Molecular and structural transmembrane determinants critical for embedding claudin-5 into tight junctions reveal distinct four helix bundle arrangement

The mechanism of tight junction (TJ) assembly and the structure of TJ strand-forming claudins (Cldn) are unclear. To identify determinants of assembly of blood brain barrier-related Cldn3 and Cldn5, chimeric mutants were analyzed by cellular reconstitution of TJ strands and live cell imaging. Based on rescue of mutants deficient for strand formation, we identified Cldn5 residues involved in claudin folding (A132, A163, I166, L174) and assembly (C128, I142). Experimental results were combined with structural bioinformatics approaches. Initially the experimentally validated previous model of the extracellular loop 2 (ECL2) of Cldn5 was extended to the flanking transmembrane segments (TM3/4). A coiled-coil interface likely caused by alternating small and large residues is supported by concomitant knob-into-hole interactions including Cldn5-specific residues identified herein. To address arrangement of the transmembrane segments in a four helix bundle data from evolutionary sequence couplings and comparative modelling of intramolecular interfaces in the transmembrane region of claudins led to a complete Cldn5 model. Our suggested claudin subtype-specific intramolecular interfaces that are formed by conserved coiled-coil motifs and non-conserved residues in distinct TM positions were confirmed by the recently released crystal structure of Cldn15. The identified molecular and structural determinants essentially contribute to assembly of claudins into TJ strands.
Source: BJ Cell - Category: Biochemistry Authors: Tags: BJ Biomolecules Source Type: research