Identification and Characterization of the X‑Dimer of Human P‑Cadherin: Implications for Homophilic Cell Adhesion Shota Kudo, † Jose M. M. Caaveiro, ‡ Shuichiro Goda, § Satoru Nagatoishi, ‡ Keisuke Ishii, ∥ Tadashi Matsuura, ∥ Yukio Sudou, ∥ Tatsuhiko Kodama, ⊥ Takao Hamakubo, ⊥ and Kouhei Tsumoto* ,†,‡,#,∇ † Department of Chemistry & Biotechnology, The University of Tokyo, Tokyo 108-8639, Japan ‡ Department of Bioengineering, The University of Tokyo, Tokyo 108-8639, Japan § Faculty of Engineering, Nagasaki University, Nagasaki 852-8521, Japan ∥ Perseus Proteomics Inc., Tokyo 153-0041, Japan ⊥ Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan # Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan ∇ Department of Medical Genome Science, The University of Tokyo, Tokyo 108-8639, Japan * S Supporting Information ABSTRACT: Cell adhesion mediated by cadherins depends critically on the homophilic trans-dimerization of cadherin monomers from apposing cells, generating the so-called strand- swap dimer (ss-dimer). Recent evidence indicates that the ss-dimer is preceded by an intermediate species known as the X-dimer. Until now, the stabilized form of the X-dimer had only been observed in E-cadherin among the classical type I cadherins. Herein, we report the isolation and characterization of the analogous X-dimer of human P-cadherin. Small-angle X-ray scattering (SAXS) and site- directed mutagenesis data indicates that the overall architecture of the X-dimer of human P-cadherin is similar to that of E- cadherin. The X-dimerization is triggered by Ca 2+ and governed by specific protein−protein interactions. The attachment of three molecules of Ca 2+ with high affinity (K d =9 μM) stabilizes the monomeric conformation of P-cadherin (ΔT m = 17 °C). The Ca 2+ -stabilized monomer subsequently dimerizes in the X-configuration by establishing protein−protein interactions that require the first two extracellular domains of the cadherin. The homophilic X-dimerization is very specific, as the presence of the highly homologous E-cadherin does not interfere with the self-recognition of P-cadherin. These data suggest that the X-dimer could play a key role in the specific cell−cell adhesion mediated by human P-cadherin. C adherins are calcium-dependent cell adhesion proteins involved in selective cell−cell recognition, cell-sorting during morphogenesis, and the development and maintenance of solid tissues. 1−3 Defects in the adhesive properties of cadherins are associated with severe malignancies such as carcinogenesis and tumor invasion. 4−6 The cadherin superfamily of proteins comprises more than a hundred different proteins. The best-characterized group, in terms of structure and function, are the classical cadherins, 7 which are divided into two groups (type I and II) according to their primary sequence and three-dimensional structure. 8 Classical cadherins display five extracellular cadherin (EC) domains, a single transmembrane domain, and an intracellular domain for the attachment to catenin. 9 Each EC domain (EC1−EC5) is composed of approximately 110 residues displaying an immunoglobulin-like fold. EC domains are connected by hinge regions containing binding sites for the essential Ca 2+ ions. 9,10 The effect of the binding of Ca 2+ on the function of cadherins has been previously studied at the molecular and cellular levels. 11−13 Mechanistically, classical cadherins promote cell−cell adhesion by trans-dimerization of their EC1 (N-terminal) ectodomains. 14,15 The so-called strand- swap dimer (ss-dimer) represents the stable dimerization complex of classical E- and N-cadherins. The ss-dimer is characterized by the exchange of a short N-terminal β-strand between the dimerizing cadherins. Binding is governed by the docking of Trp2 of EC1 in a well-defined hydrophobic pocket of the EC1 domain of the partner cadherin. Recent evidence indicates that the ss-dimerization of classical E-cadherin is preceded by an intermediate known as the X- dimer. 14,16−20 The name of this intermediate dimerization state refers to its singular shape in the crystal form. The X-dimer can be prepared in a stable form by adding an extra residue at the N-terminus of the primary sequence of E-cadherin (e.g., a Met residue), by removing the side chain of residues involved in the stabilization of the ss-dimer (i.e., W2A or E89A), or in the presence of a competitive inhibitor of the ss-dimer such as the Received: September 30, 2013 Revised: February 22, 2014 Published: February 23, 2014 Article pubs.acs.org/biochemistry © 2014 American Chemical Society 1742 dx.doi.org/10.1021/bi401341g | Biochemistry 2014, 53, 1742−1752