Cadherin Dimers Research Articles

The application of solution NMR spectroscopy to study dynamics of two-domain calcium-binding proteins

Protein dynamics due to flexible linkers connecting otherwise rigid domains may be critical for the functioning of a variety of biological systems, ranging from membrane transporters to calcium-signaling and the formation of intercellular junctions. Considering that NMR spectroscopy is extremely powerful to characterize dynamics at various time scales, this manuscript brings an overview of the main strategies that have been employed to characterize inter-domain dynamics in relevant biological systems. Emphasis was given to the calcium binding proteins: calmodulin, cadherin, and the Na+/Ca2+ exchanger calcium-sensor domain. The introduction of paramagnetic centers in diamagnetic proteins is seen as key to obtaining unambiguous information about inter-domain dynamics. This is because the self-alignment of one of the domains in multi-domain proteins avoids the problem of dealing with alignment tensor fluctuations in dynamic systems. The combination of residual dipolar couplings (RDCs) and pseudocontact shifts (PCSs) with computational strategies aiming to provide an ensemble description of protein dynamics is seen as the most powerful strategy to gain detailed atomistic information on inter-domain motions. It is noteworthy that the cadherin ectodomains and the Na+/Ca2+ exchanger calcium sensor respond in the same way upon calcium-binding: in the absence of calcium the two domains are flexibly linked to one another and may preferentially sample kinked inter-domain arrangements, while calcium binding stabilizes a rigid and extended inter-domain arrangement. It is thus remarkable that nature chose the same molecular mechanism to promote two very different biological functions that are triggered by calcium signaling: intercellular adhesion by the formation of cadherin dimers and the allosteric regulation of a membrane transporter in the case of the Na+/Ca2+ exchanger.

Multiple dimeric structures and strand-swap dimerization of E-cadherin in solution visualized by high-speed atomic force microscopy

Classical cadherins play key roles in cell-cell adhesion. The adhesion process is thought to comprise mainly two steps: X-dimer and strand-swap (SS-) dimer formation of the extracellular domains (ectodomains) of cadherins. The dimerization mechanism of this two-step process has been investigated for type I cadherins, including E-cadherin, of classical cadherins, whereas other binding states also have been proposed, raising the possibility of additional binding processes required for the cadherin dimerization. However, technical limitations in observing single-molecule structures and their dynamics have precluded the investigation of the dynamic binding process of cadherin. Here, we used high-speed atomic force microscopy (HS-AFM) to observe full-length ectodomains of E-cadherin in solution and identified multiple dimeric structures that had not been reported previously. HS-AFM revealed that almost half of the cadherin dimers showed S- (or reverse S-) shaped conformations, which had more dynamic properties than the SS- and X-like dimers. The combined HS-AFM, mutational, and molecular modeling analyses showed that the S-shaped dimer was formed by membrane-distal ectodomains, while the binding interface was different from that of SS- and X-dimers. Furthermore, the formation of the SS-dimer from the S-shaped and X-like dimers was directly visualized, suggesting the processes of SS-dimer formation from S-shaped and X-dimers during cadherin dimerization.

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.

Regulation of cadherin dimerization by chemical fragments as a trigger to inhibit cell adhesion

Many cadherin family proteins are associated with diseases such as cancer. Since cell adhesion requires homodimerization of cadherin molecules, a small-molecule regulator of dimerization would have therapeutic potential. Herein, we describe identification of a P-cadherin-specific chemical fragment that inhibits P-cadherin-mediated cell adhesion. Although the identified molecule is a fragment compound, it binds to a cavity of P-cadherin that has not previously been targeted, indirectly prevents formation of hydrogen bonds necessary for formation of an intermediate called the X dimer and thus modulates the process of X dimerization. Our findings will impact on a strategy for regulation of protein-protein interactions and stepwise assembly of protein complexes using small molecules.

Molecular Recognition between Cadherins Studied by a Coarse-Grained Model Interacting with a Coevolutionary Potential.

Studying the conformations involved in the dimerization of cadherins is highly relevant to understand the development of tissues and its failure, which is associated with tumors and metastases. Experimental techniques, like X-ray crystallography, can usually report only the most stable conformations, missing minority states that could nonetheless be important for the recognition mechanism. Computer simulations could be a valid complement to the experimental approach. However, standard all-atom protein models in explicit solvent are computationally too demanding to search thoroughly the conformational space of multiple chains composed of several hundreds of amino acids. To reach this goal, we resorted to a coarse-grained model in implicit solvent. The standard problem with this kind of model is to find a realistic potential to describe its interactions. We used coevolutionary information from cadherin alignments, corrected by a statistical potential, to build an interaction potential, which is agnostic about the experimental conformations of the protein. Using this model, we explored the conformational space of multichain systems and validated the results comparing with experimental data. We identified dimeric conformations that are sequence specific and that can be useful to rationalize the mechanism of recognition between cadherins.

Structure-Based Virtual Screening Allows the Identification of Efficient Modulators of E-Cadherin-Mediated Cell-Cell Adhesion.

Cadherins are a large family of transmembrane calcium-dependent cell adhesion proteins that orchestrate adherens junction formation and are crucially involved in tissue morphogenesis. Due to their important role in cancer development and metastasis, cadherins can be considered attractive targets for drug discovery. A recent crystal structure of the complex of a cadherin extracellular portion and a small molecule inhibitor allowed the identification of a druggable interface, thus providing a viable strategy for the design of cadherin dimerization modulators. Here, we report on a structure-based virtual screening approach that led to the identification of efficient and selective modulators of E-cadherin-mediated cell–cell adhesion. Of all the putative inhibitors that were identified and experimentally tested by cell adhesion assays using human pancreatic tumor BxPC-3 cells expressing both E-cadherin and P-cadherin, two compounds turned out to be effective in inhibiting stable cell–cell adhesion at micromolar concentrations. Moreover, at the same concentrations, one of them also showed anti-invasive properties in cell invasion assays. These results will allow further development of novel and selective cadherin-mediated cell–cell adhesion modulators for the treatment of a variety of cadherin-expressing solid tumors and for improving the efficiency of drug delivery across biological barriers.

Molecular and structural bases of interaction between extracellular domains of nectin-2 and N-cadherin.

Cell adhesion molecules such as nectins and cadherins play important role in the formation of adherens junction. While nectins interact through their extracellular domains in both homophilic and heterophilic manner among themselves, extracellular domains of cadherins participate only in homophilic fashion to mediate cell-cell adhesion. It is well established that nectins recruit cadherins in the adhesion sites through an interplay of adaptor molecules in the cytoplasmic side thereby increasing the effective concentration of both the adhesion molecules on the cell surface. This study provides molecular and structural bases of the novel interaction between extracellular domains of nectin-2 and N-cadherin, by which nectins can also recruit cadherins at the site of adherens junction through an adaptor-independent mechanism. Surface plasmon resonance study demonstrates that nectin-2 can directly recognize N-cadherin with a KD of 3.5 ± 0.6 μM which is physiologically relevant considering the affinities between other cell adhesion molecules including cadherin dimerization. Furthermore, structural analysis of currently available homodimeric structures of both nectin-2 and N-cadherin followed by molecular docking as well as complementary mutagenesis studies revealed the binding interface of this novel interaction.

The X-ray structure of human P-cadherin EC1-EC2 in a closed conformation provides insight into the type I cadherin dimerization pathway.

Cadherins are a large family of calcium-dependent proteins that mediate cellular adherens junction formation and tissue morphogenesis. To date, the most studied cadherins are those classified as classical, which are further divided into type I or type II depending on selected sequence features. Unlike other members of the classical cadherin family, a detailed structural characterization of P-cadherin has not yet been fully obtained. Here, the high-resolution crystal structure determination of the closed form of human P-cadherin EC1-EC2 is reported. The structure shows a novel, monomeric packing arrangement that provides a further snapshot in the yet-to-be-achieved complete description of the highly dynamic cadherin dimerization pathway. Moreover, this is the first multidomain cadherin fragment to be crystallized and structurally characterized in its closed conformation that does not carry any extra N-terminal residues before the naturally occurring aspartic acid at position 1. Finally, two clear alternate conformations are observed for the critical Trp2 residue, suggestive of a transient, metastable state. The P-cadherin structure and packing arrangement shown here provide new and valuable information towards the complete structural characterization of the still largely elusive cadherin dimerization pathway.

Design of a photoswitchable cadherin.

There is a growing interest in engineering proteins whose function can be controlled with the spatial and temporal precision of light. Here, we present a novel example of a functional light-triggered switch in the Ca-dependent cell–cell adhesion protein E-cadherin, created using a mechanism-based design strategy. We report an 18-fold change in apparent Ca2+ binding affinity upon illumination. Our results include a detailed examination of functional switching via linked changes in Ca2+ binding and cadherin dimerization. This design opens avenues toward controllable tools that could be applied to many long-standing questions about cadherin’s biological function in cell–cell adhesion and downstream signaling.

Dynamics of Extracellular Domains of Type I & II Cadherins: Disparity and Roles

Cadherin-mediated adhesion plays a crucial role in multicellular organisms. Dysfunction of this adhesion system has major consequences in a number of pathologies, including cancer invasion and metastasis. 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. The cadherin extracellular region is considered one of the dominant factors governing its adhesive properties, and unraveling its mechanical properties should bring a better understanding of the adhesion mechanism. Here, stretching of cadherins was explored experimentally by atomic force microscopy (AFM), and computationally by steered molecular dynamics (SMD) simulations, where the dominance of partial unfolding of the ectodomains was observed. 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 types 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.

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

Physical principles underlying cadherin‐mediated cell‐cell recognition

A variety of cell‐cell adhesion processes are mediated by the (trans) dimerization of cadherin proteins presented from apposing cell surfaces. A multiscale combined experimental/theoretical approach to this general problem will be described with particular focus on the development of a correlation between measured binding affinities at the molecular level and cell‐cell adhesive specificity. Following cell‐cell contact, cadherin dimers cluster together to form cell‐cell junctions. Since such clustering does not occur in the absence of cell‐cell contact, there is a necessary coupling between trans and lateral (cis) interactions. The molecular mechanism that underlies this coupling will be discussed and is characterized in detail through combination of x‐ray crystallography and coarse grained simulations. In addition to the insights that will be discussed in the context of cadherin function, general principles that may underlie the molecular basis of many cell‐cell adhesion processes will be proposed. This work was supported by NSF, MCB‐0918535

Prolines in βA-Sheet of Neural Cadherin Act as a Switch To Control the Dynamics of the Equilibrium between Monomer and Dimer

Neural cadherins dimerize through the formation of calcium-dependent strand-crossover structures. Dimerization of cadherins leads to cell-cell adhesion in multicellular organisms. Strand-crossover dimer forms exclusively between the first N-terminal extracellular modules (EC1) of the adhesive partners via swapping of their βA-sheets and docking of tryptophan-2 in the hydrophobic pocket. In the apo-state wild-type cadherin is predominantly monomer, which indicates that the dimerization is energetically unfavorable in the absence of calcium. Addition of calcium favors dimer formation by creating strain in the monomer and lowering the energetic barrier between monomer and dimer. Dynamics of the monomer-dimer equilibrium is vital for plasticity of synapses. Prolines recurrently occur in proteins that form strand-crossover dimer and are believed to be the source of the strain in the monomer. N-cadherins have two proline residues in the βA-sheet. We focused our studies on the role of these two prolines in calcium-dependent dimerization. Spectroscopic, electrophoretic, and chromatopgraphic studies showed that mutations of both prolines to alanines increased the dimerization affinity by ~20-fold and relieved the requirement of calcium in dimerization. The P5A and P6A mutant formed very stable dimers that required denaturation of protein to disassemble in the apo conditions. In summary, the proline residues act as a switch to control the dynamics of the equilibrium between monomer and dimer which is crucial for the plasticity of synapses.

Molecular design principles underlying β-strand swapping in the adhesive dimerization of cadherins

SummaryCell adhesion by classical cadherins is mediated by dimerization of their EC1 domains through the “swapping” of N-terminal β-strands. We use molecular simulations, measurements of binding affinities, and x-ray crystallography to provide a detailed picture of the structural and energetic factors that control the adhesive dimerization of cadherins. We show that strand swapping in EC1 is driven by conformational strain in cadherin monomers which arises from the anchoring of their short N-terminal strand at one end by the conserved Trp2 and at the other by ligation to Ca2+ ions. We also demonstrate that a conserved pro-pro motif functions to avoid the formation of an overly tight interface where affinity differences between different cadherins, crucial at the cellular level, are lost. We use these findings to design site-directed mutations which transform a monomeric EC2-EC3 domain cadherin construct, into a strand-swapped dimer.

Unraveling Two Distinct Binding Interfaces for E Cadherin Dimerization: A Structural Study Using Single-Molecule Super-Resolved Fluorescence Imaging

Cadherins are homophilic adhesion molecules mediating Ca++ dependent cell-cell adhesion. It is not very clear how exactly cadherins interact and promote cell adhesion at the molecular level. We have previously characterized cadherin interactions with single molecule FRET and single molecule AFM and found cadherins interact mostly through outermost EC1 domains (Zhang & Sivasankar et al 2009). We then developed super-resolution localization microscopy that achieved sub-nanometer precision and accuracy and applied it to measurements of the end-to-end distances of E-cadherin dimers (Pertsinidis & Zhang et al 2010). The majority of the wild type cadherin dimers exhibits an extended EC5-EC5 distance of 32nm matching a strand-swapped trans-dimer model. A smaller population of the wild type dimers shows shorter distances of around 25nm suggesting an alternative binding configuration. We extended this work further by characterizing mutations in E-cadherin that prevent formation of the the strand-swapping interface. Interestingly, we observed that W2A dimers exist in a more compact conformation, similar to the minor population in the wild type dimers. The compact conformation is consistent with a so-called X-dimer interface discovered in recent crystal structures (Ciatto et al 2010, Harrison et al 2010). These new data refine our previous induced-fit dimerization pathway (Sivasankar & Zhang et al 2009) and are consistent with the X-dimer being the initial encounter complex. Furthermore, these results provide evidence that, at near-physiological Ca++ concentrations, E-cadherins can utilize two distinct binding interfaces to facilitate cell adhesion.

Slow Disassembly of Neural-Cadherin Dimers

Cadherins are calcium dependent homophilic cell adhesive protein molecules that are critical for morphogenesis, synaptogenesis and synapse maintenance. Cadherins comprise an extracellular region, a single transmembrane region and a cytoplasmic region. The extracellular region has five tandemly repeated ectodomains (EC1-EC5), with three calcium binding sites situated between each of these domains. Cell-cell adhesion is mediated by the dimerization of cadherins presented on neighboring cell surfaces. We are focusing our studies on two members of type I classical cadherins, Neural (N) - and Epithelial (E)-cadherins. In spite of high sequence similarity between E- and N-cadherin, they have distinct physiological localization implying differences in their adhesive properties. Here, we compare the spectral characteristics, stability, calcium binding and assembly properties of the first two domains of N- and E- cadherins. Spectroscopic studies of these proteins were predictable and indicated typical β-sheet conformation with only partial exposure of tryptophans. Although both proteins are stabilized by calcium, apo-ECAD12 is less stable than apo-NCAD12. Direct calcium titrations that found the proteins bind calcium with equally high affinity (−6.2 kcal/mol). There was a striking difference between these proteins in terms of the kinetics of disassembly. Analytical size exclusion chromatography experiments showed that disassembly of ECAD12 dimers is rapid and disassembly of NCAD12 dimers is slow regardless of the calcium concentration. We observe this striking difference with constructs containing only the first 220 residues of a 700 residue protein. Thus, not only is this an interesting protein folding-function question, this remarkable difference in these cadherins may explain their segregation into different physiological niches.

Characterizing the Initial Encounter Complex in Cadherin Adhesion

Cadherins are Ca(2+)-dependent cell-cell adhesion proteins with an extracellular region of five domains (EC1 to EC5). Adhesion is mediated by "strand swapping" of a conserved tryptophan residue in position 2 between EC1 domains of opposing cadherins, but the formation of this structure is not well understood. Using single-molecule fluorescence resonance energy transfer and single-molecule force measurements with the atomic force microscope, we demonstrate that cadherins initially interact via EC1 domains without swapping tryptophan-2 to form a weak Ca(2+) dependent initial encounter complex that has 25% of the bond strength of a strand-swapped dimer. We suggest that cadherin dimerization proceeds via an induced fit mechanism where the monomers first form a tryptophan-2 independent initial encounter complex and then undergo subsequent conformational changes to form the final strand-swapped dimer.

Structure of artificial and natural VE-cadherinbased adherens junctions

In vascular endothelium, adherens junctions between endothelial cells are composed of VE-cadherin (vascular endothelial cadherin), an adhesive receptor that is crucial for the proper assembly of vascular structures and the maintenance of vascular integrity. As a classical cadherin, VE-cadherin links endothelial cells together by homophilic interactions mediated by its extracellular part and associates intracellularly with the actin cytoskeleton via catenins. Although, from structural crystallographic data, a dimeric structure arranged in a trans orientation has emerged as a potential mechanism of cell-cell adhesion, the cadherin organization within adherens junctions remains controversial. Concerning VE-cadherin, its extracellular part possesses the capacity to self-associate in solution as hexamers consisting of three antiparallel cadherin dimers. VE-cadherin-based adherens junctions were reconstituted in vitro by assembly of a VE-cadherin EC (extracellular repeat) 1-EC4 hexamer at the surfaces of liposomes. The artificial adherens junctions revealed by cryoelectron microscopy appear as a two-dimensional self-assembly of hexameric structures. This cadherin organization is reminiscent of that found in native desmosomal junctions. Further structural studies performed on native VE-cadherin junctions would provide a better understanding of the cadherin organization within adherens junctions. Homophilic interactions between cadherins are strengthened intracellularly by connection to the actin cytoskeleton. Recently, we have discovered that annexin 2, an actin-binding protein connects the VE-cadherin-catenin complex to the actin cytoskeleton. This novel link is labile and promotes the endothelial cell switch from a quiescent to an angiogenic state.

Fibrogenic fibroblasts increase intercellular adhesion strength by reinforcing individual OB-cadherin bonds

We have previously shown that the switch from N-cadherin to OB-cadherin expression increases intercellular adhesion between fibroblasts during their transition from a migratory to a fibrogenic phenotype. Using atomic force microscopy we here show that part of this stronger adhesion is accomplished because OB-cadherin bonds resist approximately twofold higher forces compared with N-cadherin junctions. By assessing the adhesion force between recombinant cadherin dimers and between native cadherins in the membrane of spread fibroblasts, we demonstrate that cadherin bonds are reinforced over time with two distinct force increments. By modulating the degree of lateral cadherin diffusion and F-actin organization we can attribute the resulting three force states to the single-molecule bond rather than to cadherin cluster formation. Notably, association with actin filaments enhances cadherin adhesion strength on the single-molecule level up to threefold; actin depolymerization reduces single-bond strength to the level of cadherin constructs missing the cytoplasmic domain. Hence, fibroblasts reinforce intercellular contacts by: (1) switching from N- to OB-cadherin expression; (2) increasing the strength of single-molecule bonds in three distinct steps; and (3) actin-promoted intrinsic activation of cadherin extracellular binding. We propose that this plasticity adapts fibroblast adhesions to the changing mechanical microenvironment of tissue under remodeling.

Stable and Unstable Cadherin Dimers: Mechanisms of Formation and Roles in Cell Adhesion

Numerous attempts to elucidate the strength of cadherin dimerization that mediates intercellular adhesion have produced controversial and inconclusive results. To clarify this issue, we compared E-cadherin dimerization on the surface of living cells with how the same process unfolds on agarose beads. In both cases, dimerization was monitored by the same site-specific cross-linking assay, greatly simplifying data interpretation. We showed that on the agarose surface under physiological conditions, E-cadherin produced a weak dimer that immediately dissociated after the depletion of calcium ions. However, either at pH 5 or in the presence of cadmium ions, E-cadherin produced a strong dimer that was unable to dissociate upon calcium depletion. Both types of dimers were W156-dependent. Remarkably, only the strong dimer was found on the surface of living cells. We also showed that the intracellular cadherin region, the clustering of which through catenins had been proposed as stabilizer of weak intercadherin interactions, was not needed, in fact, for cadherin junction assembly. Taken together, our data present convincing evidence that cadherin adhesion is based on high-affinity cadherin-cadherin interactions.