Structural Biology and Crystallization Communications Refolding, Crystallization and Preliminary X-ray Crystallographic Study of the Whole Extracellular Regions of Nectins

The nectin family of Ca 2+-independent immunoglobulin-like cell–cell adhesion molecules contains four members. Nectins, which have three Ig-like domains in their extracellular region, form cell–cell adherens junctions cooperatively with cadherins. The whole extracellular regions of nectin-1 (nectin-1-EC) and nectin-2 (nectin-2-EC) were expressed in Escherichia coli as inclusion bodies, solubilized in 8 M urea and then refolded by rapid dilution into refolding solution. The refolded proteins were subsequently purified by three chromato-graphic steps and crystallized using the hanging-drop vapour-diffusion method. The nectin-1-EC crystals belonged to space group P2 1 3 and the nectin-2-EC crystals belonged to space group P6 1 22 or P6 5 22.


Introduction
Cell adhesion molecules are the primary mediators for various types of cell-cell junctions and play essential roles in various cellular processes, including morphogenesis, differentiation, proliferation and migration (Harris & Tepass, 2010;Ogita & Takai, 2006). In polarized epithelial cells, cell-cell junctions comprise several adhesive apparatuses including tight junctions (TJs), adherens junctions (AJs), desmosomes and gap junctions. AJs regulate TJ formation and the establishment of the apical-basal polarity at cell-cell adhesion sites . Nectins, which are immunoglobulin-like cell adhesion molecules, and cadherins, which are Ca 2+ -dependent cell adhesion molecules, localize at AJs and have essential cooperative roles in AJ formation. Nectins initiate AJ formation before cadherins form cell-cell adhesions. Initial cell-cell contacts are formed between two neighbouring cells by nectins, and cadherins are then recruited to the nectin-based adhesion sites to form strong cellcell adhesions. TJs are formed at the apical side of AJs. Together, nectins and cadherins mediate TJ formation by recruiting junctional adhesion molecules (JAMs), followed by claudins and occludins, to the apical side of AJs.
To further understand how nectin-family members form cisinteractions and trans-interactions, structural information on the extracellular region of nectin-family members is fundamental. Here, we report the refolding, crystallization and preliminary X-ray crystallographic analyses of the extracellular regions of nectin-1 and nectin-2. To our knowledge, this is the first report of the crystallization of the whole extracellular regions of nectins.
The cells were cultured in Luria-Bertani broth containing 100 mg ml À1 ampicillin at 310 K until the OD 600 reached 0.5-0.7. Protein expression was induced at 298 K by the addition of isopropyl -d-1-thiogalactopyranoside to a final concentration of 0.4 mM. At 16 h post-induction the cells were harvested by centrifugation, suspended in phosphate-buffered saline containing 40 mg ml À1 hen egg-white lysozyme, subjected to two cycles of freezing and thawing and sonicated until the lysate was homogeneous. Centrifugation of these lysates at 10 000g for 20 min yielded inclusion bodies.
The inclusion bodies were washed four times with a buffer consisting of 20 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM EDTA, 0.5% Triton X-100 and 1 mM DTT and were dissolved in a buffer consisting of 50 mM MES-NaOH pH 6.0, 8 M urea, 1 mM EDTA and 1 mM DTT. This mixture was slowly rotated overnight at 277 K before centrifugation at 10 000g for 20 min to remove insoluble materials.
The yields of inclusion bodies were $0.1 g for nectin-1-EC and $0.2 g for nectin-2-EC per litre of culture.
Unfolded proteins were refolded by 300-fold dilution into refolding solution A [500 mM l-arginine, 100 mM Tris-HCl pH 9.0, 2 mM oxidized glutathione (GSSG) and 1 mM reduced glutathione (GSH)] in the case of nectin-1-EC or refolding solution B (500 mM l-arginine, 100 mM Tris-HCl pH 9.0, 10 mM GSSG, 0.1 mM GSH) in the case of nectin-2-EC, followed by incubation for 48 h at 277 K. After concentration using a 10 000 molecular-weight cutoff ultrafiltration membrane (GE Healthcare), the samples were subjected to size-exclusion chromatography on a HiLoad 16/60 Superdex 200 pg column (GE Healthcare) to separate correctly folded proteins from aggregated forms. These fractions were dialyzed against 20 mM MES pH 6.0 to precipitate almost-misfolded proteins, filtered using an Ultrafree-MC GV 0.22 mm (Millipore) and applied onto a HiTrap SP HP column (5 ml; GE Healthcare) followed by a Mono Q column (1 ml; GE Healthcare). The protein yields were $0.5 mg for nectin-1-EC and $10 mg for nectin-2-EC from 100 mg inclusion bodies.
Small-scale refolding assays (1 ml) were performed to investigate the effects of changing the l-arginine concentration from 100 to 600 mM (for nectin-1-EC), the pH from 7.0 to 9.0 (for nectin-1-EC), the GSSG:GSH ratio from 10.0:0.1 mM to 0.1:10.0 mM (for both nectin-1-EC and nectin-2-EC) and the concentration of unfolded protein from 25 to 200 mg ml À1 (for nectin-1-EC). In each case, the unfolded protein solutions were diluted at least 300-fold into each of the refolding solutions such that only one parameter was varied while the other parameters were kept at the standard conditions. The solutions were incubated at 277 K for 48 h and then subjected to size-exclusion chromatography on a Superdex 200 10/300 GL column using an Ä KTA FPLC system (GE Healthcare; Figs. 1a-1d). A peak at an elution volume of $15.5 ml corresponded to the correctly folded protein containing native intramolecular disulfide bonds. Aggregates containing intermolecular disulfide bonds eluted in the void volume ($8.5 ml).
In the presence of 0.4 M NDSB201, nectin-1-EC and nectin-2-EC crystals were obtained using both polyethylene glycol and salt conditions as the reservoir. The initial crystallization conditions for nectin-1-EC and nectin-2-EC were further refined by changing the pH, precipitant concentration and additives. The most promising crystals of nectin-1-EC were observed in drops comprised of equal volumes of nectin-1-EC solution [20 mM Tris-HCl pH 7.5, 150 mM NaCl and 6%(w/v) 1,6-hexanediol] and precipitant solution [50 mM citric acid, 50 mM bis-Tris propane and 1-3%(v/v) PEG 3350] at 296 K (Fig. 2a). Crystals of nectin-1-EC could be obtained even if the crystallization conditions contained no NDSB201, and the NDSB201 did not influence the diffraction quality of the crystals. The most promising crystals of nectin-2-EC were observed in drops comprised of equal volumes of nectin-2-EC solution (20 mM Tris-HCl pH 9.0, 150 mM NaCl and 0.35 M NDSD201) and precipitant solution (45 mM citric acid, 55 mM bis-Tris propane and 3.6 M sodium nitrate) at 296 K (Fig. 2b).

Data collection
To improve the diffraction quality of the nectin-1-EC crystals, the crystals were subjected to dehydration with increasing concentrations of PEG 300. The crystals were transferred in a large number of steps from harvesting buffer [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 6%(w/v) 1,6-hexanediol, 50 mM citric acid, 50 mM bis-Tris propane and 5%(v/v) PEG 3350] to harvesting buffer including 25%(v/v) PEG 300 at 277 K. Before freezing with liquid nitrogen, the crystals were equilibrated in the final buffer for 3 d. This procedure markedly improved the resolution limit of the crystals from $5 to $2.8 Å . The nectin-2-EC crystals were soaked in a cryoprotection solution consisting of 20 mM Tris-HCl pH 9.0, 150 mM NaCl, 0.4 M NDSB201, 45 mM citric acid, 55 mM bis-Tris propane, 4.0 M sodium nitrate and 14%(v/v) ethylene glycol by stepwise transfer at room temperature.
Diffraction data sets were collected from nectin-1-EC and nectin-2-EC crystals on the BL44XU beamline at the SPring-8 synchrotron facility (Harima, Hyogo, Japan) at 100 K using a DIP6040 imagingplate detector (MAC Science/Bruker AXS). A total of 60 frames of data were collected for nectin-1-EC in three runs with a translation of 70 mm along the rotation axis. The nectin-1-EC data-collection parameters included a crystal-to-detector distance of 540 mm, an oscillation angle of 0.5 and an exposure time of 20 s per frame at a wavelength of 0.9000 Å (Fig. 3a). For nectin-2-EC, a total of 70 frames of data were collected with a crystal-to-detector distance of 400 mm, an oscillation angle of 0.5 and an exposure time of 2 s per frame at a wavelength of 0.9000 Å (Fig. 3b). All data sets were processed and scaled with the HKL-2000 program package (Otwinowski & Minor, 1997). Data-collection statistics are summarized in Table 1.

Results and discussion
The extracellular regions of nectin-1 and nectin-2 fused with a C-terminal 6ÂHis tag were expressed as inclusion bodies in E. coli  nectin-1-EC and nectin-2-EC proteins were successfully refolded by rapid dilution with a glutathione redox couple. To increase the yields of refolded nectin-1-EC protein, the refolding conditions (i.e. pH, GSSG:GSH ratio, l-arginine concentration and nectin-1-EC concentration) were optimized in a series of small reactions (1 ml). Correct folding was assessed by size-exclusion chromatography on a Superdex 200 10/300 GL column using an Ä KTA FPLC system (GE Healthcare; Figs. 1a-1d). Notably, a lack of the C-terminal 6ÂHis tag significantly decreased the yield of correctly folded protein; we could not obtain crystals of nectin-1-EC without the tag. Based on the results for nectin-1-EC, only the GSSG:GSH ratio was optimized for nectin-2-EC (Fig. 2e). The optimized refolding solutions for nectin-1-EC and nectin-2-EC are described in x2. Nectin-1-EC crystals belonged to the cubic space group P2 1 3, with unit-cell parameters a = b = c = 164.9 Å . Nectin-2-EC crystals belonged to the hexagonal space group P6 1 22 or P6 5 22, with unit-cell parameters a = b = 79.3, c = 235.4 Å .
Molecular-replacement calculations with MOLREP and Phaser (Collaborative Computational Project, Number 4, 1994) using the structure of a homologous protein [CD155, which has the maximum sequence identity to nectin-1-EC (48.4%) and nectin-2-EC (25.3%); PDB code 3eow; Zhang et al., 2008] as a search model were unsuccessful. Therefore, heavy-atom derivatives of nectin-1-EC and nectin-2-EC crystals have been prepared for phase determination. Diffraction data sets for the heavy-atom derivatives are currently being collected on BL44XU at the SPring-8 synchrotron facility for phase determination.