Supplemental Information. The structural basis of R Spondin recognition by LGR5 and RNF43
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1 Supplemental Information The structural basis of R Spondin recognition by LGR5 and RNF43 Po Han Chen 1, Xiaoyan Chen 1, Deyu Fang 2, Xiaolin He 1* 1 Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, Illinois Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois *Correspondence to Xiaolin He, Ph.D. Phone: Fax: E mail: x he@northwestern.edu 1
2 Supplemental Materials and Methods Cell culture, cloning and BacMam expression Sf9 insect cells were maintained in SF900 II media (Invitrogen) supplemented with 8% (v/v) heat inactivated fetal bovine serum. N acetylglucosaminyltransferase I (GnTI) deficient Human Embryonic Kindey 293 (GnTI HEK293) cells were maintained in CDM4HEK293 media (HyClone) supplemented with 4% heat inactivated fetal bovine serum. The coding sequence for the whole ectodomain of human LGR5 (GenBank: AF ), RNF43 (GenBank: AB ), RSPO1 CRD (GenBank: DQ , E35 S133), and RSPO1 TSP (E35 G209) without signal peptide were PCR amplified and subcloned into the baculovirus mediated mammalian cell gene transduction (BacMam) vector pvlad627 containing a N terminal Gaussia luciferase signal peptide and a C terminal 7 histidine tag. The constructs and the BacVector 3000 baculovirus DNA (EMD Chemicals) were used to co transfect Sf9 cells in 6 well plates in the presence of the CellfectinII reagent (Invitrogen). After incubation of the transfected cells at 27 o C for 5 days, the resulting low titer virus stock was harvested, and was used to infect Sf9 cells at cell/ml for amplification. The amplified BacMam viruses were used to transduce HEK293 GnTI cells at a density of cells/ml. Rspo1 and LGR5 were co expressed for improved protein stability, and RNF43 was expressed separately. The cells were pelleted 72 hours later, and the supernatants were concentrated and buffer exchanged to HBS (10mM Hepes ph7.5, 150mM NaCl). The recombinant proteins were captured by the Talon affinity resin and eluted with 300 mm imidazole ph 7.5, glycan minimized with endoglycosidase F1 (Sigma), and treated with bovine carboxypeptidase A (Sigma) for His tag removal. RNF43 and RSPO1/LGR5 were mixed 2
3 with appropriate ratios, and proteins were further purified with size exclusion columns (Superdex 200, Amersham Biosciences) pre equilibrated and eluted with HBS. Crystallization and data collection Crystallization was performed in a 20 0 C incubator using the sitting drop vapor diffusion method. Crystals of the complex were obtained from drops composed of 0.5 µl reservoir solution and 0.5 µl protein solution (10mg/ml) equilibrated against 1 ml reservoir solution. The composition of the reservoir solution is 10% PEG4000, 0.2M ammonium sulfate, 7% Sucrose and 0.1 M TRIS ph 8.0. Prior to being flash frozen in liquid nitrogen, the crystals were transferred to a cryosolution consisting of the reservoir solution supplemented with 19% ethylene glycol. The heavy atom derivatives were prepared by quick soaking selected crystals in the cryo protectant containing 0.2 M NaI for ~60 seconds. X ray diffraction datasets were collected at 100K at the Life Science Collaborative Access Team (LS CAT) beamline 21 ID D, the Advanced Photon Source, Argonne, Illinois, USA. The data were processed with HKL3000 (Otwinowski and Minor, 1997), and the statistics are summarized in Table S1. Structure determination and refinement The structure was solved by single isomorphous replacement with anomalous scattering (SIRAS) using Iodine soaked crystals. Taking advantage of non crystallographic symmetry present in the crystal, the electron density map was furthered improved by density modification in CNS (Brunger, 2007; Brunger et al., 1998). Initial atomic models were built manually in O and COOT 3
4 (Emsley and Cowtan, 2004). The model of LGR5 was built with LRR repeats of FSHR (PDB 4AY9) as template. Due to lack of similar structures, RSPO1 was built initially by baton building when electron density map was improved to sufficient details, followed by manual adjustment of amino acids that agree best with sequence information (i.e., by locating the conserved disulfide bonds. Model of RNF43 were built by manually fitting in the aminopeptidase structure, followed by adjustment of amino acid side chains and backbones. The complex structure was refined using CNS1.3 and REFMAC5 (Collaborative Computational Project, 1994). Local NCS restraint is maintained throughout the refinement process. Isothermal Titration Calorimetry experiment Calorimetric titrations were implemented with a VP ITC calorimeter (MicroCal) at 30 C. Using gel filtration chromatography (Superdex 200, Amersham Biosciences), all proteins to be used in the titrations were buffer exchanged by into an identical lot of HBS buffer (10 mm Hepes ph7.5, 150 mm NaCl) to minimize the dilution effects of buffer heat during titration. Proteins eluted from the column were concentrated and concentrations measured by Nanophotometer P330 (IMPLEN): RNF mm, R CRD 0.07 mm, R TSP 0.02 mm, R CRD/LGR mm, and R TSP/LGR mm. The protein samples were degassed for 5 min before being loaded separately into the reaction chamber (all samples except for RNF43) and injection syringe. The proteins in the syringe (RNF43) were injected into the reaction chamber in 3µL pulses at 5 min intervals. The data were processed with MicroCal Origin 5.0 software. References 4
5 Brunger, A.T. (2007). Version 1.2 of the Crystallography and NMR system. Nature protocols 2, Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. (1998). Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta crystallographica Section D, Biological crystallography 54, Collaborative Computational Project, N. (1994). The CCP4 suite: programs for protein crystallography. Acta crystallographica Section D, Biological crystallography 50, Emsley, P., and Cowtan, K. (2004). Coot: model building tools for molecular graphics. Acta Crystallogr D 60, Otwinowski, Z., and Minor, W. (1997). Processing of X ray diffraction data collected in oscillation mode. Method Enzymol 276,
6 Supplemental Table 1 crystals Human LGR5-Rspo1-RNF43 native I-soaked Data collection Space group P P Cell parameters / / /80.07/ Resolution range ( ) ( ) Unique reflections (5121) (952) Completeness (%) 97.8 (91.9) 99.2 (99.7) I/sigma(I) 19.5 (4.2) 18.6 (6.6) Redundancy 6.2 (4.8) 16.2 (12.8) R merge (%)* 6.1 (33.8) 8.0 (38.2) phasing Number of heavy atoms 8 R iso (%) 13.4 R ano (%) 4.8 FOM 0.53 Refinement Resolution range ( ) r.m.s.d bonds (Å) r.m.s.d angles ( ) 1.67 R free (%) 23.0 R work (%) 27.8 Number of protein atoms, averaged B factor (Å 2 ) Number of glycan atoms, averaged B factor (Å 2 ) Number of waters, averaged B factor (Å 2 ) 11019, , , 48.8 Ramachandran plot (%) (favored, allowed, outliers) 76.6, 12.5, 10.9 *R merge = Σ hkl I-<I> /Σ hkl I, where I is the intensity of unique reflection hkl, and <I> is the average over symmetry-related observation of unique reflection hkl. R work =Σ F obs -F calc /ΣF obs, where F obs and F calc are the observed and the calculated structure factors, respectively. R free is calculated using 5% of reflections set aside from refinement. 6
7 Supplemental Table 2. Selected interface residues* between Rspo1 CRD and LGR5 ECD Rspo1 CRD LGR5 ECD Asp85 R144 (LRR4) Arg87 Asp146 (LRR4), Asp171 (LRR5) F106 His166, Trp168 (LRR5) F110 Val213/214 (LRR7) Lys122 Val213 (LRR7), Glu237 (LRR8) *Interface residues are listed according to PISA analysis and visual inspection of electron density map. 7
8 Supplemental Table 3. Selected interface residues* between Rspo1 CRD and RNF43 PA Rspo1 CRD RNF43 PA Ser48 Glu110 Leu64 His86, Leu88, Tyr89 Arg66 Asp97, Gln84 Asp68 Lys81, His183 Ile69 Leu82, Gln84, Lys181, Val176 Arg70 Gln84 Gln71 Gln84, His86, Asp97 *Interface residues are listed according to PISA analysis and visual inspection of electron density map. 8
9 Chen Supplemental Figure 1. Comparison between LGR5 RSPO1 and FSHR FSH. Direct view of the LRR concave face in both structures. 9
10 Supplemental Figure 2. Crystal packing of back to back dimer. The side view and top view are presented. The modeled Ni 2+ ion is shown as the gray sphere surrounded by His residues in sticks. Boxed region indicates site of RNF43 LGR5 packing in the crystal structure. 10
11 Supplemental Figure 3. Electron density surrounding the RSPO1 LGR5 interface. RSPO1 is shown in cyan and LGR5 in green. 2Fo Fc electron density map contoured at 2.0 σ. The interface consists of hydrophilic interactions (RSPO1 D85 and R87 with LGR5 R144 and D146) and hydrophobic interactions (right part of the figure, in particular RSPO1 F106 and F110 and LGR5 W168, H166, and V213). 11
12 Supplemental Figure 4. Electron density surrounding the RSPO1 β hairpin (L64 Q71) interaction with RNF43 β3. Orientation is the same as in Fig. 3. RSPO1 is shown in marine and RNF43 shown in hotpink. The 2Fo Fc map is contoured at 1.0 σ. 12
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