supplemental information structural basis for r-spondin...
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Supplemental Information
Structural basis for R-spondin recognition by
LGR4/5/6 receptors
Dongli Wang1, Binlu Huang
2, Senyan Zhang
1, Xiaojuan Yu
1, Wei Wu
2, Xinquan
Wang1
1Ministry of Education Key Laboratory of Protein Science, Center for Structural
Biology, School of Life Sciences, Tsinghua University, Beijing 100084, P. R. China
2Ministry of Education Key Laboratory of Protein Science, School of Life Sciences,
Tsinghua University, Beijing 100084, P. R. China
To whom correspondence should be addressed. E-mail:
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Supplemental Materials and Methods
Protein expression and purification
Recombinant proteins were expressed using the Bac-to-Bac baculovirus expression
system (Invitrogen). Sf9 insect cells were maintained in Insect-Xpress protein-free
medium (Lonza) without serum. RSPO1-2F (residues 34-135, NCBI Reference
Sequence accession NP_001033722.1), with an N-terminal gp67 signal peptide to
facilitate secretion and a C-terminal 6-His tag, was cloned into the pFastBac Dual
vector (Invitrogen). The construct was transformed into bacterial DH10Bac
component cells, and the extracted bacmid was then transfected into Sf9 cells in the
presence of Cellfectin II Reagent (Invitrogen). The low-titer viruses were harvested
after incubation of the transfected cells at 300 K for 7 days, and were then amplified
for two more rounds. The amplified high-titer viruses were used to infect 4L Sf9
cells at a density of 2×106 cells/ml. The supernatant of cell culture containing the
secreted RSPO1-2F was harvested 60 h after infection and concentrated and
buffer-exchanged to HBS (10 mM HEPES, pH 7.2, 150 mM NaCl). RSPO1-2F was
captured by nickel-charged resin (GE Healthcare) and eluted with 300 mM
imidazole in HBS buffer (pH 7.2), and then further purified by gel filtration
chromatography using the Superdex 200 High Performance column (GE Healthcare).
RSPO1-2F mutants, LGR4-ECD (residues 25-527, NCBI Reference Sequence
accession NP_060960.2) and ZNRF3-ECD (residues 56-219, NCBI Reference
Sequence accession NP_001193927.1) were expressed and purified in the same way.
To obtain the complex of RSPO1-2F with LGR4-ECD, baculoviruses encoding
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RSPO1-2F and LGR4-ECD were co-infected into Sf9 cells and the secreted complex
was purified as described above.
Crystallization and data collection
The purified complex of RSPO1-2F with LGR4-ECD was concentrated to about 10
mg/ml in HBS buffer. The crystals were grown using sitting drop vapor diffusion at
291 K by mixing equal volumes of protein and reservoir solution containing 0.1 M
sodium citrate, pH 6.0, and 10 % (w/v) PEG 6000. For data collection, crystals were
cryo-protected in reservoir solution supplemented with 20% (v/v) glycerol and flash
frozen in liquid nitrogen. Diffraction data were collected at the BL17U beam line of
the Shanghai Synchrotron Research Facility (SSRF). Diffraction data were indexed,
integrated, and scaled with the program HKL2000 (Otwinowski 1997).
Structural determination and refinement
The structure was determined by molecular replacement with PHASER (McCoy et al.
2007) in CCP4 suite (Collaborative Computational Project 1994). The initial
search model includes the 9 LRR modules of the Netrin-G Ligand-3 (NGL3, PDB
code 3ZYO) (Seiradake et al. 2011) and it was further processed into a poly-alanine
model with CHAINSAW (Stein 2008). The molecular replacement search by
PHASER (McCoy et al. 2007) gave a solution with RFZ of 4.6 and TFZ of 8.7. The
positions of other LRR modules of LGR4-ECD were determined by molecular
replacement search with 2 or 3 LRR modules as search model. Manual model
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adjustment including replacing side chains and adding the LRRNT and LRRCT was
performed with COOT (Emsley 2004). Iterative refinement of individual coordinates
and atomic displacement parameters with PHENIX (Adams et al. 2002) and model
adjustment yielded a model with Rwork of 33.5% and Rfree of 38.9% at a resolution of
2.5 Å. Density map improvement by atoms update and refinement at this stage with
ARP/wARP (Cohen et al. 2008; Langer 2008) showed clear extra densities for
RSPO1-2F, and automatic model extension was performed with BUCCANEER
(Cowtan 2006). At the final steps of model building and refinement, water molecules
and glycans were added based on the electron densities. Structure validation was
performed with PROCHECK (Laskowski 1993) and Molprobity server (Chen et al.
2010). All structural figures were made with PyMol (DeLano).
Affinity measurement
Interactions of LGR4-ECD with RSPO1-2F and its mutants were analyzed by
surface plasmon resonance (SPR) using Biacore T100 (GE Healthcare) at 298 K.
LGR4-ECD was immobilized on flow cell 2 of Series S sensor chip CM5 using the
standard amine-coupling method (GE Healthcare) to about 600 Response Unit (RU).
The flow cell 1 was immobilized blank as a reference. To collect data for kinetic and
affinity analysis, a concentration series of RSPO1-2F or its mutants with six
non-zero concentrations and one zero concentration in binding buffer (HBS plus
0.005% Tween-20) were injected over the chip at a flow rate of 30 μl/min. The
complex was allowed to associate for 60 s and dissociate for 60 s. Regeneration was
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accomplished by passing binding buffer over the chip surface until dissociation
completed or with a 10 s injection of 10 mM NaOH if needed. Data was analyzed
with Biacore T100 evaluation software by fitting to a 1:1 Langmuir binding fitting
model.
STF Reporter Assay
HEK293T and mouse L-cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, penicillin and streptomycin at 310 K
with 5% CO2. Mouse Wnt3a conditioned medium (CM) was produced from mouse
L cells stably transfected with Wnt3a plasmid. For the luciferase reporter assay,
HEK293T cells were seeded into 96-well plates and allowed to reach 50–60%
confluence for transfection. Plasmids used per well were 15 ng SuperTopFlash and
0.5 ng pRL-TK using VigoFect tranfection reagent (Vigorous). Protein samples were
added with triplicates at 8 h after transfection. Firefly and Renilla luciferase
activities were measured 20 h later using the Dual-Luciferase reporter assay system
(Vigorous). Renilla activity was used to normalize Firefly activity. All experiments
were repeated at least three times with similar results (Wang et al. 2010) .
References
Adams PD, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ, Moriarty
NW, Read RJ, Sacchettini JC, Sauter NK, Terwilliger TC. 2002. PHENIX:
building new software for automated crystallographic structure determination.
Acta Crystallogr D 58: 1948-1954.
Chen VB, Arendall WB, 3rd, Headd JJ, Keedy DA, Immormino RM, Kapral GJ,
Murray LW, Richardson JS, Richardson DC. 2010. MolProbity: all-atom
structure validation for macromolecular crystallography. Acta Crystallogr D
66: 12-21.
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Cohen SX, Ben Jelloul M, Long F, Vagin A, Knipscheer P, Lebbink J, Sixma TK,
Lamzin VS, Murshudov GN, Perrakis A. 2008. ARP/wARP and molecular
replacement: the next generation. Acta Crystallogr D 64: 49-60.
Collaborative Computational Project N. 1994. The CCP4 suite: programs for
protein crystallography. Acta Crystallogr D 50: 760-763.
Cowtan K. 2006. The Buccaneer software for automated model building. 1. Tracing
protein chains. Acta Crystallogr D 62: 1002-1011.
DeLano WL. Pymol Molecular Graphics System (DeLano Scientific, San
Carlos, California, USA, 2002).
Emsley P, Cowtan, K. 2004. Coot: model-building tools for molecular graphics. Acta
Crystallogr D 60: 2126-2132.
Langer G, Cohen, SX, Lamzin, VS, Perrakis, A. 2008. Automated macromolecular
model building for X-ray crystallography using ARP/wARP version 7. Nat
Protoc 3: 1171-1179.
Laskowski R, MacArthur, MW, Moss, DS, Thornton, JM. 1993. PROCHECK: a
program to check the stereochemical quality of protein structures. J Appl
Crystallogr 26: 283-291.
McCoy A, Grosse-Kunstleve R, Adams P, Winn M, Storoni L, Read R. 2007. Phaser
crystallographic software. J Appl Crystallogr 40: 658-674.
Otwinowski Z, Minor, W. 1997. Processing of X-ray diffraction data collected in
oscillation mode Method Enzymol 276: 307-326.
Seiradake E, Coles CH, Perestenko PV, Harlos K, McIlhinney RA, Aricescu AR,
Jones EY. 2011. Structural basis for cell surface patterning through
NetrinG-NGL interactions. EMBO J 30: 4479-4488.
Stein N. 2008. CHAINSAW: a program for mutating pdb files used as templates in
molecular replacement. J Appl Crystallogr 41: 641-643.
Wang Y, Fu Y, Gao L, Zhu G, Liang J, Gao C, Huang B, Fenger U, Niehrs C, Chen
YG et al. 2010. Xenopus skip modulates Wnt/beta-catenin signaling and
functions in neural crest induction. J Biol Chem 285: 10890-10901.
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Supplemental Table 1 Crystallographic statistics
Data collection
Beamline SSRF BL-17U
Wavelength 1.00 Å
Space group P32
Cell dimensions
a, b, c (Å) 91.38, 91.38, 87.27
, , () 90, 90, 120
Resolution (Å) 50-2.50 (2.56-2.50)
Rmerge (%) 12.1 (68.2)
I / σI 12.1 (2.4)
Completeness (%) 99.9 (100.0)
Redundancy 3.8 (3.8)
Refinement
Resolution (Å) 24.7 – 2.50
No. Reflections 28261
Rwork / Rfree (%) 16.0/20.9
No. atoms
Protein 4265
water 217
Glycan 42
B-factors (Å2)
Protein 42.7
Water
Glycan
39.4
97.7
R.m.s. deviations
Bond lengths (Å) 0.007
Bond angles (°) 1.160
Ramachandran plot
Most favored regions 75.8
Additional allowed regions 24.0
Generously allowed regions 0.0
Disallowed regions 0.2
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Supplemental Table 2 Selected interactions (d 3.5 Å) between RSPO1-2F and
LGR4-ECD.
RSPO1-2F LGR4-ECD
Pro77 (F3) Asn114 (LRR3)
Ser78 (F3) Gln113 (LRR3)
Asp85 (F3) Arg135 (LRR4)
Arg87 (F3) Asp137 (LRR4), Asp161 (LRR5), Asp162 (LRR5)
Pro89 (F3) Tyr234 (LRR8)
Phe106 (F4) Trp159 (LRR5)
His108 (F4) Glu252 (LRR9)
Asn109 (F4) Thr229 (LRR8)
Phe110 (F4) Val205 (LRR7)
Lys122 (F5) Gln180 (LRR6), Asn226 (LRR8)
Arg124 (F5) Lys251 (LRR9)
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Supplemental Figure 1. Properties of RSPO1-2F. (A) SPR binding analysis of
RSPO1-2F (WT and mutants) with LGR4-ECD using a Biacore T100. LGR4-ECD
was coupled directly to a Series S CM5 sensor chip and various concentrations of
analyte were injected through the flow cell. SPR curves (light magenta) were fit
kinetically using a 1:1 Langmuir binding model (black lines). (B) RSPO1-2F
exhibited a similar level of activity as the full-length RSPO1 in Wnt3a-induced
SuperTopFlash (STF) reporter assay. (C) The complex of RSPO1-2F with LGR4-ECD
was purified by gel filtration chromatography.
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Supplemental Figure 2. Electron density at the binding interface of RSPO1-2F
(green) with LGR4-ECD (blue). (A) 2Fo-Fc electron density contoured at 1.0
surrounding RSPO1 residues Asp85 and Arg87. (B) 2Fo-Fc electron density
contoured at 1.0 surrounding RSPO1 residues Phe106 and Phe110.
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Supplemental Figure 3. Sequence alignments of the LRRNT, each LRR module, and
the LRRCT of LGR4/5/6 receptors. The standard LRR motif “LxxLxLxxNxL” is
shown in the consensus sequences, and other conserved non-polar amino acids that
facilitate to form LRR (I, F, and V) are indicated as “*”. Cysteines are colored red. In
LRR11 and LRR12, the asparagine in the “LxxLxLxxNxL” motif is absent and the
replacing amino acids are colored orange. In LRR1, the first leucine in the
“LxxLxLxxNxL” motif is replaced by threonine to improve hydrophilicity of the
molecule.
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Supplemental Figure 3 (continued)
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Supplemental Figure 3 (continued)
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Supplemental Figure 4. The cysteine-rich flanking motif CF3 and the disulfide bond
pattern in the LRRCT of LGR1/2/3 and LGR4/5/6 receptors. (A) LGR1/2/3 receptors
have three conserved cysteines (red) and other conserved signature residues (blue) as
in the canonical CF3 motif. The CF3 motif in LGR4/5/6 receptors is non-canonical
because the amino acid distance between the third cysteine and the front two
consecutive cysteines is shorter than in the canonical CF3 motif. Other signature
residues of the canonical CF3 motif are also absent in LGR4/5/6 receptors. The
disulfide bonds are represented with lines connecting forming cysteine residues. (B)
The LRRCT of FSHR (LGR1) is composed of two structural elements K260-S295
and T331-I359, which are connected by a disordered linker (left panel). These two
elements are further connected by three disulfide bonds (right panel) (PDB code
4AY9).
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Supplemental Figure 5. Analysis of interactions between ZNRF3-ECD, RSPO1-2F
and LGR4-ECD by gel filtration chromatography using the Superdex 200 High
Performance column. (A) Purification of ZNRF3-ECD. (B) ZNRF3-ECD and
RSPO1-2F were mixed at a molar ratio of 1:1 and subjected to column three hours
later. ZNRF3-ECD and RSPO1-2F were eluted in separate peaks, indicating that there
is no interaction between them. (C) ZNRF3-ECD and LGR4-ECD were mixed at a
molar ratio of 2:1 and subjected to column three hours later. ZNRF3-ECD and
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LGR4-ECD were also eluted in separate peaks, indicating that there is no interaction
between them. (D) ZNRF3-ECD, RSPO1-2F and LGR4-ECD were mixed at a molar
ratio of 2:2:1 and subjected to column three hours later. The first peak is the
LGR4-ECD/RSPO1-2F/ZNRF3-ECD ternary complex, suggesting the binding of
RSPO1-2F with LGR4-ECD generates a composite surface to interact with
ZNRF3-ECD. The second peak is excessive ZNRF3-ECD and RSPO1-2F.