Elastic versus brittle mechanical responses predicted for dimeric cadherin complexes

Cadherins are a superfamily of adhesion proteins involved in a variety of biological processes that include the formation of intercellular contacts, the maintenance of tissue integrity, and the development of neuronal circuits. These transmembrane proteins are characterized by ectodomains composed of a variable number of extracellular cadherin (EC) repeats that are similar but not identical in sequence and fold. E-cadherin, along with desmoglein and desmocollin proteins, are three classical-type cadherins that have slightly curved ectodomains and engage in homophilic and heterophilic interactions through an exchange of conserved tryptophan residues in their N-terminal EC1 repeat. In contrast, clustered protocadherins are straighter than classical cadherins and interact through an antiparallel homophilic binding interface that involves overlapped EC1 to EC4 repeats. Here we present molecular dynamics simulations that model the adhesive domains of these cadherins using available crystal structures, with systems encompassing up to 2.8 million atoms. Simulations of complete classical cadherin ectodomain dimers predict a two-phased elastic response to force in which these complexes first softly unbend and then stiffen to unbind without unfolding. Simulated α, β, and γ clustered protocadherin homodimers lack a two-phased elastic response, are brittle and stiffer than classical cadherins and exhibit complex unbinding pathways that in some cases involve transient intermediates. We propose that these distinct mechanical responses are important for function, with classical cadherin ectodomains acting as molecular shock absorbers and with stiffer clustered protocadherin ectodomains facilitating overlap that favors binding specificity over mechanical resilience. Overall, our simulations provide insights into the molecular mechanics of single cadherin dimers relevant in the formation of cellular junctions essential for tissue function.     

Functional analysis of the structural basis of homophilic cadherin adhesion

The structures of many cell surface adhesion proteins comprise multiple tandem repeats of structurally similar domains. In many cases, the functional significance of this architecture is unknown, and there are several cases in which evidence for individual domain involvement in adhesion has been contradictory. In particular, the extracellular region of the adhesion glycoprotein cadherin consists of five tandemly arranged domains. One proposed mechanism postulated that adhesion involves only trans interactions between the outermost domains. However, subsequent investigations have generated several competing models. Here we describe direct measurements of the distance-dependent interaction potentials between cadherin mutants lacking different domains. By quantifying both the absolute distances at which opposed cadherin fragments bind and the quantized changes in the interaction potentials that result from deletions of individual domains, we demonstrate that two domains participate in homophilic cadherin binding. This finding contrasts with the current view that cadherins bind via a single, unique site on the protein surface. The potentials that result from interactions involving multiple domains generate a novel, modular binding mechanism in which opposed cadherin ectodomains can adhere in any of three antiparallel alignments.     

Structure and binding mechanism of vascular endothelial cadherin: a divergent classical cadherin

Vascular endothelial cadherin (VE-cadherin), a divergent member of the type II classical cadherin family of cell adhesion proteins, mediates homophilic adhesion in the vascular endothelium. Previous investigations with a bacterially produced protein suggested that VE-cadherin forms cell surface trimers that bind between apposed cells to form hexamers. Here we report studies of mammalian-produced VE-cadherin ectodomains suggesting that, like other classical cadherins, VE-cadherin forms adhesive trans dimers between monomers located on opposing cell surfaces. Trimerization of the bacterially produced protein appears to be an artifact that arises from a lack of glycosylation. We also present the 2.1-Å-resolution crystal structure of the VE-cadherin EC1-2 adhesive region, which reveals homodimerization via the strand-swap mechanism common to classical cadherins. In common with type II cadherins, strand-swap binding involves two tryptophan anchor residues, but the adhesive interface resembles type I cadherins in that VE-cadherin does not form a large nonswapped hydrophobic surface. Thus, VE-cadherin is an outlier among classical cadherins, with characteristics of both type I and type II subfamilies.     

Lifetime measurements reveal kinetic differences between homophilic cadherin bonds

Cadherins are multidomain adhesion proteins whose interactions direct cell sorting during histogenesis. They determine cell adhesion specificity, but prior studies failed to identify physical differences that could underlie cell sorting. These single molecule studies identify kinetic and strength differences between different cadherins. They further demonstrate that the modular extracellular architecture of cleavage stage C-cadherin supports a multistate binding mechanism. These multiple bonds exhibit a kinetic hierarchy of strengths that map to the different cadherin domains. The outer two N-terminal domains of C-cadherin form two bound states with dissociation rates of 3.9 and 0.02 s(-1). The latter is 25-fold slower than between the corresponding epithelial cadherin segments. In addition to the two fast bonds, the five-domain fragment (CEC1-5) forms two additional stronger, longer-lived bonds with dissociation rates of 0.00039 and 0.00001 s(-1). We further quantified the lifetimes of bonds subject to a constant force, and thus identified multiple dissociation events with rates that agree quantitatively with the force spectroscopy data. The qualitative features are similar to those reported for epithelial cadherin. However, the significant differences in the dissociation rates of the outer domains, which include the specificity-determining region, suggest that kinetic differences may determine cadherin discrimination, rather than adhesion energies.     

The crystal structure of human E-cadherin domains 1 and 2, and comparison with other cadherins in the context of adhesion mechanism

Cell adhesion mediated by type I cadherins involves homophilic "trans" interactions that are thought to be brought about by a strand exchange mechanism involving the N-terminal extracellular domain. Here, we present the high-resolution crystal structure of the N-terminal two domains of human E-cadherin. Comparison of this structure with other type I cadherin structures reveals features that are likely to be critical to facilitate dimerization by strand exchange as well as dimer flexibility. We integrate this structural knowledge to provide a model for type I cadherin adhesive interactions. Intra-molecular docking of the conserved N-terminal "adhesion arm" into the acceptor pocket in monomeric E-cadherin appears largely identical to inter-molecular docking of the adhesion arm in adhesive trans dimers. A strained conformation of the adhesion arm in the monomer, however, may create an equilibrium between "open" and "closed" forms that primes the cadherin for formation of adhesive interactions, which are then stabilized by additional dimer-specific contacts. By contrast, in type II cadherins, strain in the adhesion arm appears absent and a much larger surface area is involved in trans adhesion, which may compensate the activation energy required to peel off the intra-molecularly docked arm. It seems that evolution has selected slightly different adhesion mechanisms for type I and type II cadherins.     

Desmoglein 2 can undergo Ca2+-dependent interactions with both desmosomal and classical cadherins including E-cadherin and N-cadherin

Desmoglein (Dsg) 2 is a ubiquitously expressed desmosomal cadherin. Particularly, it is present in all cell types forming desmosomes, including epithelial cells and cardiac myocytes and is upregulated in the autoimmune skin disease pemphigus. Thus, we here characterized the binding properties of Dsg2 in more detail using atomic force microscopy (AFM). Dsg2 exhibits homophilic interactions and also heterophilic interactions with the desmosomal cadherin desmocollin (Dsc) 2, and further with the classical cadherins E-cadherin (E-Cad) and N-cadherin (N-Cad), which may be relevant for cross talk between desmosomes and adherens junctions in epithelia and cardiac myocytes. We found that all homo- and heterophilic interactions were Ca²⁺-dependent. All binding forces observed are in the same force range, i.e., 30 to 40 pN, except for the Dsg2/E-Cad unbinding force, which with 45 pN is significantly higher. To further characterize the nature of the interactions, we used tryptophan, a critical amino acid required for trans-interaction, and a tandem peptide (TP) designed to cross-link Dsg isoforms. TP was sufficient to prevent the tryptophan-induced loss of Dsg2 interaction with the desmosomal cadherins Dsg2 and Dsc2; however, not with the classical cadherins E-Cad and N-Cad, indicating that the interaction modes of Dsg2 with desmosomal and classical cadherins differ. TP rescued the tryptophan-induced loss of Dsg2 binding on living enterocytes, suggesting that interaction with desmosomal cadherins may be more relevant. In summary, the data suggest that the ubiquitous desmosomal cadherin Dsg2 enables the cross talk with adherens junctions by interacting with multiple binding partners with implications for proper adhesive function in healthy and diseased states.     

Mechanism of homophilic cadherin adhesion

Direct measurements of the distance-dependent forces between membrane-bound cadherins were used to test current models of homophilic cadherin interactions. The results reveal a complex binding mechanism in which the proteins adhere in multiple alignments that involve more than the amino-terminal domains.     

Cadherin-mediated cell sorting not determined by binding or adhesion specificity.

Cadherin adhesion molecules play important roles in the establishment of tissue boundaries. Cells expressing different cadherins sort out from each other in cell aggregation assays. To determine the contribution of cadherin binding and adhesion specificity to the sorting process, we examined the adhesion of cells to different purified cadherin proteins. Chinese hamster ovary cell lines expressing one of four different cadherins were allowed to bind to the purified cadherin extracellular domains of either human E-cadherin or Xenopus C-cadherin, and the specificity of adhesion was compared with cell-sorting assays. None of the different cadherin-expressing cells exhibited any adhesive specificity toward either of the two purified cadherin substrates, even though these cadherins differ considerably in their primary sequence. In addition, all cells exhibited similar strengthening of adhesion on both substrates. However, this lack of adhesive specificity did not determine whether different cadherin-expressing cells would sort from each other, and the tendency to sort was not predictable by the extent of sequence diversity in their extracellular domains. These results show that cadherins are far more promiscuous in their adhesive-binding capacity than had been expected and that the ability to sort out must be determined by mechanisms other than simple adhesive-binding specificity.     

Cadherin-directed actin assembly: E-cadherin physically associates with the Arp2/3 complex to direct actin assembly in nascent adhesive contacts

Cadherin cell adhesion molecules are major determinants of tissue patterning which function in cooperation with the actin cytoskeleton. In the context of stable adhesion, cadherin/catenin complexes are often envisaged to passively scaffold onto cortical actin filaments. However, cadherins also form dynamic adhesive contacts during wound healing and morphogenesis. Here actin polymerization has been proposed to drive cell surfaces together, although F-actin reorganization also occurs as cell contacts mature. The interaction between cadherins and actin is therefore likely to depend on the functional state of adhesion. We sought to analyze the relationship between cadherin homophilic binding and cytoskeletal activity during early cadherin adhesive contacts. Dissecting the specific effect of cadherin ligation alone on actin regulation is difficult in native cell-cell contacts, due to the range of juxtacrine signals that can arise when two cell surfaces adhere. We therefore activated homophilic ligation using a specific functional recombinant protein. We report the first evidence that E-cadherin associates with the Arp2/3 complex actin nucleator and demonstrate that cadherin binding can exert an active, instructive influence on cells to mark sites for actin assembly at the cell surface.     

Phylogenetic analysis of the cadherin superfamily.

Cadherins are a multigene family of proteins which mediate homophilic calcium-dependent cell adhesion and are thought to play an important role in morphogenesis by mediating specific intercellular adhesion. Different lines of experimental evidence have recently indicated that the site responsible for mediating adhesive interactions is localized to the first extracellular domain of cadherin. Based upon an analysis of the sequence of this domain, I show that cadherins can be classified into three groups with distinct structural features. Furthermore, using this sequence information a phylogenetic tree relating the known cadherins was assembled. This is the first such tree to be published for the cadherins. One cadherin subtype, neural cadherin (N-cadherin), shows very little sequence divergence between species, whereas all other cadherin subtypes show more substantial divergence, suggesting that selective pressure upon this domain may be greater for N-cadherin than for other cadherins. Phylogenetic analysis also suggests that the gene duplications which established the main branches leading to the different cadherin subtypes occurred very early in their history. These duplications set the stage for the diversified superfamily we now observe.     

Regulation of cadherin-mediated adhesion in morphogenesis

Cadherin cell-adhesion proteins mediate many facets of tissue morphogenesis. The dynamic regulation of cadherins in response to various extracellular signals controls cell sorting, cell rearrangements and cell movements. Cadherins are regulated at the cell surface by an inside-out signalling mechanism that is analogous to the integrins in platelets and leukocytes. Signal-transduction pathways impinge on the catenins (cytoplasmic cadherin-associated proteins), which transduce changes across the membrane to alter the state of the cadherin adhesive bond.     

Characterization of N-cadherin unbinding properties in non-malignant (HCV29) and malignant (T24) bladder cells

The expression of N‐cadherin, characteristic of various cancers, very often leads to changes in the cells' adhesive properties. Thus, we sought to find out if N‐cadherin expressed in various, but cancer‐related cells, differs in its functional properties that could contribute to variations in cells' phenotypes. In our work, measurements of an unbinding force of a single N‐cadherin molecule, probed with the same antibody both on a surface of living non‐malignant (HCV29) and malignant cells (T24) of bladder cancer, were carried out with the use of an atomic force microscopy. The results show the enhanced N‐cadherin level in T24 malignant cells (8.7% vs. 3.6% obtained for non‐malignant one), confirmed by the Western blot and the immunohistochemical staining. The effect was accompanied by changes in unbinding properties of an individual N‐cadherin molecule. Lower unbinding force values (26.1 ± 7.1 pN) in non‐malignant cells reveal less stable N‐cadherin complexes, as compared to malignant cells (61.7 ± 14.6 pN). This suggests the cancer‐related changes in a structure of the binding site of the antibody, located at the extracellular domain of N‐cadherin. Copyright © 2011 John Wiley & Sons, Ltd.     

Structural and functional diversity of cadherin superfamily: Are new members of cadherin superfamily involved in signal transduction pathway?

A large number of cadherins and cadherin-related proteins are expressed in different tissues of a variety of multicellular organisms. These proteins share one property: their extracellular domains consist of multiple repeats of a cadherin-specific motif. A recent structure study has shown that the cadherin repeats roughly corresponding to the folding unit of the extracellular domains. The members of the cadherin superfamily are roughly classified into two groups, classical type cadherins proteins and protocadherin type according to their structural properties. These proteins appear to be derived from a common ancestor that might have cadherin repeats similar to those of the current protocadherins, and to have common functional properties. Among various cadherins, E-cadherin was the first to be identified as a Ca²⁺-dependent homophilic adhesion protein. Recent knockout mice experiments have proven its biological role, but there are still several puzzling unsolved properties of the cell adhesion activity. Other members of cadherin superfamily show divergent properties and many lack some of the expected properties of cell adhesion protein. Since recent studies of various adhesion proteins reveal that they are involved in different signal transduction pathways, the idea that the new members of cadherin superfamily may participate in more general cell-cell interaction processes including signal transduction is an intriguing hypothesis. The cadherin superfamily is structurally divergent and possibly functionally divergent as well. © 1996 Wiley-Liss, Inc.     

Sequence and structural determinants of strand swapping in cadherin domains: do all cadherins bind through the same adhesive interface?

Cadherins are cell surface adhesion proteins important for tissue development and integrity. Type I and type II, or classical, cadherins form adhesive dimers via an interface formed through the exchange, or “swapping”, of the N-terminal β-strands from their membrane-distal EC1 domains. Here, we ask which sequence and structural features in EC1 domains are responsible for β-strand swapping and whether members of other cadherin families form similar strand-swapped binding interfaces. We created a comprehensive database of multiple alignments of each type of cadherin domain. We used the known three-dimensional structures of classical cadherins to identify conserved positions in multiple sequence alignments that appear to be crucial determinants of the cadherin domain structure. We identified features that are unique to EC1 domains. On the basis of our analysis, we conclude that all cadherin domains have very similar overall folds but, with the exception of classical and desmosomal cadherin EC1 domains, most of them do not appear to bind through a strand-swapping mechanism. Thus, non-classical cadherins that function in adhesion are likely to use different protein-protein interaction interfaces. Our results have implications for the evolution of molecular mechanisms of cadherin-mediated adhesion in vertebrates.     

Cortactin is necessary for E-cadherin-mediated contact formation and actin reorganization

Classical cadherin adhesion molecules are key determinants of cell-cell recognition during development and in post-embryonic life. A decisive step in productive cadherin-based recognition is the conversion of nascent adhesions into stable zones of contact. It is increasingly clear that such contact zone extension entails active cooperation between cadherin adhesion and the force-generating capacity of the actin cytoskeleton. Cortactin has recently emerged as an important regulator of actin dynamics in several forms of cell motility. We now report that cortactin is recruited to cell-cell adhesive contacts in response to homophilic cadherin ligation. Notably, cortactin accumulates preferentially, with Arp2/3, at cell margins where adhesive contacts are being extended. Recruitment of cortactin is accompanied by a ligation-dependent biochemical interaction between cortactin and the cadherin adhesive complex. Inhibition of cortactin activity in cells blocked Arp2/3-dependent actin assembly at cadherin adhesive contacts, significantly reduced cadherin adhesive contact zone extension, and perturbed both cell morphology and junctional accumulation of cadherins in polarized epithelia. Together, our findings identify a necessary role for cortactin in the cadherin-actin cooperation that supports productive contact formation.     

Type II cadherin ectodomain structures: implications for classical cadherin specificity

Type I and II classical cadherins help to determine the adhesive specificities of animal cells. Crystal-structure determination of ectodomain regions from three type II cadherins reveals adhesive dimers formed by exchange of N-terminal beta strands between partner extracellular cadherin-1 (EC1) domains. These interfaces have two conserved tryptophan side chains that anchor each swapped strand, compared with one in type I cadherins, and include large hydrophobic regions unique to type II interfaces. The EC1 domains of type I and type II cadherins appear to encode cell adhesive specificity in vitro. Moreover, perturbation of motor neuron segregation with chimeric cadherins depends on EC1 domain identity, suggesting that this region, which includes the structurally defined adhesive interface, encodes type II cadherin functional specificity in vivo.     

Classical cadherins.

Cadherins represent a gene family of Ca(2+)-dependent cell adhesion molecules (CAMs) identified during development and in adult organs. They generally mediate cell-cell adhesion by homotypic interaction, although heterotypic binding between different cadherin molecules is possible. Molecular cloning and sequence comparison has led to the characterization of a highly homologous group of 'classical' cadherins and more distantly related members, together composing a gene superfamily. The classical cadherins are transmembrane glycoproteins which exhibit, in addition to the structural homologies, a very similar overall protein topology. Protein sequence comparison has led to the identification of domains of common functional importance. The cytoplasmic domains of cadherins associate with peripheral cytoplasmic proteins termed catenin alpha, beta and gamma with molecular weights of 102, 88 and 80 kDa respectively. This complex formation seems to regulate the adhesive function of cadherins, most likely by connecting cadherins with actin microfilaments. Possible implications of catenins for cadherin function are discussed.     

Remarkable disparity in mechanical response among the extracellular domains of type I and II cadherins

Cadherins, a large family of calcium-dependent adhesion molecules, are critical for intercellular adhesion. While crystallographic structures for several cadherins show clear structural similarities, their relevant adhesive strengths vary and their mechanisms of adhesion between types I and II cadherin subfamilies are still unclear. Here, stretching of cadherins was explored experimentally by atomic force microscopy and computationally by steered molecular dynamics (SMD) simulations, where partial unfolding of the E-cadherin ectodomains was observed. The SMD simulations on strand-swapping cadherin dimers displayed similarity in binding strength, suggesting contributions of other mechanisms to explain the strength differences of cell adhesion in vivo. Systematic simulations on the unfolding of the extracellular domains of type I and II cadherins revealed diverse pathways. However, at the earliest stage, a remarkable similarity in unfolding was observed for the various type I cadherins that was distinct from that for type II cadherins. This likely correlates positively with their distinct adhesive properties, suggesting that the initial forced deformation in type I cadherins may be involved in cadherin-mediated adhesion.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:25     

Identification of a conserved region common to cadherins and influenza strain A hemagglutinins

Cadherins are a family of integral membrane glycoproteins that mediate homophilic, calcium-dependent cell adhesion in vertebrate species. The primary structures of six members of the cadherin family have recently been determined. The extracellular portion of these proteins is composed of five domains, the first of which is the most highly conserved among cadherins. Previous searches of protein sequence databases have revealed little or no sequence homology between cadherins and other proteins. Here we report that the first extracellular domain of cadherins exhibits substantial sequence homology with the amino termini of influenza strain A hemagglutinins. These regions of sequence homology have been shown to be functionally important in both cadherins and hemagglutinins. Our observations suggest that a functional domain of cadherins is conserved among other proteins.     

Glycosyl phosphatidylinositol--anchored T-cadherin mediates calcium-dependent, homophilic cell adhesion.

Cadherins are a family of cell adhesion molecules that exhibit calcium-dependent, homophilic binding. Their function depends on both an HisAlaVal sequence in the first extracellular domain, EC1, and the interaction of a conserved cytoplasmic region with intracellular proteins. T-cadherin is an unusual member of the cadherin family that lacks the HisAlaVal motif and is anchored to the membrane through a glycosyl phosphatidylinositol moiety (Ranscht, B., and M. T. Dours-Zimmermann. 1991. Neuron. 7:391-402). To assay the function of T-cadherin in cell adhesion, we have transfected T-cadherin cDNA into CHO cells. Two proteins, mature T-cadherin and the uncleaved T-cadherin precursor, were produced from T-cadherin cDNA. The T-cadherin proteins differed from classical cadherins in several aspects. First, the uncleaved T-cadherin precursor was expressed, together with mature T-cadherin, on the surface of the transfected cells. Second, in the absence of calcium, T-cadherin was more resistant to proteolytic cleavage than other cadherins. Lastly, in contrast to classical cadherins, T-cadherin was not concentrated into cell-cell contacts between transfected cells in monolayer cultures. In cellular aggregation assays, T-cadherin induced calcium-dependent, homophilic adhesion which was abolished by treatment of T-cadherin-transfected cells with phosphatidylinositol-specific phospholipase C. These results demonstrate that T-cadherin is a functional cadherin that differs in several properties from classical cadherins. The function of T-cadherin in homophilic cell recognition implies that the mechanism of T-cadherin-induced adhesion is distinct from that of classical cadherins.