Characterizing interactions in E-cadherin assemblages

Abstract

Cadherin intermolecular interactions are critical for cell-cell adhesion and play essential roles in tissue formation and the maintenance of tissue structures. In this study, we focus on E-cadherin, a classical cadherin that connects epithelial cells, to understand how they interact in cis and trans conformations when attached to the same cell or opposing cells. We employ coevolutionary sequence analysis and molecular dynamics simulations to confirm previously known interaction sites as well as to identify new interaction sites. The sequence coevolutionary results yield a surprising result indicating that there are no strongly favored intermolecular interaction sites, which is unusual and suggests that many interaction sites may be possible, with none being strongly preferred over others. By using molecular dynamics, we test the persistence of these interactions and how they facilitate adhesion. We build several types of cadherin assemblages, with different numbers and combinations of cis and trans interfaces to understand how these conformations act to facilitate adhesion. Our results suggest that, in addition to the established interaction sites on the EC1 and EC2 domains, an additional plausible cis interface at the EC3-EC5 domain exists. Furthermore, we identify specific mutations at cis/trans binding sites that impair adhesion within E-cadherin assemblages.

Figure 1

DCA Z scores of sequence correlations involving residues K14, V81, and G85. The sequence correlations for the entire protein are calculated using direct coupling analysis (DCA) (16,17) based on the multiple sequence alignments from the Pfam Database (PfamID: PF00028). The multiple sequence alignment contains 6,852 sequences. Each of the EC domains in cadherin corresponds to the same Pfam domain PF00028. Note that the residues have been reindexed to run from 1 to 100. The multiple sequence alignment (after removing gaps) has 100 positions The Z score is calculated using the raw DCA value minus the mean of all DCA values, then divided by the standard deviation. The highest Z scores indicate all three residues are strongly correlated with their sequence neighbors. Correlations with sequence-distant residues are relatively weak, but potentially defining of interactions, as identified by purple boxes and the specific residues are named in Table 1 also. The larger sequence-distant correlation values are listed in Table 1. Furthermore, all the mapped residues have been included in Data S4 and S5. To see this figure in color, go online.

Figure 2

Structure of one Ecad ectodomain (PDB: 3Q2V (12)) trans dimer. This structure has been used for building the larger assemblages. The dark-red spheres identify the locations of the important featured residues—L175, W2, V81, P231, and A491. Here, EC1 domains are colored in orange and EC2 domains are cyan. To see this figure in color, go online.

Figure 3

Steered MD simulations of four Ecad ectodomains interacting in trans and cis orientations, when the cis interface (V81 and L175) is pulled in different directions (±x or ±y or ±z). (A) The Ecad assemblage showing two trans interactions (purple spheres) and two cis interactions—one at EC1-EC2 (pink spheres), together with the newly discovered cis interaction between EC3 and EC5 (orange spheres). (B) Interaction energy profiles of the novel cis interface at EC3-EC5 (A491 and P231) and (C) Interaction energy profile of cis residues at EC1-EC2 (L175 and V81), respectively. At a trans interface, the W2 residues interact with neighboring hydrophobic cavities from the opposed Ecad ectodomain. (D) Here, we display the interaction energy profile of one of the W2/W2′ residues, embedded in the hydrophobic cavity; 1) interaction energy profile of W2 in hydrophobic cavity (residues 89 to 92), 2) interaction energy profile of W2′ in the hydrophobic cavity (residues 89–92). To see this figure in color, go online.

Figure 4

Protein dynamics in a large Ecad assemblage with five trans chains showing the role of interacting intermolecular residues during a production simulation of 15 ns. (A) There are five chains, comprised of ten separate cadherins in this assemblage (one chain is hidden below the plane in the present view). Results show how the interactions at the cis and trans interfaces change, particularly between the pink and purple central Ecad chains. To identify these, we have marked the specific interactions with colored dots: two trans interface pairs comprised of W2 and W2′ with dark green and orange dots; the usual cis interface at EC1-EC2 (L175 and V81) in mauve, purple, and bright green dots, respectively, and the new cis interface at EC3-EC5 (A231 and P491) marked with pink and yellow dots. (B) Interaction energies and C^α interatomic distances for one of the trans interfaces W2/W2′ with residues E89, M90, and D92 from the neighboring Ecad forming the hydrophobic cavity. (C) Interaction energies and C^α interatomic distances at the cis interaction between L175 and V81 (both pairs). (D) Interaction energies and C^α interatomic distances at the newly identified cis interaction between A231 and P491; we have highlighted the interaction energies between 5 and 15 ns with a blue box. (E) Interaction energy profile of the newly identified cis interaction between A231 and P491 between 5 and 15 ns suggests that, while interaction energies are weaker compared with the cis interaction between L175 and V81, they do persist throughout the simulation. To see this figure in color, go online. For a Figure360 author presentation of this figure, see https://doi.org/10.1016/j.bpj.2023.06.009.

Figure 5

Intermolecular distances of all trans interaction sites (W2 and W2′ with their respective hydrophobic cavities) in Ecad assemblages of the same five units as in Fig. 3, during a production MD of 15 ns, shows that they do not vary much. (A) Each Ecad trans dimer is shown in different colors for the intermolecular distances between the W2/W2′ and the hydrophobic cavity where they are embedded. (B) Intermolecular distances between the C^α atom of W2 and center of mass of the hydrophobic cavity where it is buried in the trans binding sites in this Ecad assemblage. (C) Intermolecular distances between the C^α atom of W2 and the center of mass of the hydrophobic cavity where W2 is buried for all the trans interactions in the Ecad assemblage. (D) Intermolecular distances between the C^α atom of W2′ and the center of mass of the hydrophobic cavity where W2′ is buried for all the trans interactions in the Ecad assemblage. The two tryptophans (as observed in the previous figure as well) are in symmetric positions. Overall, this suggests that the strand-swapped dimer interactions are quite stable and do not change significantly overall during the simulations. To see this figure in color, go online.

Figure 6

The effects of mutations at L175 and neighboring residues observed at a cis interface during production MD of 100 ns in the Ecad Assemblage of 2 units. (A) The Ecad assemblage of 2 chains; orange box is the part of the structure that is enlarged below. (B) G85 and F35 on neighboring Ecads is found to coevolve with L175 in the sequence data. (C) Production MD results suggest the root mean-square fluctuations increase significantly, throughout the structure, upon introduction of mutations on the Ecad assemblage. To see this figure in color, go online.