Receptor DNAM-1/CD226 Nectin-2/CD112 and Its Binding to Immune Crystal Structure of Cell Adhesion Molecule

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Crystal Structure of Cell Adhesion Molecule Nectin-2/CD112 and Its Binding to Immune Receptor DNAM-1/CD226

This information is current as Jun Liu, Xiaomin Qian, Zhujun Chen, Xiang Xu, Feng Gao, of September 26, 2021. Shuijun Zhang, Rongguang Zhang, Jianxun Qi, George F. Gao and Jinghua Yan J Immunol 2012; 188:5511-5520; Prepublished online 30 April 2012;

doi: 10.4049/jimmunol.1200324 Downloaded from http://www.jimmunol.org/content/188/11/5511

References This article cites 67 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/188/11/5511.full#ref-list-1 http://www.jimmunol.org/

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Crystal Structure of Cell Adhesion Molecule Nectin-2/CD112 and Its Binding to Immune Receptor DNAM-1/CD226

Jun Liu,*,†,1,2 Xiaomin Qian,*,‡,1 Zhujun Chen,*,x Xiang Xu,*,x Feng Gao,{ Shuijun Zhang,*,‡ Rongguang Zhang,{ Jianxun Qi,* George F. Gao,*,†,‡,‖ and Jinghua Yan*

The nectin and nectin-like molecule (Necl) family includes important cell adhesion molecules (CAMs) characterized by their Ig- like nature. Such CAMs regulate a broad spectrum of cell–cell interactions, including the interaction between NK cells and cytotoxic T lymphocytes (CTLs) and their target cells. CAM members nectin-2 (CD112) and Necl-5 (CD155) are believed to form homodimers (for nectin-2) or heterodimers in their functions for cell adhesion, as well as to interact with immune costimulatory receptor DNAX accessory molecule 1 (DNAM-1) (CD226) to regulate functions of both NK and CTL cells. However, the structural basis of the interactive mode of DNAM-1 with nectin-2 or Necl-5 is not yet understood. In this study, a soluble nectin-2 Ig-like V- Downloaded from set domain (nectin-2v) was successfully prepared and demonstrated to bind to both soluble ectodomain and cell surface-expressed full-length DNAM-1. The 1.85-A˚ crystal structure of nectin-2v displays a perpendicular homodimer arrangement, revealing the homodimer characteristics of the nectin and Necls. Further mutational analysis indicated that disruption of the homodimeric interface of nectin-2v led to a failure of the homodimer formation, as conﬁrmed by crystal structure and biochemical properties of the mutant protein of nectin-2v. Interestingly, the monomer mutant also loses DNAM-1 binding, as evidenced by cell staining with tetramers and surface plasmon resonance assays. The data indicate that interaction with DNAM-1 requires either the homodi- http://www.jimmunol.org/ merization or engagement of the homodimeric interface of nectin-2v. These results have implications for immune intervention of tumors or autoimmune diseases in the DNAM-1/nectin-2–dependent pathway. The Journal of Immunology, 2012, 188: 5511–5520.

ytotoxic lymphocytes, such as NK cells and CTLs, are hesion molecules (4–6). Among the immune costimulatory major players in cell-mediated immunity against viral receptors, attention has been focused on the Ig superfamily C infection and tumorigenesis (1–3). The recognition of member DNAX accessory molecule 1 (DNAM-1; also called nonself and abnormal self-Ags and the activation of NK cells and CD226), which is expressed on the majority of NK cells, T cells, CTLs are mediated by AgRs, a series of costimulators, and ad- monocytes, and platelets (7–9). DNAM-1 is involved in the ex- by guest on September 26, 2021 pansion, differentiation, and activation of NK cells and naive T cells (10, 11). Furthermore, DNAM-1–deficient mice display *CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of increased tumor development after transplantation of tumor cells Microbiology, Chinese Academy of Sciences, Beijing, 100101 China; †Graduate Uni- versity, Chinese Academy of Sciences, Beijing, 100049 China; ‡School of Life Sci- (12). A nonsynonymous polymorphism (Gly307Ser) of DNAM-1 ences, University of Science and Technology of China, Hefei, Anhui Province, 230026 was recently identified as a genetic risk factor for a variety of China; xCollege of Life Science, Anhui Agricultural University, Anhui, 230036 { autoimmune diseases, such as type 1 diabetes, autoimmune thy- China; National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China; and ‖Research Network of Immunity roid disease, rheumatoid arthritis, multiple sclerosis, systemic scle- and Health, Beijing Institutes of Life Science, Chinese Academy of Sciences, Bei- rosis, systemic lupus erythematosus, and psoriasis (13). Therefore, jing, 100101 China studies of DNAM-1’s interactions with its ligands are important 1 J.L. and X.Q. contributed equally to this study. for our understanding of its true physiological functions, especially 2Current address: Department of Internal Medicine, Yale University, New Haven, CT. in the immune system. Received for publication January 26, 2012. Accepted for publication March 31, 2012. Nectin-2 (also called CD112 or poliovirus receptor-related This work was supported by the 973 Project of the China Ministry of Science and protein 2) and Necl-5 (also called CD155, or poliovirus recep- Technology (Protein Science Special Project Grant 2010CB911902). G.F.G. is a lead- tor) have been identified as two ligands of DNAM-1 in both humans ing principal investigator of the Innovative Research Group of the National Natural Science Foundation of China (Grant 81021003). The funders had no role in study and mice (14–16). Evidence from in vitro and in vivo studies design, data collection and analysis, decision to publish, or preparation of the man- revealed that the interaction of DNAM-1 with its ligands is in- uscript. volved in the functions of a variety of immune cells (12, 17). For The atomic coordinates and structure factors for nectin-2v and FAMP mutant pre- instance, a strong correlation was found between the expression of sented in this article have been submitted to the Protein Data Bank (http://www.pdb. org) under accession numbers 4DFH and 4DFI, respectively. nectin-2 and Necl-5 and the lysis susceptibility of myeloid leu- Address correspondence and reprint requests to Dr. Jinghua Yan and Prof. George F. kemia by NK cells (18). Nectin-2– and Necl-5–transduced tumor Gao, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of cells are efficiently rejected in mice through the stimulation of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. E-mail ad- DNAM-1–expressing innate immunity by CD8a+ dendritic cells dresses: [email protected] (J.Y.) and [email protected] (G.F.G.) (DCs), as well as NK cells (19). NK cell-mediated lysis of DCs Abbreviations used in this article: CAM, cell adhesion molecule; DC, dendritic cell; DNAM-1, DNAX accessory molecule 1; gD, glycoprotein D; Necl, Nectin-like mol- also involves DNAM-1 and its ligands; moreover, the degree of ecule; nectin-2v, V-set domain of nectin-2; PDB, Protein Data Bank; RMSD, root contribution of DNAM-1 in this process is correlated with the mean square deviation; SynCAM, synaptic cell adhesion molecule; V, variable; WT, surface densities of nectin-2 and Necl-5 (20). Furthermore, wild type. monocyte extravasation through the endothelium is also regulated Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 by DNAM-1 and Necl-5 expressed at endothelial junctions (21). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200324 5512 STRUCTURE OF CD112 HOMODIMER AND ITS BINDING TO CD226

The interaction of eosinophil-expressed nectin-2 with DNAM-1 L-SeMet–labeled nectin-2v was also expressed in E. coli strain BL21 on mast cells contributes to costimulatory responses in allergic (DE3). Adaptive medium (20% Luria–Bertani medium and 80% M9 me- reactions (22). However, few reports have characterized the in- dium) was used to culture the cells at 37˚C to an OD600 of 0.4–0.8. Subsequently, the E. coli cells were resuspended in M9 medium and cul- teraction of DNAM-1 and its ligands at the molecular level (23). tured in restrictive medium (1% glucose) to an OD600 of 0.6–0.8 before The impact of the binding mode of DNAM-1 and nectin-2/Necl-5 induction. The medium was supplemented with 60 mg/l L-SeMet; 100 mg/l on their functions is necessary for our understanding of NK/CTL lysine, threonine, and phenylalanine; 50 mg/l leucine, isoleucine, and va- cell functions. line; and 0.5 mM isopropyl 1-thio-b-D-galactopyranoside. Induction oc- curred at 16˚C for 20 h. Finally, the L-SeMet–labeled protein was purified Nectin-2 and Necl-5 belong to the nectin and Necl family, which and stored under the same conditions as was the native protein. consists of nine members (nectin-1–4 and Necl-1–5). These The cDNA-encoding extracellular domain of human DNAM-1 from aa molecules are novel Ca2+-independent Ig-like cell adhesion mol- 19–250 was inserted into the NdeI and XhoI sites of the pET-21a ex- ecules (CAMs) (24). Nectin and Necls each contain three Ig-like pression vector. The recombinant DNAM-1 was expressed in E. coli strain domains (one V-set and two C-set domains) in their extracellular BL21 as inclusion bodies. The DNAM-1 inclusion bodies were isolated, as previously described (42), and then dissolved in denaturing buffer (100 regions (25). The distinction between nectins and Necls is that mM Tris [pH 8], 0.4 M L-arginine, 2 mM EDTA, 4 M urea, 5 mM reduced Necls lack an afadin-binding motif in their cytoplasmic domains glutathione, 0.5 mM glutathione disulfide). The mixture was dialyzed (25, 26). It is believed that nectins/Necls can form homo- or against an 8-fold volume of water for 8 h and then against an 8-fold heterodimers, contributing to cell–cell adhesion (24, 27–29). volume of 10 mM Tris [pH 8], 10 mM NaCl for an additional 8 h. The renatured protein was concentrated and further purified by gel filtration Thus, nectin/Necls play a pivotal role in the formation of cell on a HiLoad 16/60 Superdex 200 PG column (GE Healthcare). polarity and the metastasis of tumor cells (30). Nectin-2 was initially discovered as a poliovirus receptor-related protein (31). It Crystallization and data collection Downloaded from is expressed on a variety of cells and is especially overexpressed The hanging-drop vapor-diffusion technique was used to screen crystal- on a variety of tumor cells (16). Nectin-2 can stimulate the reaction growing conditions (Hampton Research) for WT nectin-2v and its FAMP of NK cells and CTLs through its interaction with DNAM-1 (32). mutant at 18˚C. After 5–7 d, crystals of WT nectin-2v were obtained within Previous studies suggested that nectins/Necls might form cis- a solution 0.2 M ammonium acetate, 0.1 M Bis-Tris (pH 5.5), and 25% dimers through their first C-set domain and consequently form PEG 3350. Similarly, crystals of the FAMP mutant were grown in 0.2 M ammonium sulfate, 0.1 M Tris (pH 8.5), and 25% (w/v) PEG 3350. trans-dimers through their V-set domain during cell–cell adhesion Diffraction data of the crystals were collected at the Shanghai Syn- http://www.jimmunol.org/ (28, 33–35). This interactive mode of nectins/Necls may be sup- chrotron Radiation Facility (SSRF) Beamline BL17U (Shanghai, China), ported by the structural determination of the Necl-1 (also known whereas anomalous x-ray diffraction data were collected at peak (l = as synaptic CAM [SynCAM]3) V-set domain in 2006, in which 0.9783 A˚ ) wavelengths from the selenomethionyl-derivative protein at the the homodimer of Necl-1 was considered a trans-dimer (36). The Advanced Photon Source (Chicago, IL). The collected intensities were indexed, integrated, corrected for absorption, scaled, and merged using crystal structure of the extracellular region of nectin-1, with three HKL2000 (43). Ig domains (including the V-set and both two C-set Ig-like domains), also displayed a dimer architecture via its V-set do- Structure determination main, which is proposed as a cis-dimer because of the perpen- Data collection and processing statistics are summarized in Table I. The dicular arrangement of the homodimer interface (37). More peak dataset was used for experimental phasing by the single-wavelength by guest on September 26, 2021 recently, the V-set domain of Necl-3 (also known as SynCAM2) anomalous dispersion method. A total of three expected heavy atoms was was also determined to be a homodimer (38). Despite these crystal located by SHELXD (44), and initial phases were calculated using Phaser structures, the true dimerization mode of nectins/Necls remains (45). Density modification was performed by DM (46). Approximately 90% of the residues were automatically traced by ARP/wARP (47). debatable. The structures of WT nectin-2v and the FAMP mutant were solved by the In this study, we generated a soluble V-set domain of nectin-2 molecular-replacement method using Phaser (37) from the CCP4 program (nectin-2v) and demonstrated that it binds to both DNAM-1– suite with the L-SeMet model. Extensive model building and restrained expressing cells and the soluble ectodomain of DNAM-1. The refinement were performed with COOT (48) and REFMAC5 (49). Further ˚ rounds of refinement were performed using the phenix.refine program crystal structure of nectin-2v at 1.85 A clearly shows a homodimer implemented in the PHENIX package (50) with isotropic ADP refinement formed by two perpendicularly arranged molecules. Disruption of and bulk solvent modeling. The stereochemical quality of the final models the dimeric interface abolishes nectin-2v binding to DNAM-1 and was assessed with the program PROCHECK (51). yields a stable nectin-2v monomer. We propose that there are two possible models for DNAM-1 binding to nectin-2: DNAM-1 Protein structure accession numbers competes against nectin-2 dimer formation in a 1:1 (DNAM-1: The atomic coordinates and structure factors for nectin-2v and FAMP nectin-2)-binding mode and DNAM-1 binds nectin-2 dimer in a mutant presented in this article have been submitted to the Protein Data 1:2 (DNAM-1:nectin-2)-binding mode. Bank (PDB) under accession numbers 4DFH (http://www.pdb.org/pdb/ search/structidSearch.do?structureId=4DFH) and 4DFI (http://www.pdb. org/pdb/search/structidSearch.do?structureId=4DFI), respectively.

Materials and Methods Gel-filtration and native gel assays Protein expression and purification A calibrated Superdex 200 column (GE Healthcare) was used for molecular Ig variable (V) domain of human nectin-2 (nectin-2v) from aa 32–158 (wild weight estimation for the WT and mutant nectin-2v. Gel-filtration fractions type [WT]) and mutant of the V domain (FAMP mutant = F145S, A143S, were subjected to SDS-PAGE or native PAGE in a Bio-Rad Mini-Protean II M89S, and P94S) encoding cDNAs were inserted into the NdeI and XhoI or Protean II (20-cm plates) slab cell (Bio-Rad, Richmond, CA), with the sites of the pET-21a expression vector (Novagen). Recombinant proteins discontinuous buffer system in 7.5% (w/v) polyacrylamide-separating gels were expressed in Escherichia coli strain BL21 (DE3) as inclusion bodies. and 5% (w/v) stacking gels. Samples were incubated at 45˚C for 30 min in Preparation, denaturation, and renaturation of the inclusion bodies were sample buffer, with a final concentration of 47 mM Tris-HCl (pH 7.8), 2% performed, as previously described (39–41). Refolded soluble proteins (w/v) SDS, 7.5% (v/v) glycerol, 40 mM DTT as the thiol-reducing agent, were first purified by gel filtration through a Superdex 75 10/300 GL and 0.002% (w/v) bromphenol blue. The supernatants were centrifuged at column with an AKTA FPLC (GE Healthcare), followed by a Resource Q 15,000 3 g for 2.5 min before loading. Native PAGE was performed in ion exchange column (GE Healthcare). Subsequently, gel filtration was 7.5% (w/v) polyacrylamide-separating gels and 4% (w/v) stacking gels, used as the final purification step, with a buffer of 20 mM Tris (pH 8) and with or without 0.1% (w/v) CHAPS. Before fractionation, the samples 50 mM NaCl, and the target protein was concentrated to 5 or 10 mg/ml for were incubated for 30 min at 45˚C in Tris-HCl (pH 7.8) buffer, with or crystallization. without 0.1% (w/v) CHAPS. The Journal of Immunology 5513

Cell culture and transfection freshly prepared HRP-DAB substrate reagent (Beyotime) for 15 min; the developing color was stopped by rinsing with PBS. The images were Human 293T cells were cultured in DMEM supplemented with 10% FCS. obtained via laser confocal microscopy (Zeiss LSM 710). The endogenous expression of nectin-2 and DNAM-1 on the cell surface was evaluated by flow cytometry with anti–nectin-2 mAb (sc-65333; Santa Surface plasmon resonance measurements Cruz) and anti–DNAM-1 mAb (FAB666F; R&D Systems) conjugated to fluorescein, respectively. The binding affinity between DNAM-1 and nectin-2v or FAMP mutant To test the interaction of DNAM-1 with nectin-2, the plasmid pDNAM-1 was analyzed at 25˚C on a Biacore 3000 machine with CM5 chips (GE was generated by inserting cDNA corresponding to full-length DNAM-1 Healthcare). HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM into modified eukaryotic expression vector pCAGGS, which contains a EDTA, 0.005% Tween 20) was used for all measurements. For surface FLAG tag sequence downstream of the multiple coding site. Recombinant plasmon resonance (SPR) measurements, both DNAM-1 and nectin-2v plasmids were transfected into 293T cells, and the overexpression of FLAG- were purified by gel filtration using a Superdex 75 column (GE Health- fusion proteins was confirmed by Western blotting with anti-FLAG Ab care). About 700 response units of nectin-2v were immobilized on the (Sigma), according to standard procedures. chip, followed by blockade with EDTA. When the data collection was finished in each cycle, the sensor surface was regenerated with 10 mM Tetramer preparation and cell staining NaOH. A series of concentrations ranging from 1.56 to 50 mMwas designed for the experiment. Sensograms were fit globally with Biacore To prepare tetramers of nectin-2v and its mutant FAMP, cDNAs corre- 3000 analysis software (BIAevaluation Version 4.1) using 1:1 Langmuir sponding to nectin-2v and the FAMP with C-terminal biotin-binding tags binding mode. The equilibrium binding constant (KD) values were calcu- were subcloned into the NdeI and XhoI sites of the pET30a prokaryotic lated using a nonlinear curve fit (y = [P1x]/[P2 + x]). expression vector. Recombinant proteins were expressed in BL21 E. coli cells as inclusion bodies and were subsequently refolded and purified, as Results described previously (52, 53). Biotinylation of proteins in vitro was ach- ieved by incubating the protein with the Bir A biotin protein ligase, The dimeric structure of nectin-2v Downloaded from D-biotin, and ATP (52). Free biotin was removed through gel filtration using The V-set domain (residue 32–158) of the extracellular portion AKTA FPLC (GE Healthcare). The efficiency of the in vitro biotinylation of nectin-2 (i.e., nectin-2v) was produced through an in vitro was determined through streptavidin-shift assays (3). The proteins dis- playing high efficiency of biotinylation were used to produce tetramers by refolding procedure (52) from E. coli-expressed inclusion bod- incubating with PE-conjugated streptavidin (Sigma). ies. The crystal structure was solved at a resolution of 1.85 A˚ The 293T cells were transiently transfected with pDNAM-1 plasmids or (Table I). Two molecules of nectin-2v were found in one asym- empty vectors. Twenty-four hours after the transfection, cells were treated metric unit (Fig. 1A), and they form a tight dimer (perpendicular http://www.jimmunol.org/ with 0.025% trypsin, resuspended in PBS, and stained with the tetramer (0.05 mg/ml) for 1 h. Subsequently, the cells were analyzed by flow head–head interaction). As expected, nectin-2v displays a typical cytometry (EasyCyte Mini; Guava). Ig V-set structure, which consists of nine antiparallel b-strands. Structural superimposition of one molecule of nectin-2v with the Histochemistry staining V domains of all of the known structures of nectin and Necl Twenty-four hours after the transfection of pDNAM-1 plasmids or empty family members, including nectin-1 (54), Necl-1 (36), Necl-3 vectors, 293T cells were washed with PBS three times and fixed for 15 min (38), and Necl-5 (55), indicated that nectin-2v retains a similar with 3% paraformaldehyde, followed by blocking with 1% BSA for 30 min. overall conformation compared with other members (Fig. 1B). Subsequently, the cells were incubated with biotinylated nectin-2v or FAMP (Structurally determined nectin-1, Necl-1, and Necl-5 are derived mutant, with a final concentration of 2 mg/ml, at 25˚C for 1 h. Cells were by guest on September 26, 2021 washed three times with PBS, incubated with HRP-conjugated streptavidin from human, whereas Necl-3 is from mouse; however, the se- solution (1:200; Beyotime, Shanghai, China) for 1 h, and incubated with quence of the visible part of the Necl-3 structure is 100% con-

Table I. Data collection, phasing, and reﬁnement statistics

Nectin-2v (Native) Nectin-2v (Se-Met) FAMP Mutant Data collection Space group P21 P41212 P212121 Cell dimensions a, b, c (A˚ ) 52.7, 43.9, 56.1 43.3, 45.8, 52.6 43.3, 45.8, 52.6 a, b, g (˚) 90.0, 118.2, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0

Peak Wavelength 1.5418 0.9793 0.9795 Resolution (A˚ ) 50–1.85 (1.92–1.85) 50–1.8 (1.86–1.80) 50–1.8 (1.86–1.80) Rsym or Rmerge (%) 3.6 (15.1) 8.9 (89.4) 11.4 (46.9) I/sI 41.7 (11.4) 35.3 (1.8) 16.6 (3.2) Completeness (%) 99.8 (98.8) 99.5 (91.8) 96.1 (88.7) Redundancy 3.6 (3.4) 16.0 (11.9) 5.9 (3.5) Reﬁnement Resolution (A˚ ) 27.97–1.85 34.54–1.80 No. reﬂections 19,468 9,250 Rwork/Rfree (%) 17.10/19.42 19.74/23.68 No. atoms Protein 1,914 952 Water 300 124 B-factors Protein 23.2 22.7 Water 38.0 28.8 RMSD Bond lengths (A˚ ) 0.011 0.016 Bond angles (˚) 0.096 1.067 Values in parentheses are for highest-resolution shell. 5514 STRUCTURE OF CD112 HOMODIMER AND ITS BINDING TO CD226 Downloaded from http://www.jimmunol.org/

FIGURE 1. Structure of nectin-2v dimer. (A) Overview of the nectin-2v dimer. Two molecules within one asymmetric unit of the nectin-2v structure by guest on September 26, 2021 clearly display a dimer conformation. Molecule 1 is denoted in green with strands and loops covering with surface. Molecule 2 is represented similarlyin orange. The presumed D-E loop in molecule 2, which has no corresponding electron density, is signified by the orange dotted line. (B) The uncommon D-E loop within the structure of nectin-2v. Structural superposition of five structure-solved nectin/Necls, including nectin-2 (green), nectin-1 (PDB: 3ALP; purple), Necl-1 (also known as SynCAM3, PDB: 1Z9M; blue), Necl-3 (also known as SynCAM2, PDB: 3M45; yellow), and Necl-5 (PDB: 3EOW; brown), demonstrating the unique protrusion of the D-E loop of nectin-2. (C) Structure-based sequence alignment of nectin-2 and other structure-determined nectin and Necls. Black arrows denote b-strands. Loops above the alignment indicate a-helices. The residues in blue boxes are highly conserved (.80%), and the residues highlighted in red are completely conserved. The green number “1” denotes residues that form disulfide bonds. Residues that were selected for mutation in the monomeric FAMP mutant of nectin-2v are highlighted with purple asterisks. The sequence alignment was generated with Clustal X (67) and ESPript (68). served between mouse and human.) However, the alignment of of these four dimeric proteins (Fig. 2B), we found that the second these molecules clearly shows that nectin-2v contains an unusual molecule (molecule 2 in the asymmetric unit) of nectin-2v is lo- extended loop (D-E loop, covering residue 112–117 aligned with cated in the same position as nectin-1 molecule 2 but is different Necl-5) between the D and E strands (Fig. 1B). The D-E loops of from the position of molecule 2 of Necl-1 and Necl-3. the two molecules of the nectin-2v dimer protrude with the same Detailed analyses of the interface of the two molecules in the orientation (to the upper side of the dimeric interface) and display nectin-2v dimer indicated that the formation of the dimer depends a flexible conformation (the flexibility can be derived from the fact on highly hydrophobic interactions between the two molecules. that there is no electronic density observed for molecule 2 of the The interface area of the two nectin-2v molecules is 861.0 A˚ 2, nectin-2v dimer). More importantly, the uncommon loop of nectin- which is larger than that of nectin-1 (841.0 A˚ 2), Necl-1 (729.9 A˚ 2), 2v is due to the addition of residues in the protein sequence of and Necl-3 (698.0 A˚ 2). Moreover, the interface is mainly formed nectin-2 relative to other nectin and Necl family molecules (Fig. 1C), by hydrophobic amino acids, including Tyr64, Leu67, Ala84, Met89, indicating that this unique loop is an intrinsic feature of nectin-2. Pro94, Ala143, and Phe145 (Figs. 1C, 2C). These residues within the The structures of nectin-1 (three Ig domains) (54), Necl-1 (V interface area of the two molecules have a total exposed hydro- domain alone) (36), and Necl-3 (V domain alone) (38) reveal that phobic area of 470.1 A˚ 2, occupying 55.9% of the total interface these molecules also form dimers through the interaction of their area of the two molecules. The unconventional large hydrophobic V domains. By superimposing nectin-2 on nectin-1, we found that interface of nectin-2v dimer is the consequence of a combination the overall conformation of the dimer formed by nectin-2v is of nectin-2v characteristic residues (unique residues Tyr64, Leu67, similar to nectin-1, with the root mean square deviation (RMSD) Ala84, and Pro94 in nectin-2 compared with other nectins/Necls in of 1.472 A˚ (Fig. 2A), much smaller than the RMSD when aligning these positions). In addition to the hydrophobic interaction, the with Necl-1 (RMSD = 8.388 A˚ ) or Necl-3 (RMSD = 8.109 A˚ ). two molecules form hydrogen bonds to enhance dimer formation. When we superimposed the first molecules in the asymmetric unit The generally symmetric hydrogen bonds are formed between The Journal of Immunology 5515

FIGURE 2. The dimer formation of nectin-2v. (A) Alignment of nectin-2 (green) with nectin-1 (purple) shows that the two molecules in these dimers form perpendicular or even acute angles. (B) Nectin-2v homodimer formation is similar to nectin-1 but different from Necl-1 and Necl-3. When superimposing molecule 1 of these four structures, molecule 2 of nectin-2 (green) is located in the same position as molecule 2 of nectin-1 (purple), but molecule 2 of Necl-1 (blue) and Necl-3 (yellow) occupies a different position. (C) Large hydrophobic interface of the nectin-2v dimer. The vacuum electrostatic potential surface based on Amber 99 charges of molecule 1 of nectin-2v shows the hydrophobic area of the dimer interface. Seven major hydrophobic residues whose side chains point to the Downloaded from interface are represented by purple sticks. The four residues in the yellow ellipses were mutated to generate the monomeric mutant of nectin-2v (FAMP mutant). (D) Hydrogen bond network between molecule 1 (green) and molecule 2 (orange) of the nectin-2v dimer. http://www.jimmunol.org/

Ser66, Asn71, Asn80, His86, Ser92, Glu141, Pro146, Ser149, and Phe145 of nectin-2v have been replaced by four serines (Fig. 3B). Arg151 in molecule 1 and Ser66, Asn71, Gln81, His86, Ser92, Pro146, Further analysis of the hydrophobicity of the altered surface of Ser149, and Arg151 in molecule 2 (Fig. 2D). the FAMP mutant (where the interface of the homodimer of native nectin-2v is located) indicated that mutation of the four by guest on September 26, 2021 The role of the hydrophobic interface in nectin-2v dimer residues dramatically reduced the area of the hydrophobic information terface of nectin-2v (Fig. 3C, 3D), as expected. Transformation of Structural analysis indicated that the dimer conformation of nectin- the nectin-2v dimer into a monomer of the FAMP mutant is 2v mainly depends on the hydrophobic interface between the two highly correlated with the destruction of the hydrophobic inter- V domains. To further verify the pivotal role of the hydrophobic face of nectin-2v, indicating the pivotal role of the hydrophobic interface in the homodimer of nectin-2v and elucidate the key interface in the formation of nectin-2v homodimers. Thus, the residues within the interface, we generated a mutant of nectin-2v, hydrophobic interface and the ability of nectin-2v to form a tight termed the FAMP mutant. Four hydrophobic amino acids (Met89, dimer may have important roles in its physiological function Pro94,Ala143,andPhe145) in the dimer interface of nectin-2v were (i.e., DNAM-1 binding). selected for their nondirect van der Waals interactions with the To further verify the monomeric FAMP mutant compared with corresponding residues in the other dimer molecule and replaced the dimeric status of nectin-2v, we performed a series of biochemi- with the nucleophilic amino acid serine in the FAMP mutant. To cal assays. First, nectin-2v and the FAMP mutant were subjected to observe whether mutagenesis of the hydrophobic interface can ab- size-exclusion chromatography (Superdex 200 10/300 GL column). late the nectin-2v homodimer, we determined the structure of the Nectin-2v was eluted as a single peak at an elution volume of 12 ml, FAMP mutant. Indeed, we found only one FAMP molecule in the corresponding to a globular protein with an estimated molecular asymmetric unit and no dimer as the one in the structure of nectin-2v mass ∼30 kDa. However, the FAMP mutant was eluted at a volume is formed through the crystal packing or symmetric operation, in- of 14 ml, corresponding to a much smaller molecular mass ∼13 dicating that the FAMP mutant is a monomeric mutant of nectin-2v. kDa (Fig. 4A). Subsequently, we further analyzed the status of the The overall structure of the FAMP mutant is similar to both two proteins by performing native PAGE (Fig. 4B); the results molecules 1 and 2 in the homodimer of nectin-2v, with RMSDs demonstrated that nectin-2v and the FAMP mutant carried different of 0.524 and 0.496 A˚ , respectively. When we superimposed the negative charges in the native PAGE gel. Both of the proteins FAMP mutant structure onto molecule 1 of nectin-2v, we found moved from the negative electrode to the positive, but the rate of that the major differences between the two structures involve a nectin-2v migration was much slower than that of the FAMP mu- conformational shift of the loops on the borderline of the in- tant, indicating the distinct status of the two proteins. terface of the nectin-2v homodimer (Fig. 3A). These loops in the Taken together, both the structural and biochemical results in- FAMP mutant ﬂoat away from the direction of the dimer interface dicate that destruction of the hydrophobic interface of nectin-2v and adopt a loose conformation compared with the molecules in abolishes its dimerization. Moreover, the successful mutation the homodimer of nectin-2v. Comparison of the electronic den- assays guided by the structure analyses revealed that the bacterially sity of the mutated residues from the FAMP mutant with WT produced nectin-2 protein, which was used to determine the nectin-2v clearly demonstrates that Met89,Pro94,Ala143,and structure, has a naturally folded conformation. 5516 STRUCTURE OF CD112 HOMODIMER AND ITS BINDING TO CD226

assess the binding of nectin-2 to DNAM-1. Cultured 293T cells were used to generate a cell line transiently expressing DNAM-1. First, to test the expression status of nectin-2 and its receptor (DNAM-1) on the surface of the 293T cells, we stained the untrans- fected cells with anti–nectin-2 and anti–DNAM-1 mAbs (Fig. 5A). Flow cytometry analysis revealed that 293T cells express nectin-2, but not DNAM-1, on the cell surface, as previously reported (15). Nectin-2v tetramers were also used to stain the 293T cells. As expected, no binding of nectin-2v to the 293T cells was observed (Fig. 5A). These results indicate that 293T cells can act as a model to study the interaction of nectin-2 and its receptor DNAM-1. Subsequently, 293T cells were transfected with pDNAM-1 plasmid harboring full-length DNAM-1, and the expression of DNAM-1 on the transfected cells was veriﬁed with ﬂow cytometry using the anti–DNAM-1 mAb (Fig. 5B), as well as Western blotting (data not shown). The cells were then used to analyze the binding of nectin-2 to DNAM-1. Notably, tetramers of nectin-2v bound to the surface of the DNAM-1–transfected cells (Fig. 5B).

Thus, we concluded that recombinant, soluble nectin-2v has the Downloaded from potential to perform an in vivo biological function, indicating a natural three-dimensional structural conformation of this protein. To further elucidate the binding proﬁle of nectin-2v with DNAM-1, we also prepared tetramers of the monomeric FAMP mutant of nectin-2v. DNAM-1–expressing 293T cells were then

stained with the tetramers of the FAMP mutant in comparison http://www.jimmunol.org/ FIGURE 3. Structural comparison of monomer mutant (FAMP mutant) with the tetramers of WT nectin-2v (Fig. 5B). The results of flow with WT nectin-2v. (A) Structural alignment of the FAMP mutant (cyan) 89 cytometry demonstrated that, although the nectin-2v tetramer with molecule 1 (green) of the nectin-2v dimer. The four residues (Met , binds to the DNAM-1–expressing cells (25% positive cells with Pro94, Ala143, and Phe145) in the hydrophobic interface of the nectin-2v high fluorescence), tetramers of the FAMP mutant cannot stain the dimer and the corresponding substituted serines in the FAMP mutant are represented by purple and red sticks, respectively, in the boxes. The con- cells (no positive cells with high fluorescence). We also performed formational shift of the loops on the edge of the dimer interface of nectin-2v in situ staining of the DNAM-1–expressing 293T cells using is observed when comparing the two structures. These loops in the FAMP biotinylated nectin-2v and its FAMP mutant (Fig. 5C); the in situ monomer shift away (as the orientation of black arrows) from the direction coloring of the stained cells was developed via HRP. In contrast to of the dimer interface compared with molecule 1 in the nectin-2v cis-di- efficient staining of the DNAM-1–expressing cells by nectin-2v, by guest on September 26, 2021 mer. (B) The electron density at the 1s contour level clearly shows that the cells could not be stained by FAMP mutant. The disruption of 89 94 143 145 residues Met , Pro , Ala , and Phe in nectin-2v (purple residues, left the dimerization interface of nectin-2 leads to a dramatic loss of panels) were mutated to four serines in the FAMP mutant (red residues, the binding of nectin-2 to DNAM-1, indicating that this unusual C D right panel). ( and ), Deconstruction of the hydrophobic interface of hydrophobic interface between the two molecules within the nectin-2v cis-dimer. After mutation of the four hydrophobic residues in the nectin-2 homodimer plays a role (direct or indirect) in the inter- dimer interface, the corresponding vacuum electrostatic potential surface within the area of the four residues (yellow circles) obviously changed. (C) action with DNAM-1. WT nectin-2v. (D) FAMP mutant. Binding-kinetics analysis of DNAM-1 to nectin-2v and FAMP mutant measured by BIAcore Soluble nectin-2v, but not the monomer mutant, binds to cell Binding affinity of DNAM-1 to nectin-2v was performed using SPR surface-expressed DNAM-1 technology. When DNAM-1 was injected over WT nectin-2v at To test the physiological activity of our recombinant nectin-2v, we 25˚C, a fast binding kinetics was observed (Kon and Koff are too prepared a tetramer of nectin-2v using a biotin-streptavidin system to fast and are beyond the BIAcore detection sensitivity) (Fig. 6A).

FIGURE 4. Biochemical conﬁrmation of nectin-2v mutant (FAMP mutant) as a monomer. (A) Size-exclusion chromatographs of nectin-2v (cyan) and the FAMP mutant (purple) on a Superdex 200 column. Compared with the early elution of WT nectin-2v at 12 ml, the peak of the FAMP mutant is at 14 ml, with the expected molecular mass ∼30 kDa (nectin-2v) and 13 kDa (FAMP mutant). The approximate positions of the molecular mass standards of 67.0, 43.0, and 13.7 kDa are marked. (B) Analysis of the conformational status of nectin- 2v and FAMP by native PAGE. The results in (A) and (B) are representative of three independent experiments. The Journal of Immunology 5517

FIGURE 5. Soluble nectin-2v, but not its monomer mutant (FAMP mutant), binds to cell-expressed DNAM-1. (A) Cultured 293T cells stained with anti– DNAM-1, anti–nectin-2 Abs, and tetramers of nectin- 2v (blue lines). The cyan lines denote the control cells without any staining. Nectin-2, but not DNAM-1, expression is clearly shown on the 293T cell surface. (B) Left panel, DNAM-1–transfected 293T cells could clearly be stained by anti–DNAM-1 Ab (red line). Blue line represents the control cells stained with anti– DNAM-1 after the transfection of empty vectors. DNAM-1–transfected 293T cells are stained with nectin-2v (middle panel)orFAMP(right panel)tetramers (red lines). The control cells, which were transfected with empty vector pcDNA3.0, were stained with these tetramers as negative controls (blue lines). (C) In situ staining of DNAM-1–transfected 293T cells Downloaded from with biotinylated nectin-2v or FAMP (2 mg/ml ﬁnal concentration). Original magniﬁcation 3400. HRP- labeled streptavidin and HRP-DAB substrate reagents were used to stain the cells. Cells transfected with pcDNA3.0 were stained by biotinylated nectin-2v as negative controls. The results shown are representative

of three independent experiments. http://www.jimmunol.org/

The KD value was calculated as 8.97 mM, using a least-squares fit Discussion for the data, assuming a 1:1 interaction in the steady-state affinity In this study, a series of biochemical, crystallographic, and cell model (Fig. 7), which was confirmed by a Scatchard plot (Fig. biological analyses characterized the homodimer formation and the

6B). To compare the binding capacity of WT nectin-2v and the binding of nectin-2 to its immune costimulatory receptor DNAM-1. by guest on September 26, 2021 FAMP mutant to DNAM-1, DNAM-1 (200 mM) was injected over A perpendicular homodimer structure of nectin-2 V-set was solved. WT nectin-2v and the FAMP mutant at the same time. The results Furthermore, the pivotal role for the hydrophobic interface, in- show that WT nectin-2v binds to DNAM-1 well, but the FAMP cluding the key residues, was veriﬁed in the dimerization of mutant loses its binding capacity to DNAM-1 (Fig. 6C). nectin-2. The disruption of the hydrophobic interface also abol-

FIGURE 6. BIAcore SPR analysis of DNAM-1 binding to nectin-2v and its FAMP monomer mutant. (A) The kinetic proﬁle of DNAM-1 binding to nectin-2v is shown. A series of concentrations of DNAM-1 (ranging from 1.56 to 50 mM) was used to measure the binding kinetics, with nectin-2v immobilized on the CM5 chip. (B) Response units were plotted against protein concentration calculated

from the BCA Kit. KD was calculated as 8.97 mM using a least-squares fit to the data, assuming a 1:1 interaction, which was confirmed by a Scatchard plot. (C) A total of 200 mM DNAM-1 was injected through flow cells, with nectin-2v and the FAMP mutant immobilized on the same CM5 chip. Clearly, WT nectin-2v can bind to DNAM-1 well, but the FAMP mutant loses its binding capacity. 5518 STRUCTURE OF CD112 HOMODIMER AND ITS BINDING TO CD226

The structure of nectin-2v clearly displays a tight homodimer formed through a large hydrophobic interface. The recently determined structure of another nectin family member, nectin-1, conﬁrms the cis-homodimer binding, mainly through a network of hydrogen bonds (54). Via analysis of the nectin-1 dimer, Narita et al. (54) proposed that the trans-interaction of nectins may de- pend on the upper side of the nectin cis-dimer. However, in our study, tetramers of recombinant nectin-2v did not bind to 293T cells, which express nectin-2 on their surface (Fig. 5A). One interpretation of this phenomenon is that soluble recombinant nectin-2v exists as a cis-dimer in solution. The nonbinding of this cis-dimer to cell surface-expressed nectin-2 indicates that trans- interaction of nectin-2 expressed on different cell surfaces may also use a similar hydrophobic interface as in the cis-dimer. This implies that both cis- and trans-interaction of nectin-2 may occur via a similar manner through the dimerization of the ﬁrst Ig-like V-set domain (Fig. 7A, 7B). In contrast, on the surface of the same cell, nectin-2 would have two statuses, monomer and cis-dimer,

that exist in equilibrium. The monomers can trans-bind to each Downloaded from other through their first V-set domain. This mode of trans-interaction of nectin-2 can also be applied to the heterointeraction of different nectins. There is also a possibility that nectin-2 on dif- FIGURE 7. Proposed model of nectin-2 dimer formation and the ferent cells behaves differently (i.e., as either a monomer or cis- binding to DNAM-1. (A and B) Two dimer models based on the structure dimer). All of these questions remain to be answered. In addition of nectin-2v determined in our study. Cell surface (blue double mem- to the contribution of V-set domain in the dimer formation, pre- http://www.jimmunol.org/ brane)-expressed nectin-2 forms trans-dimer (A) and/or cis-dimer (B) vious cell biological studies indicated that the C-set domains of through the interaction of V domains. The C1 and C2 domains (purple and nectin or nectin-like molecules are involved in cis-interaction yellow circles) may contribute to cis-dimer formation (33, 35). (C and D) (Fig. 7A) (33–35). Related studies from different groups indepen- Two possible binding models of nectin-2 with DNAM-1. (C) DNAM-1 dently demonstrated that the C-set domains of nectin-2 and Necl-2 (green ellipses) expressed on cell surfaces (red double membrane) binds to (SynCAM1) on the surface of the cells are responsible for the D nectin-2 through the same interface as in the nectin-2v dimer. ( )DNAM-1 lateral clustering of the cis-interaction, which could complement interacts with the cis-dimer of nectin-2. or even promote the trans-interaction of these molecules (33, 35). Nectin-2v, produced through the prokaryotic expression system in our study, has no posttranslational modified residues. How- by guest on September 26, 2021 ishes the binding of nectin-2 to DNAM-1. Thus, these results may ever, it was demonstrated that the posttranslational modification be helpful for the interpretation of the properties of homodime- of adhesion molecules can modulate their homophilic adhesion rization of nectin-2 and the interactive mode of nectin-2 with and other functions (36). The KD value of nectin-2 binding to DNAM-1. DNAM-1 determined in our study is slightly lower than the KD Nectin and Necls are believed to function in both homophilic and value reported by Tahara-Hanaoka and colleagues (23). They pro- heterophilic dimers, as well as cis- and trans-dimers, in a variety of duced the nectin-2 proteins through an eukaryotic expression sys- cells, including epithelium, immune cells, and neurologic system tem featured with postmodifications of the protein. This may in- cells (24, 27–29). In addition, they interact with a broad spectrum dicate that postmodified residues of nectin-2 also contribute to the of ligands/counterreceptors to execute their functions (14–16, 24, nectin-2/DNAM-1 interaction. Fogel et al. (38) determined that N- 56, 57). More importantly, they can also be used as receptors for glycosylation within the V domains of Necls, also known as virus entry (31, 58–60). However, with these diverse functions, it SynCAMs, differentially affects Necl properties. The N-glycans is less well understood how the same molecule functions in dif- at Asn70/Asn104 of Necl-2 (SynCAM1) increase its homophilic ferent cells, especially with regard to the ability of the same interactions, whereas N-glycosylation of Necl-3 (SynCAM2) at molecule to bind different ligand/counterreceptors. Structural in- Asn60 hinders the homodimer formation. It was determined that formation about nectin and Necls is very limited. Only three Necl-2 V domain accommodates the glycosylation sites Asn70/ homodimers (three Ig domains of nectin-1, one Ig domain of Necl-1 Asn104 flanking the homo-binding interface, which may favor and Necl-3) and one monomer (two Ig domains of Necl-5) of dimer formation by limiting the conformational space available to nine members of this family have been described but with dif- the V domain. In contrast, an N-glycan on residue Asn60 of Necl-3 ferent conclusions (36, 38, 54, 55). The structure of nectin-1 is was identified within the adhesive interface of its V domain, which regarded as a cis-dimer, but Necl-1 is considered a trans-dimer may interfere with homodimer formation. When we analyzed the (36, 38, 54). There is no structural report of any heterodimers for potential N-glycosylation sites within the V domain of nectin-2 these family members. Therefore, the true scenario of the struc- through prediction (63), we found that Asn107 may be N-glyco- tural basis for the diverse binding properties of nectins/Necls sylated. Based on our structure of nectin-2v, Asn107 locates out of remains a big issue. Recently, our group (61) and Di Giovine the homodimer-binding interface and near the V/C-set domain et al. (62) independently discovered that human HSV-1 glyco- interface, which indicates that N-glycosylation may augment protein D (gD) unexpectedly precluded the nectin-1 dimer from homodimer formation of nectin-2. entering host cells (i.e., the binding interface of nectin-1 to gD In our study, the abolition of nectin-2 binding to DNAM-1, by is almost the same as the nectin-1 dimer interface), revealing altering the dimer-binding interface of nectin-2, indicates two the monomeric nectin-1 binding mode to gD, rather than the sus- possibilities of the binding mode between nectin-2 and DNAM-1 pected nectin-1 dimeric binding. (Fig. 7C, 7D): the binding of DNAM-1 to nectin-2, like HSV-1 gD The Journal of Immunology 5519 binding to nectin-1, also occupies the dimer interface, disrupting 13. Dieude´, P., M. Guedj, M. E. Truchetet, J. Wipff, L. Revillod, G. Riemekasten, M. Matucci-Cerinic, I. Melchers, E. Hachulla, P. Airo, et al. 2011. Association of dimer formation or cis-dimerization of nectin-2 is necessary for its the CD226 Ser(307) variant with systemic sclerosis: evidence of a contribution interaction with the DNAM-1 receptor. Our current work cannot of costimulation pathways in systemic sclerosis pathogenesis. Arthritis Rheum. rule out either of these two models, and future work should focus 63: 1097–1105. 14. Tahara-Hanaoka, S., A. Miyamoto, A. Hara, S. Honda, K. Shibuya, and on this. However, Tahara-Hanaoka et al. (23) found that DNAM-1 A. Shibuya. 2005. Identification and characterization of murine DNAM-1 binding to nectin-2–transfected cells was augmented substantially (CD226) and its poliovirus receptor family ligands. Biochem. Biophys. Res. when the cells are pretreated with the anti–nectin-2 mAb (R2.477.1), Commun. 329: 996–1000. 15. Bottino, C., R. Castriconi, D. Pende, P. Rivera, M. Nanni, B. Carnemolla, which recognizes an epitope at one of the C set Ig-like domains of C. Cantoni, J. Grassi, S. Marcenaro, N. Reymond, et al. 2003. Identification of nectin-2 and blocks homophilic binding (64, 65). This suggested PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule. J. Exp. Med. 198: 557–567. that DNAM-1 binding to nectin-2 on the cell surface may compete 16. Pende, D., C. Bottino, R. Castriconi, C. Cantoni, S. Marcenaro, P. Rivera, for the nectin-2 homophilic binding site, which supports the first G. M. Spaggiari, A. Dondero, B. Carnemolla,N.Reymond, et al. 2005. PVR mode that we proposed (Fig. 7C). (CD155) and Nectin-2 (CD112) as ligands of the human DNAM-1 (CD226) activating receptor: involvement in tumor cell lysis. Mol. Immunol. 42: 463– Elucidation of the molecular mechanism of the homodimer 469. formation of nectin-2 and its interaction with immune costimulator 17. Gilfillan, S., C. J. Chan, M. Cella, N. M. Haynes, A. S. Rapaport, K. S. Boles, receptor DNAM-1 will help us to understand cell–cell adhesions D. M. Andrews, M. J. Smyth, and M. Colonna. 2008. DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and and several other immune processes, such as the cytotoxicity of tumors. J. Exp. Med. 205: 2965–2973. NK and CTL cells toward tumor cells (18, 19), the interaction 18. Pende, D., G. M. Spaggiari, S. Marcenaro, S. Martini, P. Rivera, A. Capobianco, M. Falco, E. Lanino, I. Pierri, R. Zambello, et al. 2005. Analysis of the receptor- between NK cells and DCs, the elimination of stimulated T cells ligand interactions in the natural killer-mediated lysis of freshly isolated myeloid by NK cells (66), monocyte extravasation through the endothe- or lymphoblastic leukemias: evidence for the involvement of the Poliovirus re- Downloaded from lium (21), and the interaction of eosinophils with mast cells (22). ceptor (CD155) and Nectin-2 (CD112). Blood 105: 2066–2073. 19. Tahara-Hanaoka, S., K. Shibuya, H. Kai, A. Miyamoto, Y. Morikawa, Our findings increase the general understanding of DNAM-1 and N. Ohkochi, S. Honda, and A. Shibuya. 2006. Tumor rejection by the poliovirus its ligands in immune recognition and activity, as well as pave the receptor family ligands of the DNAM-1 (CD226) receptor. Blood 107: 1491– way for immune diagnosis and therapy of tumors or autoimmune 1496. 20. Pende, D., R. Castriconi, P. Romagnani, G. M. Spaggiari, S. Marcenaro, diseases through DNAM-1 and nectin-2/Necl5–dependent inter- A. Dondero, E. Lazzeri, L. Lasagni, S. Martini, P. Rivera, et al. 2006. Expression vention. of the DNAM-1 ligands, Nectin-2 (CD112) and poliovirus receptor (CD155), on http://www.jimmunol.org/ dendritic cells: relevance for natural killer-dendritic cell interaction. Blood 107: 2030–2036. Acknowledgments 21. Reymond, N., A. M. Imbert, E. Devilard, S. Fabre, C. Chabannon, L. Xerri, We thank Dr. Fuliang Chu, Dr. Beiwen Zheng, Dr. Guangwen Lu, Dr. Zheng C. Farnarier, C. Cantoni, C. Bottino, A. Moretta, et al. 2004. DNAM-1 and PVR regulate monocyte migration through endothelial junctions. J. Exp. Med. 199: Fan, Shihong Zhang, Dr. Yi Shi, and Qun Yan for excellent assistance and 1331–1341. suggestions throughout the project. Assistance from the staff at the Shanghai 22. Bachelet, I., A. Munitz, D. Mankutad, and F. Levi-Schaffer. 2006. Mast cell Synchrotron Radiation Facility (SSRF Beamline BL17U) (Shanghai, China) costimulation by CD226/CD112 (DNAM-1/Nectin-2): a novel interface in the and the Argonne National Laboratory (Chicago, IL) is acknowledged. allergic process. J. Biol. Chem. 281: 27190–27196. 23. Tahara-Hanaoka, S., K. Shibuya, Y. Onoda, H. Zhang, S. Yamazaki, A. Miyamoto, S. Honda, L. L. Lanier, and A. Shibuya. 2004. Functional char-

Disclosures acterization of DNAM-1 (CD226) interaction with its ligands PVR (CD155) and by guest on September 26, 2021 The authors have no financial conflicts of interest. nectin-2 (PRR-2/CD112). Int. Immunol. 16: 533–538. 24. Takai, Y., J. Miyoshi, W. Ikeda, and H. Ogita. 2008. Nectins and nectin-like molecules: roles in contact inhibition of cell movement and proliferation. Nat. Rev. Mol. Cell Biol. 9: 603–615. References 25. Takai, Y., K. Irie, K. Shimizu, T. Sakisaka, and W. Ikeda. 2003. Nectins and 1. Bryceson, Y. T., M. E. March, H. G. Ljunggren, and E. O. Long. 2006. Acti- nectin-like molecules: roles in cell adhesion, migration, and polarization. Cancer vation, coactivation, and costimulation of resting human natural killer cells. Sci. 94: 655–667. Immunol. Rev. 214: 73–91. 26. Fuchs, A., and M. Colonna. 2006. The role of NK cell recognition of nectin and 2. Cerwenka, A., and L. L. Lanier. 2001. Natural killer cells, viruses and cancer. nectin-like proteins in tumor immunosurveillance. Semin. Cancer Biol. 16: 359– Nat. Rev. Immunol. 1: 41–49. 366. 3. Liu, J., S. Zhang, S. Tan, B. Zheng, and G. F. Gao. 2011. Revival of the iden- 27. Kurita, S., H. Ogita, and Y. Takai. 2011. Cooperative role of nectin-nectin and tification of cytotoxic T-lymphocyte epitopes for immunological diagnosis, nectin-afadin interactions in formation of nectin-based cell-cell adhesion. J. Biol. therapy and vaccine development. Exp. Biol. Med. (Maywood) 236: 253–267. Chem. 286: 36297–36303. 4. Lanier, L. L. 2005. NK cell recognition. Annu. Rev. Immunol. 23: 225–274. 28. Miyahara, M., H. Nakanishi, K. Takahashi, K. Satoh-Horikawa, K. Tachibana, 5. Dunn, G. P., C. M. Koebel, and R. D. Schreiber. 2006. Interferons, immunity and and Y. Takai. 2000. Interaction of nectin with afadin is necessary for its clus- cancer immunoediting. Nat. Rev. Immunol. 6: 836–848. tering at cell-cell contact sites but not for its cis dimerization or trans interaction. 6. Vivier, E., E. Tomasello, M. Baratin, T. Walzer, and S. Ugolini. 2008. Functions J. Biol. Chem. 275: 613–618. of natural killer cells. Nat. Immunol. 9: 503–510. 29. Togashi, H., K. Kominami, M. Waseda, H. Komura, J. Miyoshi, M. Takeichi, and 7. Shibuya, A., D. Campbell, C. Hannum, H. Yssel, K. Franz-Bacon, Y. Takai. 2011. Nectins establish a checkerboard-like cellular pattern in the T. McClanahan, T. Kitamura, J. Nicholl, G. R. Sutherland, L. L. Lanier, and auditory epithelium. Science 333: 1144–1147. J. H. Phillips. 1996. DNAM-1, a novel adhesion molecule involved in the cy- 30. Ogita, H., and Y. Takai. 2006. Nectins and nectin-like molecules: roles in cell tolytic function of T lymphocytes. Immunity 4: 573–581. adhesion, polarization, movement, and proliferation. IUBMB Life 58: 334–343. 8. Chen, L., X. Xie, X. Zhang, W. Jia, J. Jian, C. Song, and B. Jin. 2003. The 31. Mendelsohn, C. L., E. Wimmer, and V. R. Racaniello. 1989. Cellular receptor for expression, regulation and adhesion function of a novel CD molecule, CD226, on poliovirus: molecular cloning, nucleotide sequence, and expression of a new human endothelial cells. Life Sci. 73: 2373–2382. member of the immunoglobulin superfamily. Cell 56: 855–865. 9. Kojima, H., H. Kanada, S. Shimizu, E. Kasama, K. Shibuya, H. Nakauchi, 32. Xu, Z., and B. Jin. 2010. A novel interface consisting of homologous immu- T. Nagasawa, and A. Shibuya. 2003. CD226 mediates platelet and megakaryo- noglobulin superfamily members with multiple functions. Cell. Mol. Immunol. 7: cytic cell adhesion to vascular endothelial cells. J. Biol. Chem. 278: 36748– 11–19. 36753. 33. Momose, Y., T. Honda, M. Inagaki, K. Shimizu, K. Irie, H. Nakanishi, and 10. Dardalhon, V., A. S. Schubart, J. Reddy, J. H. Meyers, L. Monney, C. A. Sabatos, Y. Takai. 2002. Role of the second immunoglobulin-like loop of nectin in cell- R. Ahuja, K. Nguyen, G. J. Freeman, E. A. Greenfield, et al. 2005. CD226 is cell adhesion. Biochem. Biophys. Res. Commun. 293: 45–49. specifically expressed on the surface of Th1 cells and regulates their expansion 34. Yasumi, M., K. Shimizu, T. Honda, M. Takeuchi, and Y. Takai. 2003. Role of and effector functions. J. Immunol. 175: 1558–1565. each immunoglobulin-like loop of nectin for its cell-cell adhesion activity. 11. Shibuya, K., J. Shirakawa, T. Kameyama, S. Honda, S. Tahara-Hanaoka, Biochem. Biophys. Res. Commun. 302: 61–66. A. Miyamoto, M. Onodera, T. Sumida, H. Nakauchi, H. Miyoshi, and 35. Fogel, A. I., M. Stagi, K. Perez de Arce, and T. Biederer. 2011. Lateral assembly A. Shibuya. 2003. CD226 (DNAM-1) is involved in lymphocyte function- of the immunoglobulin protein SynCAM 1 controls its adhesive function and associated antigen 1 costimulatory signal for naive T cell differentiation and instructs synapse formation. EMBO J. 30: 4728–4738. proliferation. J. Exp. Med. 198: 1829–1839. 36. Dong, X., F. Xu, Y. Gong, J. Gao, P. Lin, T. Chen, Y. Peng, B. Qiang, J. Yuan, 12. Iguchi-Manaka, A., H. Kai, Y. Yamashita, K. Shibata, S. Tahara-Hanaoka, X. Peng, and Z. Rao. 2006. Crystal structure of the V domain of human Nectin- S. Honda, T. Yasui, H. Kikutani, K. Shibuya, and A. Shibuya. 2008. Accelerated like molecule-1/Syncam3/Tsll1/Igsf4b, a neural tissue-specific immunoglobulin- tumorgrowthinmicedeficientinDNAM-1receptor.J. Exp. Med. 205: 2959–2964. like cell-cell adhesion molecule. J. Biol. Chem. 281: 10610–10617. 5520 STRUCTURE OF CD112 HOMODIMER AND ITS BINDING TO CD226

37. Read, R. J. 2001. Pushing the boundaries of molecular replacement with max- 54. Narita, H., Y. Yamamoto, M. Suzuki, N. Miyazaki, A. Yoshida, K. Kawai, imum likelihood. Acta Crystallogr. D Biol. Crystallogr. 57: 1373–1382. K. Iwasaki, A. Nakagawa, Y. Takai, and T. Sakisaka. 2011. Crystal Structure of 38. Fogel, A. I., Y. Li, J. Giza, Q. Wang, T. T. Lam, Y. Modis, and T. Biederer. 2010. the cis-Dimer of Nectin-1: implications for the architecture of cell-cell junctions. N-glycosylation at the SynCAM (synaptic cell adhesion molecule) immuno- J. Biol. Chem. 286: 12659–12669. globulin interface modulates synaptic adhesion. J. Biol. Chem. 285: 34864– 55. Zhang, P., S. Mueller, M. C. Morais, C. M. Bator, V. D. Bowman, S. Hafenstein, 34874. E. Wimmer, and M. G. Rossmann. 2008. Crystal structure of CD155 and electron 39. Chen, Y., P. Liu, F. Gao, H. Cheng, J. Qi, and G. F. Gao. 2010. A dimeric microscopic studies of its complexes with polioviruses. Proc. Natl. Acad. Sci. structure of PD-L1: functional units or evolutionary relics? Protein Cell. 1: 153– USA 105: 18284–18289. 160. 56. Fuchs, A., M. Cella, E. Giurisato, A. S. Shaw, and M. Colonna. 2004. Cutting 40. Li, X., J. Liu, J. Qi, F. Gao, Q. Li, X. Li, N. Zhang, C. Xia, and G. F. Gao. 2011. edge: CD96 (tactile) promotes NK cell-target cell adhesion by interacting with Two distinct conformations of a rinderpest virus epitope presented by bovine the poliovirus receptor (CD155). J. Immunol. 172: 3994–3998. major histocompatibility complex class I N*01801: a host strategy to present 57. Yu, X., K. Harden, L. C. Gonzalez, M. Francesco, E. Chiang, B. Irving, I. Tom, featured peptides. J. Virol. 85: 6038–6048. S. Ivelja, C. J. Refino, H. Clark, et al. 2009. The surface protein TIGIT sup- 41. Liu, J., L. Dai, J. Qi, F. Gao, Y. Feng, W. Liu, J. Yan, and G. F. Gao. 2011. presses T cell activation by promoting the generation of mature immunoregu- Diverse peptide presentation of rhesus macaque major histocompatibility com- latory dendritic cells. Nat. Immunol. 10: 48–57. plex class I Mamu-A 02 revealed by two peptide complex structures and insights 58. Warner, M. S., R. J. Geraghty, W. M. Martinez, R. I. Montgomery, into immune escape of simian immunodeficiency virus. J. Virol. 85: 7372–7383. J. C. Whitbeck, R. Xu, R. J. Eisenberg, G. H. Cohen, and P. G. Spear. 1998. A 42. Qian, X., J. Qi, F. Chu, J. Liu, Q. Li, and J. Yan. 2009. Crystallization and cell surface protein with herpesvirus entry activity (HveB) confers susceptibility preliminary X-ray analysis of the V domain of human nectin-2. Acta Crystallogr. to infection by mutants of herpes simplex virus type 1, herpes simplex virus type Sect. F Struct. Biol. Cryst. Commun. 65: 615–617. 2, and pseudorabies virus. Virology 246: 179–189. 43. Otwinowski, Z., and W. Minor. 1997. Processing of X-ray diffraction data col- 59. Mu¨hlebach, M. D., M. Mateo, P. L. Sinn, S. Pru¨fer, K. M. Uhlig, V. H. Leonard, lected in oscillation mode. Methods Enzymol. 276: 307–326. C. K. Navaratnarajah, M. Frenzke, X. X. Wong, B. Sawatsky, et al. 2011. 44. Schneider, T. R., and G. M. Sheldrick. 2002. Substructure solution with Adherens junction protein nectin-4 is the epithelial receptor for measles virus. SHELXD. Acta Crystallogr. D Biol. Crystallogr. 58: 1772–1779. Nature 480: 530–533. 45. McCoy, A. J., R. W. Grosse-Kunstleve, P. D. Adams, M. D. Winn, L. C. Storoni, 60. Noyce, R. S., D. G. Bondre, M. N. Ha, L. T. Lin, G. Sisson, M. S. Tsao, and and R. J. Read. 2007. Phaser crystallographic software. J. Appl. Cryst. 40: 658– C. D. Richardson. 2011. Tumor cell marker PVRL4 (nectin 4) is an epithelial Downloaded from 674. cell receptor for measles virus. PLoS Pathog. 7: e1002240. 46. Cowtan, K. 1994. Joint CCP4 and ESF-EACBM Newsletter on Protein Crys- 61. Zhang, N., J. Yan, G. Lu, Z. Guo, Z. Fan, J. Wang, Y. Shi, J. Qi, and G. F. Gao. tallography. 31: 34–38. 2011. Binding of herpes simplex virus glycoprotein D to nectin-1 exploits host 47. Perrakis, A., M. Harkiolaki, K. S. Wilson, and V. S. Lamzin. 2001. ARP/wARP cell adhesion. Nat. Commun. 2: 577. and molecular replacement. Acta Crystallogr. D Biol. Crystallogr. 57: 1445– 62. Di Giovine, P., E. C. Settembre, A. K. Bhargava, M. A. Luftig, H. Lou, 1450. G. H. Cohen, R. J. Eisenberg, C. Krummenacher, and A. Carfi. 2011. Structure of 48. Emsley, P., and K. Cowtan. 2004. Coot: model-building tools for molecular herpes simplex virus glycoprotein D bound to the human receptor nectin-1. PLoS

graphics. Acta Crystallogr. D Biol. Crystallogr. 60: 2126–2132. Pathog. 7: e1002277. http://www.jimmunol.org/ 49. Murshudov, G. N., A. A. Vagin, and E. J. Dodson. 1997. Refinement of mac- 63. Blom, N., T. Sicheritz-Ponteń, R. Gupta, S. Gammeltoft, and S. Brunak. 2004. romolecular structures by the maximum-likelihood method. Acta Crystallogr. D Prediction of post-translational glycosylation and phosphorylation of proteins Biol. Crystallogr. 53: 240–255. from the amino acid sequence. Proteomics 4: 1633–1649. 50. Adams, P. D., P. V. Afonine, G. Bunkoćzi, V. B. Chen, I. W. Davis, N. Echols, 64. Lopez, M., F. Cocchi, L. Menotti, E. Avitabile, P. Dubreuil, and G. Campadelli- J. J. Headd, L. W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, et al. 2010. Fiume. 2000. Nectin2alpha (PRR2alpha or HveB) and nectin2delta are low- PHENIX: a comprehensive Python-based system for macromolecular structure efficiency mediators for entry of herpes simplex virus mutants carrying the solution. Acta Crystallogr. D Biol. Crystallogr. 66: 213–221. Leu25Pro substitution in glycoprotein D. J. Virol. 74: 1267–1274. 51. Laskowski, R. A., M. W. Macarthur, D. S. Moss, and J. M. Thornton. 1993. 65. Lopez, M., M. Aoubala, F. Jordier, D. Isnardon, S. Gomez, and P. Dubreuil. PROCHECK: a program to check the stereochemical quality of protein struc- 1998. The human poliovirus receptor related 2 protein is a new hematopoietic/ tures. J. Appl. Cryst. 26: 283–291. endothelial homophilic adhesion molecule. Blood 92: 4602–4611. 52. Liu, J., Y. Sun, J. Qi, F. Chu, H. Wu, F. Gao, T. Li, J. Yan, and G. F. Gao. 2010. 66. Ardolino, M., A. Zingoni, C. Cerboni, F. Cecere, A. Soriani, M. L. Iannitto, and

The membrane protein of severe acute respiratory syndrome coronavirus acts as A. Santoni. 2011. DNAM-1 ligand expression on Ag-stimulated T lymphocytes by guest on September 26, 2021 a dominant immunogen revealed by a clustering region of novel functionally and is mediated by ROS-dependent activation of DNA-damage response: relevance structurally deﬁned cytotoxic T-lymphocyte epitopes. J. Infect. Dis. 202: 1171– for NK-T cell interaction. Blood 117: 4778–4786. 1180. 67. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 53. Liu, J., P. Wu, F. Gao, J. Qi, A. Kawana-Tachikawa, J. Xie, C. J. Vavricka, 1997. The CLUSTAL_X windows interface: ﬂexible strategies for multiple se- A. Iwamoto, T. Li, and G. F. Gao. 2010. Novel immunodominant peptide pre- quence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876–4882. sentation strategy: a featured HLA-A*2402-restricted cytotoxic T-lymphocyte 68. Gouet, P., X. Robert, and E. Courcelle. 2003. ESPript/ENDscript: Extracting and epitope stabilized by intrachain hydrogen bonds from severe acute respiratory rendering sequence and 3D information from atomic structures of proteins. syndrome coronavirus nucleocapsid protein. J. Virol. 84: 11849–11857. Nucleic Acids Res. 31: 3320–3323.