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Supplemental Information
The structural basis of R‐Spondin recognition by LGR5 and RNF43
Po‐Han Chen1, Xiaoyan Chen1, Deyu Fang2, Xiaolin He1*
1Department of Molecular Pharmacology and Biological Chemistry, Northwestern University
Feinberg School of Medicine, Chicago, Illinois 60611
2Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago,
Illinois 60611
*Correspondence to
Xiaolin He, Ph.D.
Phone: 312‐503‐8030
Fax: 312‐503‐5349
E‐mail: x‐he@northwestern.edu
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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: AF062006.1), RNF43 (GenBank: AB081837.1),
RSPO1‐CRD (GenBank: DQ318235.1, 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 27oC for 5 days, the
resulting low titer virus stock was harvested, and was used to infect Sf9 cells at 2 × 106 cell/ml
for amplification. The amplified BacMam viruses were used to transduce HEK293 GnTI− cells at
a density of 1.5‐2 × 106 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
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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 200C 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 cryo‐
solution 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
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(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): RNF43 1.4 mM, R‐CRD 0.07 mM, R‐TSP 0.02 mM, R‐CRD/LGR5 0.03 mM, and R‐
TSP/LGR5 0.02 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
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Brunger, A.T. (2007). Version 1.2 of the Crystallography and NMR system. Nature protocols 2, 2728‐2733. 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, 905‐921. Collaborative Computational Project, N. (1994). The CCP4 suite: programs for protein crystallography. Acta crystallographica Section D, Biological crystallography 50, 760‐763. Emsley, P., and Cowtan, K. (2004). Coot: model‐building tools for molecular graphics. Acta Crystallogr D 60, 2126‐2132. Otwinowski, Z., and Minor, W. (1997). Processing of X‐ray diffraction data collected in oscillation mode. Method Enzymol 276, 307‐326.
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Supplemental Table 1
crystals Human LGR5-Rspo1-RNF43
native I-soaked Data collection
Space group P212121 P212121
Cell parameters 104.575/120.971/181.009 71.17/80.07/164.06
Resolution range 39.5-2.5 (2.6-2.5) 50-3.0 (3.04-3.00)
Unique reflections 74020 (5121) 19693 (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)
Rmerge (%)* 6.1 (33.8) 8.0 (38.2)
phasing
Number of heavy atoms 8
Riso (%) 13.4
Rano (%) 4.8
FOM 0.53
Refinement
Resolution range 39.5-2.5 (2.6-2.5)
r.m.s.d bonds (Å) 0.009
r.m.s.d angles (°) 1.67
Rfree (%)† 23.0
Rwork (%)† 27.8
Number of protein atoms, averaged B factor (Å2)
11019, 77.2
Number of glycan atoms, averaged B factor (Å2)
28, 67.9
Number of waters, averaged B factor (Å2)
255, 48.8
Ramachandran plot (%) (favored, allowed, outliers)
76.6, 12.5, 10.9
*Rmerge= Σhkl|I-<I>|/ΣhklI, where I is the intensity of unique reflection hkl, and <I> is the average over symmetry-related observation of unique reflection hkl.
†Rwork=Σ|Fobs-Fcalc|/ΣFobs, where Fobs and Fcalc are the observed and the calculated structure factors, respectively. Rfree is calculated using 5% of reflections set aside from refinement.
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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.
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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.
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Supplemental Figure 1. Comparison between LGR5‐RSPO1 and FSHR‐FSH. Direct view of the
LRR concave face in both structures.
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Supplemental Figure 2. Crystal packing of back‐to‐back dimer. The side view and top view are
presented. The modeled Ni2+ 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.
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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).
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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.
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