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.
Classical cadherins constitute a subfamily of calcium-dependent cell–cell adhesion proteins that belong to the large and phylogenetically diverse cadherin superfamily. The various members of the classical cadherin subfamily show a tissue-dependent expression profile as well as a high sequence and structure homology. In the different tissues, 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 also a physical link between cadherins and the actin cytoskeleton machinery [3]. The dynamic adhesive interface of the extracellular portion of E-cadherin and other classical cadherins has been revealed by several crystal structures, which so far have captured only some of the numerous conformational states of the protein [4–8]. In essence, dimerization has been shown to involve mainly the two most membrane-distal domains, EC1 and EC2. In order to form the so-called ‘strand-swapped dimer’, two E-cadherin molecules mutually exchange their N-terminal sequence (the A*-strand or adhesion arm), anchoring the aromatic side chain of their Trp2 residue into each other’s binding pocket. Interestingly, 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]. Beside its broad-ranging effects on physiological tissue organization, classical cadherin dysfunction is often correlated with cancer progression and metastasis [11]. Despite Epithelial (E) - to Neural (N-) cadherin switching being considered a molecular hallmark of epithelial to mesenchymal transition of cancer cells, carcinomas and distal metastases often retain E-cadherin expression [12], as observed for instance in late stage tumors in epithelial ovarian cancer (EOC) [13–15]. In this context, targeting the E-cadherin adhesive interface with small molecules could have a therapeutic and diagnostic value. Linear and cyclic peptides based on the His79-Ala80-Val81 conserved sequence of the EC1 domain (not belonging to the swap dimer interface) have been previously developed to inhibit N-cadherin mediated processes. The most studied cyclic peptide is ADH-1 (Ac-CHAVC-NH2), which has been shown to be an anti-angiogenic agent able to cause cell apoptosis [16]. Peptidomimetics of ADH-1 have also been identified [17]. Recently [18], we reported the first library of small peptidomimetic molecules targeting the strand-swapped dimer interfaces of E- and N-cadherin. These compounds are mimics of the tetrapeptide sequence Asp1-Trp2-Val3-Ile4 (DWVI) of the adhesion arm, modified by replacing the central dipeptide unit (WV) with various scaffolds, all of them bearing a benzyl group that is intended to mimic the indole moiety of Trp2. The most promising compounds identified by a docking protocol were synthesized and were shown to inhibit cadherin homophilic adhesion in EOC cells at low millimolar concentrations. Of these, compound 1 (a. k. a. FR159) (Fig 1), was co-crystallized with a mutant of the E-cadherin EC1-EC2 fragment lacking the first two residues of the adhesion arm (Asp1 and Trp2), while attempts to co-crystallize both compounds 1 and 2 with intact E-cadherin-EC1-EC2 did not yield results [19]. In the X-ray complex structure (PDB code: 4ZTE), compound 1 binds to a dimeric conformation of E-cadherin, the so-called ‘X-dimer’, which is a key intermediate along the E-cadherin dimerization trajectory leading from the monomeric to the strand swap dimer conformation. Unexpectedly, the ligand was found to occupy a hydrophobic pocket that is formed at the X-dimer interface and not in the Trp2 cavity for which it had been designed. This novel hydrophobic pocket formed by the side chains of residues Ile4, Pro5, Ile7, and Val22 from both cadherin molecules does not overlap with the swap dimer interface. However, in the course of the complex cadherin dimerization mechanism the protein undergoes large conformational changes (such as for instance the opening of the adhesion arm that leads to strand swap dimer formation) and goes through different intermediate steps. Hence, it is conceivable that during the process the ligand may bind transiently and via variable moieties also to different surface areas of the protein. Here, to investigate the extent by which variable ligand-cadherin interactions may form over time in solution and to provide a possible dynamic picture of the binding event, we have applied a combination of NMR (Nuclear Magnetic Resonance) and computational techniques to the complexes of two peptidomimetic inhibitors from our library (Fig 1) and human E-cadherin-EC1-EC2. We used ligand-based NMR techniques (Saturation Transfer Difference, STD, and transferred NOE, tr-NOESY) [20,21] to assess binding occurrence as well as to identify the binding epitope of the ligands and to estimate the dissociation constant of the complexes. The experiments were performed using wild type (wt) human E-cadherin-EC1-EC2 at three different temperatures (283,290 and 298 K). Furthermore, we analysed both ligands in the presence of the EC1-EC2 mutant that was used for the X-ray study and that lacks the first two N-terminal residues (E-cadherin- (Val3) -EC1-EC2). Then, in order to rationalize the atomic details of peptidomimetic-cadherin interaction, the NMR data obtained in the presence of wt E-cadherin-EC1-EC2 were analyzed computationally. Indeed, based on NMR data, docking calculations into the EC1 domain of E-cadherin were carried out first. Then, to take into account the temperature factor and introduce protein flexibility, Molecular Dynamics (MD) simulations were also performed. Two temperatures, 300 K and 320 K, were used starting from the different docking poses of 1 and 2 into the E-cadherin model. Of note, ligands binding seems to be highly dependent on the protein flexibility, particularly of the adhesive arm. Overall, the dynamic data described herein will help the design of novel cadherin inhibitors that may bind more efficiently and more selectively into the Trp2 binding pocket.
STD-NMR is a consolidated technique [20,21] based on Overhauser effect that is used to study the interactions between small ligands and macromolecules [22–24]. The method relies on the selective irradiation of the protein, which allows magnetization to be transferred to the bound ligand (which is in great excess in solution compared to the protein concentration). The saturated ligand is displaced in solution due to the binding equilibrium and the observation of the ligand signals in the NMR spectrum provides an indication of the interaction. Those ligand protons that are nearest to the protein are more likely to become highly saturated, and therefore show the strongest signal in the mono-dimensional STD spectrum. Owing to the efficiency of the saturation process, the modulation of the ligand signal intensity is used as an epitope-mapping method to describe the target-ligand interactions. In fact, the intensity of the STD signal (expressed as absolute STD percentage) reflects the proximity of the ligand to the protein surface. Therefore, the group epitope mapping obtained provides information about the nature of the chemical moieties of the ligand that are crucial for molecular recognition in the binding site. We also used the STD amplification factors (STD-AF) to derive the dissociation constant (KD) of the ligand-protein complexes [25–27]. Moreover, we performed tr-NOESY experiments in order to determine the preferred bound conformation of the ligands. In solution, the EC1-EC2 domain fragment can adopt several conformations depending on protein and Ca2+ ion concentration. At 1 mM calcium concentration and at less than 40 μM protein concentration, a monomeric species is observed predominantly, while dimeric forms or even oligomers are present at higher protein concentrations in solution (600 μM) [28]. The calcium ions provide a rigidification of the linker region connecting the EC1 and EC2 domains in the monomer, thus making the dimerization surface available. STD–NMR and tr-NOESY spectra were acquired in 20 mM phosphate buffer at pH 7. 4 (with 150 mM NaCl and 1 mM CaCl2) and 40 μM EC1-EC2 fragments of E-cadherin. In this condition, a monomeric form is predominant in solution [28]. We performed STD-NMR experiments at 283K, 290K and 298K since lowering the temperature helps to shorten the rotational correlation time of the receptor and to increase the effective magnetization transfer. Interestingly, for both compounds we observed different binding epitopes in relation to temperature variations. Furthermore, we also studied the binding modes of the ligands in the presence of the truncated mutant (E-cadherin- (Val3) -EC1-EC2). The NMR interaction data of peptidomimetics 1 and 2 with the EC1 domain of E-cadherin were further investigated computationally. In the X-ray structure of compound 1 bound to the deleted E-cadherin- (Val3) -EC1-EC2 construct missing the Asp1-Trp2 N-terminal residues, the ligand fits into a novel site at the interface of the X-dimer assembly rather than occupying the Trp2 hydrophobic pocket as initially assumed based on their design strategy. However, according to the NMR results discussed above, the ligands show different binding epitopes depending on the protein construct used, suggesting that the adhesive arm may indeed also be involved in stabilizing the ligand-cadherin binding. To clarify this further, we performed MD simulations starting from the docking poses of 1 and 2 generated into the Trp2 binding pocket of wt E-cadherin model. To take into account temperature modulation, the MD runs were carried out at 300 K and 320 K. For each ligand, a detailed analysis of ligand protonation states and conformational geometries was carried out (see SI) in order to select the most relevant conformations and ionization forms for docking calculations.
In this study, we evaluated the binding properties of two peptidomimetic ligands to the extracellular portion of E-cadherin using NMR spectroscopy combined to molecular docking and molecular dynamics calculations. Both ligands have been previously shown to act as mM inhibitors of E-cadherin-mediated cell adhesion [18] and to the best of our knowledge, they are the first peptidomimetics developed from the N-terminal DWVI sequence of the E-cadherin EC1 domain. In these compounds, the central dipeptide unit (Trp2-Val3) of the tetrapeptide motif have been replaced by two different scaffolds bearing an aromatic group that, in our docking model, inserts into the hydrophobic cavity of Trp2, thus preventing swap dimer formation. In accordance with this hypothesis, a reconstruction of the free energy profile of the conformational transition of the E-cadherin monomer from its closed inactive state (with the Trp2 indole moiety intramolecularly docked) to its open form (indole moiety solvent exposed), has shown that in solution the monomer could significantly populate both the open and closed states, which are almost iso-energetic [34]. However, the recent crystallographic structure of 1 in complex with a deleted form of E-cadherin EC1-EC2 domain (lacking the N-terminal Asp1-Trp2 residues) showed also a novel possible mechanism of action based on a different adhesive interface [19]. Indeed, in the X-ray structure the peptidomimetic compound binds to the X-dimer conformation of the protein, a crucial kinetic intermediate of the cadherin dimerization pathway, by inserting into a novel hydrophobic cavity that is formed at the interface of the two interacting cadherins. Clearly, owing to the complexity of the E-cadherin homo-dimerization process and to the dynamic behaviour of the target itself, which undergoes major conformational changes as part of its substrate recognition mechanism, further investigations of ligand-cadherin binding are needed. In this work, to sample a set of possible interactions of compound 1 and 2 with E-cadherin, we carried out STD-NMR experiments in the presence of two slightly different E-cadherin species, the wt-E-cadherin-EC1-EC2 and the E-cadherin- (Val3) -EC1-EC2 fragments. Depending on the protein constructs, 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 sequence over time and among the set of possible binding events. Prompted by these considerations, the ligand binding mode proposed by the docking model that places the compound in the Trp2 cavity making interactions with the adhesive arm portion was selected for further investigations. MD simulations were performed starting from the corresponding docking poses of 1 and 2 into a wt-E-cadherin EC1 model. To assess the temperature modulation, MD runs were performed at 300 K and 320 K and the results compared to the STD spectra. The stability of the docking poses and also the interactions with the E-cadherin pocket, including the adhesive arm, were evaluated and compared at the different temperatures. In the input structures, both ligands insert the aromatic group into the Trp2 pocket. The aromatic moiety remains docked into the hydrophobic pocket also during MD runs at 300 K and 320 K, supporting the presence of the aromatic hydrogens STD signal (detected at all temperatures). In general, the starting ligand binding mode is quite conserved during MD run while the protein displays major fluctuations. In particular, for compound 1 protein flexibility increases with temperature, especially for the adhesive arm residues. As a consequence, at 320 K the contacts and the hydrogen bonds of the STD amide protons with the adhesive arm are not present and only the percentage of NHIle contacts with the pocket residues is enhanced compared to 300 K (Table 1). At 300 K, NH1 resulted to be more engaged by the adhesive arm residues, followed by NH10 and NHIle and this behavior agrees with the STD spectrum at 283 K. At 320 K the population of the ligand NHIle contacts with the protein increased while the other amide protons lost their contacts with the adhesive arm (too flexible) and also reduced the interactions with the residues of the binding site. This trend is in good agreement with the STD spectrum at 298 K where the NHIle signal replaces those of NH1 and NH10. During MD simulations of compound 2, only the binding mode type A is stable and the ligand remains bound to the receptor. At 300 K, the interaction of NH19 proton with E-cadherin is stronger than NH2 and this behaviour is in agreement with the STD spectra at 283 K and 290 K, showing higher intensity of NH19 signals compared to NH2. At 320 K, we observed a slight decrease of NH19 contacts (Asp1 not present) and, despite losing the interaction with Trp2, an increase of NH2 contacts population with the pocket residues. This trend is consistent with the intensity variation observed in the corresponding STD spectra. In conclusion, our MD results on ligand-cadherin binding are in agreement with STD spectra. The simulations at different temperatures could also explain the different ligand binding epitope observed in the presence of wt E-cadherin. In particular, the modulation of the signals seems to be dependent on the protein flexibility, especially the adhesive arm, an active part of the binding site that participate in the interaction with the ligand. Based on these results, the design of novel cadherin inhibitors to target more efficiently and selectively the Trp2 binding pocket, will be focused on the stabilization of ligand interactions towards both the hydrophobic cavity and the adhesive arm residues. Moreover, further NMR studies with labelled E-cadherin constructs (wt vs. deleted forms) will be carried out to map the protein residues involved in the interaction with the inhibitors and clarify their mechanism of action.
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.
protons
chemical bonding
protein interactions
cadherins
materials science
protein structure
hydrogen bonding
physical chemistry
cell adhesion
proteins
chemistry
salt bridges
nucleons
molecular biology
adhesives
physics
biochemistry
biochemical simulations
cell biology
nuclear physics
electrochemistry
biology and life sciences
materials
physical sciences
computational biology
macromolecular structure analysis
Exploring E-cadherin-peptidomimetics interaction using NMR and computational studies