Structural and energetic determinants of adhesive binding specificity in type I cadherins

Abstract

Type I cadherin cell-adhesion proteins are similar in sequence and structure and yet are different enough to mediate highly specific cell-cell recognition phenomena. It has previously been shown that small differences in the homophilic and heterophilic binding affinities of different type I family members can account for the differential cell-sorting behavior. Here we use a combination of X-ray crystallography, analytical ultracentrifugation, surface plasmon resonance and double electron-electron resonance (DEER) electron paramagnetic resonance spectroscopy to identify the molecular determinants of type I cadherin dimerization affinities. Small changes in sequence are found to produce subtle structural and dynamical changes that impact relative affinities, in part via electrostatic and hydrophobic interactions, and in part through entropic effects because of increased conformational heterogeneity in the bound states as revealed by DEER distance mapping in the dimers. These findings highlight the remarkable ability of evolution to exploit a wide range of molecular properties to produce closely related members of the same protein family that have affinity differences finely tuned to mediate their biological roles.cadherin dimerization | protein family design | entropy contribution I n metazoans, the elaboration and maintenance of multicellular architectures relies upon the ability of cells to specifically adhere to one another. Cadherins constitute a superfamily of singlepass transmembrane proteins that can confer such specific adhesive properties to cells (1). In particular, the classical type I and type II cadherins, which are only found in vertebrates and are characterized by an extracellular region comprised of five extracellular cadherin (EC) domains, have been shown to help drive cell-patterning behavior in numerous settings: for example, in morphogenesis (2-4) and in neural patterning (5, 6). Cells expressing the same classical cadherin on their surface generally aggregate through homophilic interactions, whereas cells expressing different cadherins segregate into distinct layers that, in at least some instances, remain in contact with each other through heterophilic binding (7-9).Cell adhesion by classic cadherins is mediated by the dimerization of cadherin extracellular domains emanating from apposed cell surfaces through an interface confined to the N-terminal EC1 domain (Fig. 1A). Numerous crystal structures have revealed the atomic details of the trans (i.e., between cells) dimerization interface for three type I cadherins: C-, E-, and N-cadherins (10-13). In all three cases, the dimer partner molecules swap their N-terminal β-strand (the A*-strand), whose conserved Trp2 residues provide an "anchor" for the adhesive interface by docking into a complementary hydrophobic pocket in the partner protomer (Fig. 1A). A second dimerization interface that can form in the trans orientation has been observed in crystal structures of mutants of both type I and type II classical cadherins. Specifically, numerous mu...

Structural basis of adhesive binding by desmocollins and desmogleins

“…S1), yielding heterophilic K D s ranging between 3.6 and 43.9 μM (Fig. 1C), similar to the affinities of other tight-binding cell adhesion molecules (22)(23)(24)(25)(26)(27)(28). Interestingly, the strongest-binding pairs (Dsg1:Dsc1 and Dsg4:Dsc1) correspond to those expressed in the outermost layers of human skin, and the weakest (Dsg3:Dsc3) to those restricted to basal layers (1,10), consistent with a potential role for differential affinities in maintenance of cell arrangement in stratified epithelia (1).…”

Molecular determinants of cadherin ideal bond formation: Conformation-dependent unbinding on a multidimensional landscape

“…Next, we used MD simulations to investigate the molecular determinants of cadherin conformational interconversion. Since only the EC1-EC2 domains are involved in trans binding, we used four EC1-EC2 trans dimer crystal structures in our simulations: (i) WT cadherin (PDB ID code 2QVF), (ii) mutant W2A (PDB ID code 3LNH), which replaces the swapped Trp with an Ala and traps cadherin in an X-dimer conformation (12), (iii) mutant K14E (PDB ID code 3LNE), which eliminates a key salt bridge in the X-dimer interface and traps cadherin in an S-dimer structure (12), and (iv) W2F, "conformational-shuttling" mutant (PDB ID code 4NUQ), which relieves the strain in the swapped N-terminal β-strand and forms weak binding affinity S-dimers (37); previous DEER experiments showed that this W2F mutant construct populates an equilibrium ensemble of X-dimers and S-dimers (21). All structures used in these simulations were from Ecad except the W2F mutant, which was from N-cadherin (Ncad), a closely related type I classical cadherin that shares a high sequence identity (∼77%) of its N-terminal β-strand with Ecad (∼53% sequence identity over the whole EC1-EC2 dimer structure), with conserved amino acids forming the swapping interface.…”

“…The change in torsional angle between the EC1 domains in the W2F mutant was more abrupt (Fig. 4D, black line), likely due to both the smaller Phe2 side chain and fewer interactions at the dimer interface (21,37). In comparison, the bigger Trp2 indole ring occupied a larger surface area in the hydrophobic pocket of WT-Ecad dimers and was additionally stabilized by a hydrogen bond formed between its side chain NH group and the Asp90 carbonyl group in the hydrophobic pocket (10,17,37).…”

“…NMR studies show that trans dimer formation is a slow process that occurs over a ∼1-s timescale (20); this slow dimerization process presumably enables cadherins to sample metastable, intermediate conformations along their reaction coordinate. Furthermore, double electron-electron resonance (DEER) experiments (21) showed that classical cadherins exist in an equilibrium ensemble of multiple conformational states and that the dynamic exchange between conformations has important effects on cell-cell adhesion. However, the pathway by which cadherins interconvert between X-dimers and S-dimers and its effect on adhesion is not well understood.…”

Divergence of structural strategies for homophilic E-cadherin binding among bilaterians

“…The classical cadherins have a homophilic binding property that allows homogeneous cells to be organized into an independent solid tissue such as an epithelium (Nose et al, 1990;Takeichi, 1991;Vendome et al, 2014). Within the tissue, the extracellular bonds formed between the paired cadherins are thought to transmit forces originating from intracellular actomyosin activities that are required for epithelial homeostasis and morphogenesis (Borghi et al, 2012;Guillot and Lecuit, 2013;Heisenberg and Bellaïche, 2013;Takeichi, 2014).…”