Domain-swapped Dimerization of the Second PDZ Domain of ZO2 May Provide a Structural Basis for the Polymerization of Claudins

Jiawen Wu,Yinshan Yang,Jiahai Zhang,Peng Ji,Wenjing Du,Peng Jiang,Dinghai Xie,Hongda Huang,Mian Wu,Guangzhao Zhang,Jihui Wu,Yunyu Shi

doi:10.1074/jbc.m703826200

Abstract

Zonula occludens proteins (ZOs), including ZO1/2/3, are tight junction-associated proteins. Each of them contains three PDZ domains. It has been demonstrated that ZO1 can form either homodimers or heterodimers with ZO2 or ZO3 through the second PDZ domain. However, the underlying structural basis is not well understood. In this study, the solution structure of the second PDZ domain of ZO2 (ZO2-PDZ2) was determined using NMR spectroscopy. The results revealed a novel dimerization mode for PDZ domains via three-dimensional domain swapping, which can be generalized to homodimers of ZO1-PDZ2 or ZO3-PDZ2 and heterodimers of ZO1-PDZ2/ZO2-PDZ2 or ZO1-PDZ2/ZO3-PDZ2 due to high conservation between PDZ2 domains in ZO proteins. Furthermore, GST pulldown 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 tight junctions.

Highlights

Including tight junctions (TJs),3 adherens junctions, and desmosomes (1)
Sparky3, University of California, San Their Self-associations—It has been demonstrated that ZO1
These results confirmed that ZO1-PDZ2 and ZO2-PDZ2 both exist as dimers, in agreement with the results of gel-filtration chromatography

Summary

MATERIALS AND METHODS

Oligonucleotides—The sequences of the oligonucleotides for target cDNA PCR amplification used in this study are listed as follows: P1, 5Ј-CATATGACTAAAGTCACACTGGTGAAATC-3Ј; P2, 5Ј-CTCGAGTTCATCTCTTTGAACTACC-3Ј; P3, 5Ј-CATATGATCGGGGTCCTCCTGATGAAAAG-3Ј; P4, 5Ј-CTCGAGGCTGTCTCTCAACACCACTAGCTG-3Ј; P5, 5Ј-GCGAATTCTATGACTAAAGTCACACTGGTG-3Ј; and P6, 5Ј-GCGAATTCTATGATCGGGGTCCTCCTGATG-3Ј. Proteins were eluted from beads by 5-min boiling in sample buffer (Bio-Rad) and separated on 15% denatured SDSPAGE gel, followed by Coomassie Brilliant Blue staining. The supernatants were collected and incubated with EZviewTM Red ANTI-FLAG௡ M2 affinity gel for 4 h at 4 °C, bound proteins were recovered by boiling the washed beads in SDS sample buffer and analyzed by immunoblot. These samples were made from a 1:1 mixture of uniformly (H-bond) restraints were generated by a combination of the H/D exchange experiment and the standard secondary structure of the protein based on NOE patterns. Ples were solubilized in sample buffer (Bio-Rad), boiled, and centrifuged at 13,200 rpm for 5 min His fusion proteins were separated on 15% SDS-PAGE gel, followed by Coomassie Brilliant Blue staining.

RESULTS

Heavy atoms

DISCUSSION