Crystal structures of Drosophila N-cadherin PNAS PLUS ectodomain regions reveal a widely used class of Ca2þ-free interdomain linkers Xiangshu Jina,b,1, Melissa A. Walkera,1, Klára Felsövályia,b,c,1, Jeremie Vendomea,b,c,1, Fabiana Bahnaa,b, Seetha Mannepallia, Filip Cosmanescua, Goran Ahlsena, Barry Honiga,b,c,2, and Lawrence Shapiroa,d,2 aDepartment of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032; bHoward Hughes Medical Institute, Columbia University, New York, NY 10032; cCenter for Computational Biology and Bioinformatics, Columbia University, New York, NY 10032; and dEdward S. Harkness Eye Institute, Columbia University, New York, NY 10032 Contributed by Barry Honig, October 27, 2011 (sent for review August 17, 2011) 2þ Vertebrate classical cadherins mediate selective calcium-dependent in otherwise nonadhesive cells induces Ca -dependent cell cell adhesion by mechanisms now understood at the atomic level. aggregation (13, 15). Although DN- and DE- cadherins perform However, structures and adhesion mechanisms of cadherins from biological roles roughly orthologous to those assumed by classical invertebrates, which are highly divergent yet function in similar cadherins in vertebrate species, their ectodomains differ mark- roles, remain unknown. Here we present crystal structures of edly from their vertebrate counterparts both in size and sequence three- and four-tandem extracellular cadherin (EC) domain seg- features. Compared to vertebrate counterparts, DN- and DE- ments from Drosophila N-cadherin (DN-cadherin), each including cadherins include a larger number of EC domains, some of which the predicted N-terminal EC1 domain (denoted EC1’) of the mature are highly diverged from vertebrate counterparts, and a mem- protein. While the linker regions for the EC1’-EC2’ and EC3’-EC4’ brane-proximal region consisting of EGF-like and laminin G do- pairs display binding of three Ca2þ ions similar to that of vertebrate mains. While the precise number of EC domains in DN-cadherin cadherins, domains EC2’ and EC3’ are joined in a “kinked” orienta- is not known with certainty because various prediction methods 2þ tion by a previously uncharacterized Ca -free linker. Biophysical currently available identify different numbers ranging from 9 to BIOPHYSICS AND analysis demonstrates that a construct containing the predicted 16, it is unclear how as many as 16 EC domains per cadherin COMPUTATIONAL BIOLOGY N-terminal nine EC domains of DN-cadherin forms homodimers could be arranged at intercellular junctions, because intercellular with affinity similar to vertebrate classical cadherins, whereas distances in both vertebrate and invertebrate tissues are similar deleting the ninth EC domain ablates dimerization. These results (20–30 nm) (16, 17) and can be spanned by only five EC domains suggest that, unlike their vertebrate counterparts, invertebrate per molecule in vertebrate species. cadherins may utilize multiple EC domains to form intercellular Here we report crystal structures of DN-cadherin ectodomain þ adhesive bonds. Sequence analysis reveals that similar Ca2 -free regions corresponding to the predicted N-terminal four EC linkers are widely distributed in the ectodomains of both verte- domains in the mature protein. While the linker regions between brate and invertebrate cadherins. domains 1 and 2 and domains 3 and 4 display binding of three Ca2þ ions similar to that of vertebrate cadherins, domains 2 and ell-cell adhesion is a distinguishing feature of metazoan spe- 3 are joined in a “kinked” orientation by a Ca2þ-free linker pre- Ccies essential to the development and maintenance of solid viously uncharacterized in cadherins. The orientations of do- tissues (1). In vertebrates, calcium-dependent cell adhesion is mains 2 and 3 defined by this Ca2þ-free interdomain linker are mediated primarily by members of the cadherin superfamily (2). similar in all three crystal structures. The DN-cadherin fragments Cadherins are defined as proteins containing “extracellular containing the predicted N-terminal four EC domains are mono- cadherin” (EC) domains (3–6), protein modules of ∼110 amino meric both in crystals and in solution, whereas a larger construct acids, which adopt a β-sandwich fold with a Greek key topology that includes the N-terminal nine EC domains forms homodimers similar to that of immunoglobulin (Ig) domains. The best char- with a dissociation constant of ∼0.35 μM. These data, taken acterized cadherins are vertebrate classical cadherins, a family of together, suggest that in contrast to vertebrate classical and proteins which share similar domain structures, each consisting T-cadherins, DN-cadherin and related cadherins function in of an ectodomain with five tandem EC domains, a single trans- intercellular adhesion through binding interfaces that are not membrane region, and a conserved cytoplasmic tail (7). The con- localized to their distal N-termini. Rather, it is more likely that nections between each set of successive EC domains are rigidified DN-cadherin and related cadherins form a globular structure 2þ by the stereotyped binding of three Ca ions (8, 9). Classical cad- with adhesive interfaces involving several EC domains, perhaps herins have been shown to function in intercellular adhesion by thematically similar to the arrangement of multiple Ig domains binding through their ectodomains, which are in turn linked to the required for Dscam binding (18). Finally, based on the unique actin cytoskeleton through associations of the cytoplasmic domains Ca2þ-free interdomain linkage found in the DN-cadherin crystal with catenin adaptor proteins (reviewed in ref. 10 and 11). The cadherin superfamily is also broadly represented in inver- tebrates. Analysis of the Drosophila genome has revealed 17 Author contributions: X.J., B.H., and L.S. designed research; X.J., M.A.W., K.F., J.V., F.B., S.M., F.C., and G.A. performed research; X.J., M.A.W., K.F., and J.V. analyzed data; and genes that encode proteins containing EC-like domains (12). X.J., K.F., J.V., B.H., and L.S. wrote the paper. Three of these molecules, DN-cadherin encoded by CadN, DE- The authors declare no conflict of interest. cadherin encoded by Shg, and DN-cad2 encoded by CadN2, Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, which appears likely to be a partial duplication product of the www.pdb.org [PDB ID codes 3UBF (EC1’-EC3’ crystal form I), 3UBG (EC1’-EC3’ crystal CadN gene, contain catenin binding sites in their cytoplasmic form II), and 3UBH (EC1’-4’)]. regions, and have been shown to interact with the Drosophila 1X.J., M.A.W., K.F., and J.V. contributed equally to this work. β – -catenin homolog armadillo (12 14). DN- and DE-cadherins 2To whom correspondence may be addressed. E-mail: [email protected] or lss8@ serve cell adhesion and tissue patterning functions analogous to columbia.edu. their vertebrate counterparts (13, 14). Also, like vertebrate clas- This article contains supporting information online at www.pnas.org/lookup/suppl/ sical cadherins, overexpression of DN-cadherin or DE-cadherin doi:10.1073/pnas.1117538108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1117538108 PNAS Early Edition ∣ 1of8 Downloaded by guest on October 5, 2021 structures, we present bioinformatic analyses of the entire cad- model to determine the remaining two structures by molecular herin superfamily that reveal the widespread presence of similar replacement. Data and refinement statistics are listed in Table S1. Ca2þ-free linkers between successive EC domains in a large num- The overall architecture of DN-cadherin EC1’-EC4’ fragment ber of cadherins. does not resemble any of the previously determined structures of vertebrate cadherin ectodomains, which adopt an elongated Results curved structure (8, 9) (Fig. 1). Instead, DN-cadherin EC1’-EC4’ DN-Cadherin EC Domains and N Terminus. Prior to this work, the fragment adopts a V-shaped structure imparted by a prominent number of EC domains and domain boundaries for DN-cadherin “kink” between domains EC2’ and EC3’ with a ∼80° angle be- have not been known with certainty, as sequence analyses have tween the long axes of these two domains. In all three structures, predicted a composition of 9 to 16 EC domains and different each EC domain presents a seven-stranded β-sandwich fold with boundaries for each domain (Fig. S1). Moreover, the precise a Greek key topology seen in other structures of cadherin ecto- N terminus of the mature DN-cadherin has not been determined domain fragments published to date (Fig. 1A). The linker regions experimentally. between the EC1’ and EC2’ domains, and that between the To determine the precise N terminus of the mature ectodo- EC3’ and EC4’ domain pairs contain three calcium ions bound in main as well as the EC domain boundaries of DN-cadherin, a way similar to that seen in classical cadherins (Fig. 1 A and B): we first used the XPXF/W motif, a marker of the beginning of an Specifically, the three calcium ions are coordinated by a DXNDX EC domain (5), to divide the sequence of the extracellular region (Asp-X-Asn-Asp-X) linker between the two successive EC do- of DN-cadherin. Using this approach, we identified 19 segments mains, a DR/YE motif and a single glutamate (E) residue con- of approximately 110 amino acids that can be considered as tributed from the prelinker domain, a DXD (Asp-X-Asp) motif putative EC domains in the extracellular region of DN-cadherin. and a single aspartate (D) residue contributed from the postlin- We next used PSI-BLAST to search for a set of DN-cadherin re- ker domain. The EC1’-EC2’ interdomain linkage in the EC1’- lated proteins that include ectodomains of similar length with at EC3’ structure determined in crystal form I is an exception in least 60% sequence identity and a cytoplasmic region with a con- that the conserved calcium coordination patterns were disrupted sensus β-catenin interaction motif.