The mutual interactions of the extracellular domains of tight junction proteins

Tight junctions (TJs) are complex multiprotein structures found in epithelial and endothelial cells that serve as selective barriers and regulate the diffusion of small molecules and ions through the intercellular space (Alberts et al., 2015). TJs are composed of strands that encircle the cells like a belt-like network, and thus control the unlimited diffusion of permeants. The backbone of the TJ strands is composed of the family of proteins called claudins. The crystal structure of claudin 15 (Suzuki et al., 2014) and of fragments of other members of the claudin family complexed with toxins (Saitoh et al., 2015; Shinoda et al., 2016) have been resolved. However, the molecular organization of the TJ strands is unknown but is essential to understanding normal physiological function (Zihni, Mills, Matter, & Balda, 2016) as well as dysfunction in pathological states, including viral interactions. Furthermore, the development of new therapeutic strategies will rely on a molecular level understanding of the TJ barrier mechanism. Here we investigate the behaviour of the extracellular loops of claudin 1, which is essential for the epidermal barrier (Kirschner, Houdek, Fromm, Moll, & Brandner, 2010). We have employed atomistic and coarse-grained simulations of the extracellular loop domains restrained on a 2-d plane to mimic their natural occurrence in a lipid bilayer. The systems comprise large grids of the protein loop domains (8x8) randomly rotated and restrained on a plane, surrounded by water and counterions. Their cis (in the same lipid bilayer) and trans (between opposing bilayers) interactions were simulated and characterised. The domains reveal a tendency to form linear structures, though no specific cis-interaction appears to dominate. The individual loops do not show any particular regions with strong binding affinities. The residues with similar physical properties (i.e. either strongly positively or negatively charged, or non-polar) are dispersed throughout the loop structures and do not occur in local contiguous regions. The binding energy of interaction between the most frequent dimers observed in our simulations was also characterised by umbrella sampling. The lack of a specific, strong interaction underpinning any organisational motif for the cis- interaction suggests that the loops interactions are not the determinants of the molecular organisation of TJs. The next step is to investigate the self-assembly of the whole claudin monomers embedded in lipid bilayers with a view to identifying the key interactions and packing that gives rise to the formation of TJ strands