Structural Basis of a Key Factor Regulating the Affinity between the Zonula Occludens First PDZ Domain and Claudins

Julian Nomme,Aleksandar Antanasijevic,Michael Caffrey,Christina M Van Itallie,James M Anderson,Alan S Fanning,Arnon Lavie

doi:10.1074/jbc.m115.646695

Abstract

The molecular seal between epithelial cells, called the tight junction (TJ), is built by several membrane proteins, with claudins playing the most prominent role. The scaffold proteins of the zonula occludens family are required for the correct localization of claudins and hence formation of the TJ. The intracellular C terminus of claudins binds to the N-terminal PDZ domain of zonula occludens proteins (PDZ1). Of the 23 identified human claudin proteins, nine possess a tyrosine at the -6 position. Here we show that the claudin affinity for PDZ1 is dependent on the presence or absence of this tyrosine and that the affinity is reduced if the tyrosine is modified by phosphorylation. The PDZ1 β2-β3 loop undergoes a significant conformational change to accommodate this tyrosine. Cell culture experiments support a regulatory role for this tyrosine. Plasticity has been recognized as a critical property of TJs that allow cell remodeling and migration. Our work provides a molecular framework for how TJ plasticity may be regulated.

Highlights

The interaction between the C terminus of claudin proteins and the ZO-1 PDZ1 domain regulates tight junction assembly
Crystal Structures of the ZO-1 PDZ1 Domain with and without Claudin Ligands—For the purpose of studying the structure of the human ZO-1 PDZ1 domain in complex with the C terminus of human claudin-1 and -2, we attempted both co-crystallization and soaking methods using heptapeptides corresponding to the respective claudin sequences
We took advantage of a previously described strategy in which the claudin C-terminal residues are fused at the C terminus of the PDZ domain via a tri-glycine linker [22, 28, 29], such that the residues corresponding to the claudin would bind to a PDZ1 domain of a neighboring molecule

Summary

Background

The interaction between the C terminus of claudin proteins and the ZO-1 PDZ1 domain regulates tight junction assembly. The mechanisms that regulate claudin function remain poorly defined but of considerable interest Their localization at the barrier, turnover, trafficking, and selective barrier properties might be influenced by the specificity and affinity of binding to cytoplasmic scaffolding proteins, such as ZO-1. The tyrosine residue present in claudin-2 at PϪ6 intercalates between the ␤2-␤3 loop of the core PDZ1 domain This observation suggested that due to the more extensive interaction, claudin-2 would bind more tightly than claudin-1 to PDZ1. The modulation of claudin-2 binding to PDZ1 by phosphorylation state of Tyr-224 suggests a potential mechanism for physiologic regulation of affinity These results can be applied to a better understanding of the regulation of claudin function at the tight junction and potentially be generalized to PDZ-ligand interactions outside the tight junction

Experimental Procedures

Number of reflections Observed Unique

Results and Discussion