Structural studies of DN-cadherin, an invertebrate classical cadherin, and other, non-classical cadherins.

摘要

Classical cadherins, defined as those that interact with catenins via their cytoplasmic domains, are cell surface molecules required for the formation and maintenance of solid tissues in both vertebrate and invertebrate species. In vertebrates, classical cadherins mediate calcium-dependent cell-cell adhesion through a "strand-swapping" mechanism that involves reciprocal binding of the N-terminal strand from the first extracellular cadherin (EC) domain of each protomer into a receptor pocket of the partner molecule. This interaction and its resultant biophysical properties have likely evolved in response to biological constraints imposed by the development of complex tissues. Significantly, strand-swapping can only occur after a prodomain has been removed from the N-terminus of vertebrate classical cadherins by subtilisin-like proprotein convertases.;By contrast, though previous work has established the role of catenin-binding cadherins in cell-cell adhesion in invertebrate species, the adhesive mechanism of invertebrate classical cadherins, including Drosophila Neural (DN)-cadherin is unknown. Structure-guided sequence comparisons highlight several important differences between DN-cadherin and vertebrate classical cadherins. DN-cadherin and related proteins have significantly larger ectodomains (up to 19 extracellular cadherin [EC] repeats) than vertebrate classical cadherins (five EC repeats). The significance of this is unclear given that cadherins in vertebrate and invertebrate species span similar intercellular distances. Significantly, DN-cadherin and its invertebrate orthologs do not contain residues known to be necessary for strand-swapping, and their mechanism of adhesion remains unknown. Finally, it is not known whether DN-cadherin requires furin-like processing to achieve its mature adhesive form.;In this dissertation, we have undertaken bioinformatics, structural, and biophysical experiments to better understand DN-cadherin EC domain structure and the mechanism through which DN-cadherin and related proteins mediate intercellular adhesion in invertebrate species. We have used Zn-SAD to determine the structures of recombinant fragments of DN-cadherin that we predict to correspond to regions including the mature N-terminus. The structures reveal a "concatenated" cadherin domain arrangement created by a novel, non-calcium binding hairpin linker which confers a remarkable bend between EC domains 2' and 3' not observed in structures of vertebrate classical cadherins. We then employed bioinformatics analysis to determine whether this concatenated domain arrangement occurs in other cadherin proteins. Results of this analysis suggest that one or more concatenated cadherin domain regions similar to those observed in DN-cadherin occur in other non-classical cadherins, including FAT tumor suppressor, dachsous, and flamingo/CELSR. Finally, we used analytical ultracentrifugation (AUC) to test homophilic binding of the DN-cadherin three and four EC domain fragments used for crystallization. They were found to be monomeric, which prompted us to express larger protein regions and to test their ability to bind homophillically. Results from other AUC experiments show that recombinant proteins predicted to constitute the ten N-terminal most cadherin domains of mature DN-cadherin do in fact dimerize with affinity in the micromolar range.;The novel cadherin interdomain linker observed in the crystal structures described here provides new clues to understanding the architecture of the DN-cadherin ectodomain, and also that of other, related molecules. Specifically, the predicted presence of multiple concatenated EC domain regions---2 in DN-cadherin, 3-7 in Fat tumor suppressors, 3-5 in dachsous---presents the possibility that these molecules do not bridge the intercellular space by a linear arrangement of cadherin repeats as seen in vertebrate classical cadherins but may instead adopt globular quaternary structures that could in principle present binding surfaces involving more than one domain. Consistent with this, our AUC results implicate a larger protein region (≥ 4 EC domains) in DN-cadherin dimerization, several more than are known to be required in vertebrate classical cadherins. A multi-domain adhesive interface is consistent with observations that essential residues involved in the well-characterized strand-swap dimerization of vertebrate classical cadherins are absent from their invertebrate.