| Cell junctions exist within the tissue of a multicellular organism as a specialized structure. They are especially abundant in epithelial tissues. In vertebrates, there are three major types of cell junctions, which are tight jucntion (TJ), adherent junction (AJ) and gap junction (GJ). All the three junctions differ from each other in their morphous, location and function. TJs locate most apical in the lateral membrane, they connect the two adjencent cells tightly. TJ function as gate to regulate the ion and small molecule to pass through intercellular pathway. Furthermore, TJ maintain the polarity of epithelial cells by separating transmembrane proteins. AJ locates near TJ in epithelial cells, and will leave a 10-20 nm space between the adjacent cells. GJ is constituted by two anchored connexon which contains 6 connexins. As a channel, GJ allows the sharing of signals and nutrition between connecting cells.Zonular occludens 1(ZO1) was found in TJs, and later in AJs and GJs. As a scaffolding protein, ZO1 binds directly with caludins, occlundin and JAMs to regulate TJs. Also ZO1 associats with actin and ZONAB, which implied ZO1 was invovled in cell signaling, proliferation and differentiation. In cells which lack of TJs, ZO1 was found in AJs interacting with catenin. In GJs, many connexins have been reported to interact with ZO1. For examply, Cx43 intheract with ZO1 through its very C-terminal, and the interaction regulate the balance of the amount of Cx43 between GJ plaques and nonjunctional pools.In our work, we determined the solution structure of the second PDZ domain of ZO1 (ZO1PDZ2) which exhibited a highly stable domain-swapped homodimer form. 20 residues in the N-terminal in one chain swapped into the other chain. The homodiemr is very stable and reserved the hydrophobic pocket for PDZ binding ligands.By NMR chemical shift perturbation issue and ITC issue, ZO1PDZ2 was found to bind the C-terminal of connexins in a way of type II PDZ domain. Cx25, Cx45 and Cx59 were found to interact with ZO1PDZ2 for the first time, which need to be investigated more by in vivo experiments.ZO1PDZ2 could be converted to a monomeric form by inserting three or more residues in the middle of its secondβstrand, which implied the mechanism of the domain-swapping in ZO1PDZ2. FPLC issue and SV-AUC issue were employed to measure the molecular weight of ZO1PDZ2 and its mutants. the monomer mutant could bind the C-terminal peptide of Cx43, but in a weaker way. Furthermore, the interaction between ZO1PDZ2 and ZO2PDZ2 or ZO3PDZ2 was also abolished by the insertion mutation.A ZO1 mutant with an insertion into its PDZ2 domain was generated and in vivo investigations were carried out using transepithelial electrical resistance experiments in Madin-Darby canine kidney cells. The result indicated that, unlike expression of wild-type ZO1, expression of the ZO1 mutant could not rescue the delay of TJ assembly caused by endogenous ZO1 knockdown. This is the first direct experimental evidence for the important role of the ZO1 PDZ2 domain in the formation of TJs and these data strongly suggest that the domain-swapping of PDZ2 in vivo is essential for ZO1 functionality in TJ assembly.
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