E-cadherin is an extracellular protein transmembrane and calcium-dependent of cell-cell adhesion. E-cadherin has an important function in formation of adherens junction in the intercellular junction of biological barriers such as the intestinal mucosa and the blood brain barrier (BBB). Peptides derived from the conserved region of EC1 have highly potential activity to modulate the adherens junctions in BBB. Peptides have shown their ability to deliver the drug molecules across the BBB and overcome drug delivery issues in BBB such as metabolism enzymes, tight junction and efflux pump that prevent drug transport from the systemic circulation into the brain. In modulating the BBB, peptides inhibit cadherin-cadherin interactions. The selective peptides are necessary required to improve E-cadherin modulation. Elucidation of EC1 domain structure would provide information in designing the peptides selectively to modulate E-cadherin. In atomic level structure, solution NMR spectroscopy is a valuable tool to examine the changes in structure, conformation and dynamics. In this study, we have successfully applied the three-dimensional NMR techniques to determine the ¹H, ¹³C, and ¹⁵N backbone assignments of EC1 in the ¹³C-¹⁵N-labeled EC1 solution structure study.  The EC1 backbone assignment then was used as a fingerprint to find the resonance chemical shift of possible EC1-peptide binding sites by titrating the peptide in solution. Titration using HAV6 (Ac-SHAVSS-NH₂) peptide and ADTC5 (Ac-CDTPPVC-NH₂) into ¹⁵N-labeled-EC1 solution have been undertaken by two-dimensional NMR to resolve the binding site of the EC1-peptide complex by monitoring perturbations in the backbone amides. Residue Ile-4 and Asp-103 of EC1 domain showed the most changes over other residues based on CSP value. By transforming CSP value to docking study we could find that residue Ile-4 and Asp-103 showed distinct conformations with favorable binding modes, chemical bonds involved and the best affinities with HAV6 and ADTC5 peptides. Furthermore, the result will be used as model to design small peptides that have higher binding capability to the EC1 for improving drug delivery through the intestinal mucosa and the blood brain barrier.

Keywords: EC1 domain, E-cadherin, Blood brain barrier, HAV6, ADTC5,  ¹H-¹³C- and ¹⁵N-backbone assignment, peptide titration, CSP, docking