible for the interactions between ZO proteins. The association between ZO1 and ZO2/ZO3 is through their second PDZ domains. Utepbergenov's group showed significant amount of ZO1 homodimers in MDCK cells and demonstrated that the second PDZ domain was both necessary and sufficient for the dimerization. The SH3/GK domain was also reported to be important in ZO1 dimerization, similarly as of in other MAGUK proteins, such as PSD-95 and Dlg/SAP90/SAP102.Dimerization or oligomerization of ZO proteins plays a pivotal role in determining the activity of their binding partners. Claudins are thought to constitute the backbone of TJ strands. They can form homo- and hetero- oligomers during their engagement in the formation of paracellular channels or pores. ZOs bind to claudin YV carboxyl terminals through their first PDZ domain. The dimerization of ZO1 (probably also ZO2) not only initiates the polymerization of claudins namely the formation of TJ strands, but also directs the correct localization of TJ strands.In chapter II, the second PDZ domains of human proteins ZO1 and ZO2 were cloned, expressed and purified, and the solution structure of the second PDZ domain of ZO2 was determined by NMR spectroscope. Unexpectedly, ZO2-PDZ2 forms very tight two-fold symmetric homodimers via a three-dimensional (3D) domain swapping assembly mode. The ZO2-PDZ2 dimer structure is ideally suited for the tight assembly of ZOs in TJs.The unique amino acid sequence within theβ2a andβ2b region of the ZO2-PDZ2 is likely responsible for the formation of the domain swapped dimer. As revealed by the multiple sequence alignment analysis, ZO2-PDZ2 lacks the long loop betweenβ2 strand andβ3 strand that can be found in other PDZ domains. This means that the linker betweenβ2a andβ2b of ZO2-PDZ2 is not long enough forβ2b to swing back to form an antiparallelβ-sheet withβ2a strand, hence facilitates the swapping structure. Due to the high conservation of PDZ2 domains in ZO proteins, we suppose that domain swapping structure also applies for the ZO1-PDZ2 and ZO3-PDZ2 homodimers or ZO1-PDZ2/ZO2-PDZ2 and ZO1-PDZ2/ZO3-PDZ2 heterodimers. The tight dimeric structure of ZO2-PDZ2 (and likely ZO1-PDZ2) is due to extensive inter-subunit hydrogen bonds and hydrophobic interactions formed via swapping of the N-terminal twoβ-strands of each subunit.Furthermore, the in vitro GST pull-down experiments and in vivo immunoprecipitation study have been utilized to define the interactions between ZO1-PDZ2 and ZO2-PDZ2. We demonstrated that ZO1-PDZ2 and ZO2-PDZ2 interact directly both in vitro and in vivo; concomitantly, both PDZ2 domains undergo self-associations. Using chemical cross-linking and dynamic laser light scattering method, we concluded that both Z01-PDZ2 and ZO2-PDZ2 exist as a combination of dimers and oligomers in solution. The in vitro self-association of and the interactions between ZO1-PDZ2 and ZO2-PDZ2 should be caused by their homo-and hetero-oligomerization, since both ZO1-PDZ2 and ZO2-PDZ2 are tight dimers. It is likely that the unique surface-charge potential property of the homodimers afford them the ability to aggregate. We presume that for the oligomerization or multimerization of ZO proteins in vivo, both PDZ2 domain and SH3/GK module may be necessary.