Occludin binds to the SH3-hinge-GuK unit of zonula occludens protein 1: potential mechanism of tight junction regulation

Abstract

The interaction between tight junction proteins occludin and zona occludens protein 1 (ZO-1) was clarified. The sequence cc1 within the hinge region of ZO-1, connecting its SH3 and GuK domains, was identified as a new association site for the occludin C-terminus, core binding area GLRSSKRNLRKSR (mouse ZO-1(606-618)). Occludin also bound to the sequence H2 within GuK, core area HKLRKNNH (ZO-1(759-766)). In occludin, the binding core was ELSRLDKELDDYREESEEY (mouse occludin(455-473)). Helicity of the sequences was suggested by circular dichroism. Because basic residues in ZO-1, acidic residues in occludin (underlined), coiled-coil helix-forming leucine heptad motifs (bold) in occludin and, probably, in cc1 were essential, we conclude that interactions were both helical and ionic. Moreover, the GuK domain bound other GuK molecules, suggesting oligomerization of ZO-1. Generally, the assumption is supported that the SH3-hinge-GuK region represents a functional and regulatory unit in ZO-1 forming a multiprotein tight junction complex with occludin.

Molecular Determinants of the Interaction between Clostridium perfringens Enterotoxin Fragments and Claudin-3

“…For comparison of Cld3-(140 -159) and Cld3-(140 -159)N148D/L150T, 500 and 250 resonance units of Cld3 peptides were immobilized, respectively. Binding experiments were performed as described earlier (22). The analytes GST-CPE-(116 -319), GST-CPE-(290 -319), and GST (control) were injected in concentrations between 0.01 and 20 M in running buffer (PBS, pH 7.4, supplemented with 2 mM dithiothreitol).…”

Epithelial barrier assembly requires coordinated activity of multiple domains of the tight junction protein ZO-1

“…The cytosolic tail of occludin binds to distinct structural elements within the U5 motif and GUK domain in vitro (Fanning et al, 1998;Furuse et al, 1994;Schmidt et al, 2004), but whether direct interaction with either of these domains is necessary or sufficient for incorporation of occludin into the AJC has not been directly addressed. To address this issue, we first examined the interaction of the cZNA transgenes with a GST occludin fusion protein (Fig.…”

Tight junction-based epithelial microenvironment and cell proliferation

“…The key connecting points for this issue are provided by the PDZ-binding motifs of claudins, which bind to PDZ-containing membrane scaffolding proteins such as ZO-1/2/3 and MUPP1 (Itoh et al, 1999;Hamazaki et al, 2002). Occludin and JAM-1 were shown to be associated with ZO-1 through both non-PDZ and PDZ domains Schmidt et al, 2004). Recent studies on ZO-1/2/3-deficient cultured epithelial cells have revealed unequivocally that ZO-1/2 determines where and when the claudins are polymerized to form TJ strands with barrier functions (Umeda et al, 2006).…”