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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:

xinquanwang@mail.tsinghua.edu.cn

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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.