title: Structure of the conserved cytoplasmic C-terminal domain of occludin: identification of the ZO-1 binding surface.
abstract: Occludin is a transmembrane protein localized at tight junctions whose functions are complex yet poorly understood. Current evidence supports a role for occludin in both the formation of the paracellular barrier and in cell signaling. While the N-terminal extracellular domains of occludin mediate homotypic adhesion, the distal C-terminal cytoplasmic domain of occludin controls protein targeting and endocytosis. The C terminus can also bind to the scaffolding proteins ZO-1, ZO-2, ZO-3, cingulin, the membrane trafficking protein VAP33, and the cytoskeletal protein F-actin, suggesting an important role for this domain. This domain is highly homologous to an important functional domain in the C terminus of the ELL family of RNA polymerase II transcription factors. To explore the function of occludin, we determined the high-resolution crystal structure of its C-terminal distal cytoplasmic domain. The structure comprises three helices that form two separate anti-parallel coiled-coils and a loop that packs tightly against one of the coiled-coils. Using in vitro binding studies and site-directed mutagenesis, we have identified a large positively charged surface that contains the binding site for ZO-1, and this surface is required for proper localization of occludin to cell-cell junctions. On the basis of sequence conservation, we predict that occludin domains from different species and the C-terminal domain of the ELL transcription factors share a very similar structure. Our results provide a model to further test the function of occludin and its binding to other proteins.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA.
authors.forename: Yuanhe
authors.initials: Y
authors.lastname: Li
authors.forename: Alan S
authors.initials: AS
authors.lastname: Fanning
authors.forename: James M
authors.initials: JM
authors.lastname: Anderson
authors.forename: Arnon
authors.initials: A
authors.lastname: Lavie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D018360:Crystallography, X-Ray; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D062826:Zonula Occludens-1 Protein
chemical_list: D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein