Exploring E-cadherin-peptidomimetics interaction using NMR and computational studies

Cadherins are homophilic cell-cell adhesion molecules whose aberrant expression has often been shown to correlate with different stages of tumor progression. In this work, we investigate the interaction of two peptidomimetic ligands with the extracellular portion of human E-cadherin using a combination of NMR and computational techniques. Both ligands have been previously developed as mimics of the tetrapeptide sequence Asp1-Trp2-Val3-Ile4 of the cadherin adhesion arm, and have been shown to inhibit E-cadherin-mediated adhesion in epithelial ovarian cancer cells with millimolar potency. To sample a set of possible interactions of these ligands with the E-cadherin extracellular portion, STD-NMR experiments in the presence of two slightly different constructs, the wild type E-cadherin-EC1-EC2 fragment and the truncated E-cadherin-(Val3)-EC1-EC2 fragment, were carried out at three temperatures. Depending on the protein construct, a different binding epitope of the ligand and also a different temperature effect on STD signals were observed, both suggesting an involvement of the Asp1-Trp2 protein sequence among all the possible binding events. To interpret the experimental results at the atomic level and to probe the role of the cadherin adhesion arm in the dynamic interaction with the peptidomimetic ligand, a computational protocol based on docking calculations and molecular dynamics simulations was applied. In agreement with NMR data, the simulations at different temperatures unveil high variability/dynamism in ligand-cadherin binding, thus explaining the differences in ligand binding epitopes. In particular, the modulation of the signals seems to be dependent on the protein flexibility, especially at the level of the adhesive arm, which appears to participate in the interaction with the ligand. Overall, these results will help the design of novel cadherin inhibitors that might prevent the swap dimer formation by targeting both the Trp2 binding pocket and the adhesive arm residues. Author summary Classical cadherins are the main adhesive proteins at the intercellular junctions and play an essential role in tissue morphogenesis and homeostasis. A large number of studies have shown that cadherin aberrant expression and/or dysregulation often correlate with pathological processes, such as tumor development and progression. Notwithstanding the emerging role played by cadherins in a number of solid tumors, the rational design of small inhibitors targeting these proteins is still in its infancy, likely due to the challenges posed by the development of small drug-like molecules that modulate protein-protein interactions and to the structural complexity of the various cadherin dimerization interfaces that constantly form and disappear as the protein moves along its highly dynamic and reversible homo-dimerization trajectory. In this work, we study the interaction of two small molecules with the extracellular portion of human E-cadherin using a combination of spectroscopic and computational techniques. The availability of molecules interfering in the cadherin homophilic interactions could provide a useful tool for the investigation of cadherin function in tumors, and potentially pave the way to the development of novel alternative diagnostic and therapeutic interventions in cadherin-expressing solid tumors.