Nature Structural & Molecular Biology: doi: /nsmb.1969
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9 Supplementary Methods Structure determination All the diffraction data sets were collected on BL-41XU (using ADSC Quantum 315 HE CCD detector) at SPring8 (Harima, Japan) or on BL5A (using ADSC Quantum 315 CCD detector) or BL17A (using ADSC Quantum 270 CCD detector) at Photon Factory (Tsukuba, Japan), and were processed and scaled with the HKL2000 package 56. The 3.15 Å resolution structure was solved by molecular replacement with Phaser 57 using the previously reported structure of the MV-H (PDB ID: 2ZB6) as a search model. The Cα trace of the β-sheet structure of CD48 (PDB ID: 2DRU) was manually fitted into the residual electron density. Further model refinement procedures were carried out with Phenix 58 and Refmac 59 coupled with the model correction function of Lafire 60. Iterative manual model building and correction were done using COOT 61. The final structure was refined to the R free and R factors of 29.2% and 23.1%, respectively, with a root mean square deviation of Å in bond length and 1.5 in bond angles. This model was checked using the program Procheck 62. Of 2059 resides in the model, 72.8% are in the most favored regions and 23.3% are in additional allowed regions in the Ramachandran plot. The location of MV-H in the 4.5 Å resolution structure was solved by molecular replacement with Phaser. The MV-H structure from the 3.15 Å resolution structure was used as a search model. Both of 2Fo-Fc and Fo-Fc maps enabled us to place the maslam-v domain clearly, and it was confirmed that the MV-H-SLAM interaction is identical between the 3.15 Å and 4.5 Å resolution structures. To perform subsequent structure refinement for the 4.5 Å resolution structure, the protocol with Phenix was 1
10 limited to the rigid body refinement and TLS refinement of individual domains due to the low resolution limit. The final structure has R free and R of 33.8% and 32.6%, respectively. The 3.55 Å resolution structure was also determined by molecular replacement using the 3.15 Å resolution structure as a search model. The structure was further refined to R free and R of 28.3% and 25.0%, respectively, using CNS 63 and Phenix. Detailed data collection and crystallographic statistics are summarized in Table 1. Figures were produced using PyMOL (DeLano Scientific LLC, Palo Alto, CA, USA. Binding analysis using SPR. The MV-H head domain (residue ), the ectodomain of hslam and its mutants were expressed in HEK293 cells, purified and dissolved in HBS-P buffer. SPR experiments were performed with a BIAcore3000 (Biacore AB). Proteins were immobilized on the CM5 sensor chip, onto which streptavidin had been covalently attached by amine-coupling using an amine-coupling kit (Biacore AB). For coupling, the samples were injected at μg ml -1 for 1 10 min in HBS-P buffer. For MV-H-SLAM binding assay, MV-H was injected over the immobilized SLAM proteins. The binding response at each concentration (0.125, 0.25, 0.5, 1.0, 2.0, 4.0, and 6.0 μm) was calculated by subtracting the equilibrium response measured in the control flow cell from the response in each sample flow cell. Kinetic constants were derived by using the curve-fitting facility of the BIAevaluatin version 4.1 software (GE healthcare) and the simple 1:1 Langmuir binding model (A + B AB). For SLAM-SLAM homodimer binding assay, SLAM (4.6875, 9.375, 18.75, 37.5, 2
11 75, 150, and 300 μm) was injected over the immobilized SLAM mutant proteins. The data were analyzed using BIAevaluatin version 4.1 and ORIGIN version 7 (MicroCal Inc). K d values were determined by equilibrium analyses using nonlinear curve fitting of the Langmuir binding isotherm. MV entry. CHO cells were plated in 48-well plates and transfected with 500 ng of the plasmid DNA encoding wild-type or mutant SLAM proteins using a Lipofectamine 2000 reagent (Invitrogen Life Technologies). At 24 h after transfection, the cells were infected with 50 μl of serially diluted EGFP-expressing recombinant MV 64. A fusion block peptide (Peptide Institute) was added 2 h after infection to prevent the second round infection by progeny virions 65. Expressions of SLAM proteins on transfected cells were examined by a FACScan machine (Becton-Dicinson) using anti-ha tag monoclonal antibody 12CA5 (Roche Diagnostics), and comparable levels of cell surface expressions were confirmed. Infectious titers of MV were determined by counting the numbers of EGFP-expressing cells at 48 h after infection, and are expressed as relative values compared with that for the wild-type SLAM. MV infection. HEK293 cells were plated in 24-well plates and transfected with 1 μg of the plasmid DNA encoding wild-type or mutant SLAM proteins, using a Lipofectamine 2000 reagent. At 24 h after transfection, the cells were infected with 100 μl of serially diluted EGFP- or Renilla luciferase-expressing recombinant MV 18. EGFP autofluorescence in MV-infected cells were observed with a fluorescence microscope at 3
12 48 h after infection. Luciferase activities in MV-infected cells were quantified at 24 h post infection. Native PAGE and immunoblotting. HEK293T cells were plated in 12-well plates and transfected with 1 μg of the plasmid DNA encoding the full-length MV-H protein (the wild-type IC-B strain or the Edmonston vaccine strain) 66, using a polyethyleneimine reagent. At 48 h after transfection, the cells were washed with PBS and then the proteins were extracted with 0.7% (v/v) digitonin in PBS. MV-H proteins were separated by BN-PAGE using NativePAGE TM Novex Bis-Tris Gel System (3 12 % Bis-Tris gel, Invitrogen). After BN-PAGE, the proteins were detected by immunoblotting with a rabbit polyclonal antibody against MV-H (a gift from T. Kohama), followed by alkaline phosphatase-conjugated secondary antibody (Thermo scientific) using SigmaFAST TM BCIP /NBT (SIGMA). 51. Wang, J. H. et al. Structure of a heterophilic adhesion complex between the human CD2 and CD58 (LFA-3) counterreceptors. Cell 97, (1999). 52. Jones, E. Y., Davis, S. J., Williams, A. F., Harlos, K. & Stuart, D. I. Crystal structure at 2.8 A resolution of a soluble form of the cell adhesion molecule CD2. Nature 360, (1992). 53. Ikemizu, S. et al. Crystal structure of the CD2-binding domain of CD58 (lymphocyte function-associated antigen 3) at 1.8-A resolution. Proc. Natl. Acad. 4
13 Sci. U S A 96, (1999). 54. Yan, Q. et al. Structure of CD84 provides insight into SLAM family function. Proc. Natl. Acad. Sci. U S A 104, (2007). 55. Russell, R. J. et al. The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design. Nature 443, (2006). 56. Otwinowski, Z. & Minor, W. Processing of X-ray Diffraction Data Collected in Oscillation Mode. Methods Enzymol. 276, (1997). 57. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, (2007). 58. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, (2010). 59. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, (1997). 60. Yao, M., Zhou, Y. & Tanaka, I. LAFIRE: software for automating the refinement process of protein-structure analysis. Acta Crystallogr. D Biol. Crystallogr. 62, (2006). 61. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, (2004). 62. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein 5
14 structures.. J. Appl. Crystallogr. 26, (1993). 63. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, (1998). 64. Hashimoto, K. et al. SLAM (CD150)-independent measles virus entry as revealed by recombinant virus expressing green fluorescent protein. J. Virol. 76, (2002). 65. Richardson, C. D., Scheid, A. & Choppin, P. W. Specific inhibition of paramyxovirus and myxovirus replication by oligopeptides with amino acid sequences similar to those at the N-termini of the F1 or HA2 viral polypeptides. Virology 105, (1980). 66. Tahara, M., Takeda, M., Seki, F., Hashiguchi, T. & Yanagi, Y. Multiple amino acid substitutions in hemagglutinin are necessary for wild-type measles virus to acquire the ability to use receptor CD46 efficiently. J. Virol. 81, (2007). 6
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