Abstract
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.
Highlights
Classical cadherins constitute a subfamily of calcium-dependent cell–cell adhesion proteins that belong to the large and phylogenetically diverse cadherin superfamily
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
We study the interaction of two small molecules with the extracellular portion of human E-cadherin using a combination of spectroscopic and computational techniques
Summary
Classical cadherins constitute a subfamily of calcium-dependent cell–cell adhesion proteins that belong to the large and phylogenetically diverse cadherin superfamily. They are mostly localized at the adherens junctions, where they promote cell–cell adhesion through the homodimeric engagement of ectodomains protruding from neighbouring cells [1,2] This process, which involves cadherin clusterization on the cell membrane, results in the formation of tight cell-cell adhesion interfaces. The extracellular portion of classical cadherins features five tandemly arranged immunoglobulin-like domains (EC1-EC5), whose relative orientation is controlled and rigidified through the coordination of calcium ions at the interdomain level. The interaction of their cytoplasmic tail with catenins allows a number of cell signalling and trafficking processes, providing a physical link between cadherins and the actin cytoskeleton machinery [3]. To reach the strand swap dimer conformation, which is the reversible endpoint of the dimerization trajectory, two monomeric cadherins must first go through an X-dimer conformation, which lowers the energy of the strand exchange process by firmly placing the two adhesion arms in close physical proximity [9,10]
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