Molecular Crosstalk Between Adherens Junction Proteins, E-cadherin and Nectin-4.

Cell-cell junctions formed by the association of cell adhesion molecules facilitate physiological events necessary for growth and development of multicellular organisms. Among them, cadherins and nectins organize and assemble to form adherens junction, which thereby mechanically couples interacting cells. A detailed understanding of the crosstalk involving these cell adhesion molecules is fundamental to the study of the various developmental processes. Although, cadherins and nectins can recruit each other in the adherens junction through an interplay of cytoplasmic adaptor molecules, here, we report a direct interaction between N-terminal extracellular domains of E-cadherin and nectin-4 as demonstrated by surface plasmon resonance (SPR) and Atomic Force Microscopy (AFM)-based single molecule force spectroscopy (SMFS). Kinetic studies using SPR demonstrate the binding between the ectodomains of E-cadherin and nectin-4 with a KD of 3.7 ± 0.7 µM and KD of 5.4 ± 0.2 µM (reciprocal experiment). AFM-based SMFS experiments also support interaction between the ectodomains of E-cadherin and nectin-4 with the koff value of 31.48 ± 1.53 s-1 and the lifetime of the complex of 0.036 ± 0.0026 s. We thus propose a cell adhesion mechanism mediated by E-cadherin and nectin-4, which can have functional significance in early embryogenesis as evident from the expression pattern of both the proteins during early development.

Molecular mimicry of host short linear motif-mediated interactions utilised by viruses for entry.

Viruses are obligate intracellular parasites that depend on host cellular machinery for performing even basic biological functions. One of the many ways they achieve this is through molecular mimicry, wherein the virus mimics a host sequence or structure, thereby being able to hijack the host's physiological interactions for its pathogenesis. Such adaptations are specific recognitions that often confer tissue and species-specific tropisms to the virus, and enable the virus to utilise previously existing host signalling networks, which ultimately aid in further steps of viral infection, such as entry, immune evasion and spread. A common form of sequence mimicry utilises short linear motifs (SLiMs). SLiMs are short-peptide sequences that mediate transient interactions and are major elements in host protein interaction networks. This work is aimed at providing a comprehensive review of current literature of some well-characterised SLiMs that play a role in the attachment and entry of viruses into host cells, which mimic physiological receptor-ligand interactions already present in the host. Considering recent trends in emerging diseases, further research on such motifs involved in viral entry can help in the discovery of previously unknown cellular receptors utilised by viruses, as well as help in the designing of targeted therapeutics such as vaccines or inhibitors directed towards these interactions.

Heterophilic recognition between E-cadherin and N-cadherin relies on same canonical binding interface as required for E-cadherin homodimerization.

Cadherins are a family of cell surface glycoproteins that mediate Ca2+-dependent cell to cell adhesion. They organize to form large macromolecular assemblies at the junctions of cells in order to form and maintain the integrity of tissue structures, thereby playing an indispensable role in the multicellular organization. Notably, a large body of research on E- and N-cadherin, the two most widely studied members of the cadherin superfamily, suggest for homophilic associations among them to drive cell adhesion. Interestingly, latest studies also highlight for direct crosstalk among these two classical cadherins to form heterotypic connections in physiological as well as in disease environment. However, the molecular details for the heterophilic association of E-cadherin and N-cadherin has not been investigated yet, which we aimed to address in this work. Using surface plasmon resonance and flow cytometry based biophysical studies we observed heterophilic interaction between E- and N-cadherin mediated through the membrane distal ectodomains. Further, the heterodimeric interface of E-cadherin and N-cadherin was mapped using structure-guided mutational studies followed by complementary biophysical analyses to identify the important interface residues involved in the interaction. The results obtained imply significant resemblance in the interface residues of E-cadherin that are crucial for homophilic recognition of E-cadherin and heterophilic recognition of N-cadherin as well.

Emerging roles of the nectin family of cell adhesion molecules in tumour-associated pathways.

Tumour cells achieve maximum survival by modifying cellular machineries associated with processes such as cell division, migration, survival, and apoptosis, resulting in genetically complex and heterogeneous populations. While nectin and nectin-like cell adhesion molecules control development and maintenance of multicellular organisation in higher vertebrates by mediating cell-cell adhesion and related signalling processes, recent studies indicate that they also critically regulate growth and development of different types of cancers. In this review, we detail current knowledge about the role of nectin family members in various tumours. Furthermore, we also analyse the seemingly opposing roles of some members of nectin family in tumour-associated pathways, as they function as both tumour suppressors and oncogenes. Understanding this functional duality of nectin family in tumours will further our knowledge of molecular mechanisms regulating tumour development and progression, and contribute to the advancement of tumour diagnosis and therapy.

Cadherin-mediated host-pathogen interactions.

Cell adhesion molecules mediate cell-to-cell and cell-to-matrix adhesions and play an immense role in a myriad of physiological processes during the growth and development of a multicellular organism. Cadherins belong to a major group of membrane-bound cell surface proteins that, in coordination with nectins, drive the formation and maintenance of adherens junctions for mediating cell to cell adhesion, cellular communication and signalling. Alongside adhesive function, the involvement of cadherins in mediating host-pathogen interactions has been extensively explored in recent years. In this review, we provide an in-depth understanding of microbial pathogens and their virulence factors that exploit cadherins for their strategical invasion into the host cell. Furthermore, macromolecular interactions involving cadherins and various microbial factors such as secretory toxins and adhesins lead to the disintegration of host cell junctions followed by the entry of the pathogen or triggering downstream signalling pathways responsible for successful invasion of the pathogenic microbes are discussed. Besides providing a comprehensive insight into some of the structural complexes involving cadherins and microbial factors to offer the mechanistic details of host-pathogen interactions, the current review also highlights novel constituents of various cell signalling events such as endocytosis machinery elicited upon microbial infections.

Immunoglobulin-fold containing bacterial adhesins: molecular and structural perspectives in host tissue colonization and infection.

Immunoglobulin (Ig) domains are one of the most widespread protein domains encoded by the human genome and are present in a large array of proteins with diverse biological functions. These Ig domains possess a central structure, the immunoglobulin-fold, which is a sandwich of two ß sheets, each made up of anti-parallel ß strands, surrounding a central hydrophobic core. Apart from humans, proteins containing Ig-like domains are also distributed in a vast selection of organisms including vertebrates, invertebrates, plants, viruses and bacteria where they execute a wide array of discrete cellular functions. In this review, we have described the key structural deviations of bacterial Ig-folds when compared to the classical eukaryotic Ig-fold. Further, we have comprehensively grouped all the Ig-domain containing adhesins present in both Gram-negative and Gram-positive bacteria. Additionally, we describe the role of these particular adhesins in host tissue attachment, colonization and subsequent infection by both pathogenic and non-pathogenic Escherichia coli as well as other bacterial species. The structural properties of these Ig-domain containing adhesins, along with their interactions with specific Ig-like and non Ig-like binding partners present on the host cell surface have been discussed in detail.

An evolutionarily conserved charged residue dictates the specificity of heterophilic interactions among nectins.

Nectins are a family of four cell surface glycoproteins belonging to the immunoglobulin superfamily that mediate cell-cell adhesion and associated signalling pathways, thereby regulating several physiological processes including morphogenesis, growth and development of multicellular organisms. Nectins interact among themselves through their extracellular domains from the adjacent cells in both homophilic and heterophilic fashions to support cell-cell adhesion. Although nectins form homodimers as demonstrated in experimental set-ups, only the specific heterophilic interactions among nectins are physiologically relevant as shown by in vivo studies. It has been hypothesised that a conserved charged residue present at the binding interface acts as the molecular switch for heterophilic nectin-nectin recognitions. In this work, we have analysed the energetics of homophilic and heterophilic interactions of nectins, followed by surface plasmon resonance-based binding studies and complementary in silico analyses. Our findings confirm that the conserved charged residues at the binding interfaces dictate the specificity of the nectin-nectin heterophilic interactions. Furthermore, these residues also play a role in conferring higher affinity to the heterophilic interactions, thereby making them physiologically more prevalent compared to homophilic interactions. Thus, this work reveals the molecular basis of heterophilic recognitions among nectins that contribute to their physiological functions.

Structural Insights into N-terminal IgV Domain of BTNL2, a T Cell Inhibitory Molecule, Suggests a Non-canonical Binding Interface for Its Putative Receptors.

T cell costimulation is mediated by the interaction of a number of receptors and ligands present on the surface of the T cell and antigen-presenting cell, respectively. Stimulatory or inhibitory signals from these receptor-ligand interactions work in tandem to preserve immune homeostasis. BTNL2 is a type-1 membrane protein that provides inhibitory signal to T cells and plays an important role in several inflammatory and autoimmune diseases. Therefore, manipulation of the molecular interaction of BTNL2 with its putative receptor could provide strategies to restore immune homeostasis in these diseases. Hence, it is imperative to study the structural characteristics of this molecule, which will provide important insights into its function as well. In this study, the membrane-distal ectodomain of murine BTNL2 was expressed in bacteria as inclusion bodies, refolded in vitro and purified for functional and structural characterization. The domain is monomeric in solution as demonstrated by size-exclusion chromatography and analytical ultracentrifugation, and also binds to its putative receptor on naïve B cells and activated T cell subsets. Importantly, for the first time, we report the structure of BTNL2 as determined by solution NMR spectroscopy and also the picosecond-nanosecond timescale backbone dynamics of this domain. The N-terminal ectodomain of BTNL2, which was able to inhibit T cell function as well, exhibits distinctive structural features. The N-terminal ectodomain of BTNL2 has a significantly reduced surface area in the front sheet due to the non-canonical conformation of the CC' loop, which provides important insights into the recognition of its presently unknown binding partner.

Tracing the evolution of nectin and nectin-like cell adhesion molecules.

Nectin and nectin-like cell adhesion molecules (collectively referred as nectin family henceforth) are known to mediate cell-cell adhesion and related functions. While current literature suggests that nectins are prevalent in vertebrates, there are no in-depth analyses regarding the evolution of nectin family as a whole. In this work, we examine the evolutionary origin of the nectin family, using selected multicellular metazoans representing diverse clades whose whole genome sequencing data is available. Our results show that this family may have appeared earlier during metazoan evolution than previously believed. Systematic analyses indicate the order in which various members of nectin family seem to have evolved, with some nectin-like molecules appearing first, followed by the evolution of other members. Furthermore, we also found a few possible ancient homologues of nectins. While our study confirms the previous grouping of the nectin family into nectins and nectin-like molecules, it also shows poliovirus receptor (PVR/nectin-like-5) to possess characteristics that are intermediate between these two groups. Interestingly, except for PVR, the other nectins show surprising sequence conservations across species, suggesting evolutionary constraints due to critical roles played by these proteins.

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.