Figure S1 Alpha-carbon backbones of components from FSH-FSHRHB complex. a, Stereo view of FSHRHB (red) with every 10 th residue marked.

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Clarissa Perry
6 years ago
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1 a b Figure S1 Alpha-carbon backbones of components from FSH-FSHRHB complex. a, Stereo view of FSHRHB (red) with every 10 th residue marked. Cyan highlights regions of the receptor involved in hormone binding. The view is the same as in Fig. 2b. b, Stereo view of FSH (α in green; β in cyan) with every 10 th residue marked. Red highlights regions of the hormone involved in receptor binding. The view is the same as in Fig. 2b.

3 a FSHR LHR TSHR GCHHRICHCSNRVFLCQESK-VTEIPSDLPRNAIELRFVLTKLRVIQKGAFSGFGDLEKIEISQNDVLEVIEADV EALCP-EPCNCVPDG ALRCPG-PTAGLTRLSLAYLPVKVIPSQAFRGLNEVIKIEISQIDSLERIEANA GMGCSSPPCECHQEEDFRVTCKDIQRIPS-LPPSTQTLKLIETHLRTIPSHAFSNLPNISRIYVSIDVTLQQLESHS FSHR LHR TSHR FSNLPKLHEIRIEKANNLLYINPEAFQNLPNLQYLLISNTGIKHLPDVHKIHSLQ-K VLLDIQDNINIHTIERNSFVGLSFESVILWLNKN FDNLLNLSEILIQNTKNLRYIEPGAFINLPRLKYLSICNTGIRKFPDVTKVFSSESNFILEICDNLHITTIPGNAFQGMNNESVTLKLYGN FYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGLKMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNN FSHR LHR TSHR GIQEIHNCAFNGTQLDELNLSDNNNLEELPNDVFHGA-SGPVILDISRTRIHSLPSYGLENLKKLRARSTYNLKKLPTLEKLVALMEAS GFEEVQSHAFNGTTLTSLELKENVHLEKMHNGAFRGA-TGPKTLDISSTKLQALPSYGLESIQRLIATSSYSLKKLPSRETFVNLLEAT GFTSVQGYAFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLKELIARNTWTLKKLPLSLSFLHLTRAD b FSHβ TSHβ CGβ LHβ NS-----CELTNITIAIEKEECRFCISINTTWCAGYCYTRDLVYKD--PARPKIQKTCTFKELVYETVRVPGCAHHA F CIPTEYTMHIERRECAYCLTINTTICAGYCMTRDINGKLFLPKYALSQDVCTYRDFIYRTVEIPGCPLHV SKEPLRPRCRPINATLAVEKEGCPVCITVNTTICAGYCPTMTRVLQG--VLPALPQVVCNYRDVRFESIRLPGCPRGV SREPLRPWCHPINAILAVEKEGCPVCITVNTTICAGYCPTMMRVLQA--VLPPLPQVVCTYRDVRFESIRLPGCPRGV FSHβ DSLYTYPVATQCHCGKCDSDSTDCTVRGLGPSYCSFGEM KE TSHβ APYFSYPVALSCKCGKCNTDYSDCIHEAIKTNYCTKPQK SYLVG------FSV CGβ NPVVSYAVALSCQCALCRRSTTDCGGPKDHPLTCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ LHβ DPVVSFPVALSCRCGPCRRSTSDCGGPKDHPLTCDHP QLSG-----LLFL c FSHα APDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKS Figure S3 Summary of the interactions found in the second complex (FSH'-FSHRHB') of the asymmetric unit. This is the counterpart of Fig. 2c showing FSH-FSHRHB interactions. a-c, Sequence alignment. Secondary-structure assignments are shown as arrows for β- strands and cylinders for α-helices. For each residue buried at the receptor-ligand interface or the receptor dimer interface, the fractional solvent accessibility is indicated by an open circle if it is greater than 0.4, a half-filled circle if it is 0.1 to 0.4, and a filled circle if it is less than 0.1. a, Human FSHR, LHR and TSHR sequences in the region of the hormone binding domain. β-strands located at the concave face of FSHR are colored red, and strands on the convex face are in pink. FSHRHB' residues buried at the receptor-ligand interface by FSH'α alone (green), FSH'β alone (cyan), or both FSH'α and β (pink) are highlighted. FSHRHB and FSHRHB' residues buried at the receptor dimer interface are boxed in orange. N-linked glycosylation is marked by a black triangle. b, Human FSH, CG, LH and TSH β-chain sequences. FSH'β residues buried at the receptor-ligand interface are highlighted in cyan. c, The common human α-chain sequence. FSH'α residues buried at the receptorligand interface are highlighted in green.

5 a Repeat b Repeat R N G I G N D K K R N G I G N D K K T N N N T A N T S T N N N T A N T S R D K D N K Q L E R D K D N K Q L E S S N Q S E S V Q S S N Q S E S V Q A I L L I I I I F C A I L L I I I I F C R D N W D L R E R L R D N W D L R E R L L L L L L L I I L F L L L L L L I I L F K I E I L Y E K E V K I E I L Y E K E V Identical One Difference All Different Figure S5 Specificity of glycoprotein hormone and receptor recognition as analyzed by sequence variation. In each panel, the concave face of the FSHRHB structure is shown as an unrolled sheet. Residues are identified by the single letter code on filled circles for side chains that point toward the hydrophobic core of the structure and by open circles for those that are solvent exposed. a, Residues of FSHRHB involved in direct contact with the hormone are colored according to sequence variation among the three human glycoprotein hormone receptors (FSHR, LHR and TSHR). Green indicates that the residue is identical in all three receptors, pink corresponds to one difference and red means the residues in the three receptors are all different. b, Residues of FSHRHB involved in direct contact with the hormone are colored according to sequence variation at their interaction partners in the hormone. Green indicates that the FSHRHB residue contacts the common α-chain and/or residues of FSHβ that are conserved among all human glycoprotein hormone β-chains (FSHβ, CGβ/LHβ and TSHβ). Cyan represents one sequence difference found at the contacted β-chain residues. Blue indicates that the β-chain residues contacted by FSHRHB are different in all human glycoprotein hormones. Red stars in both panels mark the residues of FSHRHB where sequence variations in the partners account for specificity in the interaction (see text).

6 Table S1_a. Interactions between FSHR HB and FSH Hydrogen bonds and salt bridges 1 FSHR FSH Distance (Å) Glu34 Oε2 Tyr103 (β) OH 2.2 Lys74 Nζ Ser85 (α) Oγ 3.4 Nζ Thr86 (α) Oγ1 3.3 Gln79 Oε1 Ser43 (α) Oγ 2.5 Asp81 Oδ1 Lys45 (α) O 3.3 Asp81 Oδ2 Arg42 (α) Nη1 3.0 Oδ2 Arg42 (α) Nη2 3.3 Glu99 Oε1 Tyr88 (α) OH 2.6 Lys104 Nζ Asp93 (β) Oδ2 3.3 Asn106 Nδ2 Lys45 (α) O 3.2 Asn129 Oδ1 Leu48 (α) N 3.4 Oδ1 Val49 (α) N 3.1 Asp150 Oδ2 Lys91 (α) Nζ 3.1 Gln152 Oε1 Lys91 (α) Nζ 3.2 Asp153 Oδ2 Lys51 (α) Nζ 3.4 Lys179 Nζ Ser89 (β) Oγ 2.9 Nζ Ser89 (β) O 3.3 Nζ Asp90 (β) Oδ1 2.9 Hydrogen bonds through water molecule 1 Wat-23 O Tyr124 (FSHR) OH 3.2 Wat-23 O Asp150 (FSHR) Oδ2 2.8 Wat-23 O Lys91 (FSHα) Nζ 2.9 Hydrogen bonds through sulfate ion 1 SO O3 Arg52 (FSHR) Nη1 3.2 SO O3 Arg52 (FSHR) Nη2 3.2 SO O2 Arg97 (FSHβ) Nη1 3.3 Non-hydrogen bonding polar interactions 2 FSHR Glu50 Glu76 Gln79 Asp81 Glu99 Ala105 Asn106 Asn129 Lys243 Hydrophobic contacts 3 FSHR Val54 Leu55 Gln79 Arg101 Lys104 Tyr124 Asn129 Thr130 Gly131 Leu148 Asp153 Ile222 Lys242 FSH Arg97 (β) Arg97 (β) Ser43 (α), Thr46 (α) Arg42 (α), Thr46 (α) Thr86 (α) Thr46 (α) Thr46 (α), Leu48 (α) Val49 (α) Ala43 (β) FSH Leu99 (β) Arg42 (α), Leu99 (β), Y103 (β) Thr46 (α) Val96 (β) Thr46 (α), Met47 (α) Tyr88 (α) Met47 (α) Leu48 (α) Leu48 (α) Tyr88 (α) Val49 (α) Pro45 (β) Ala43 (β) 1 Hydrogen bond distances are in the range of Å. 2 Polar interactions have contact distances between 3.4 and 4.0 Å. 3 Carbon-carbon contacts are within 4.0 Å.

7 Table S1_b. Interactions between FSHR HB and FSH (second complex) Hydrogen bonds and salt bridges 1 FSHR FSH Distance (Å) Glu34 Oε1 Tyr103 (β) OH 3.3 Glu34 Oε2 Tyr103 (β) OH 2.9 Lys74 Nζ Ser85 (α) Oγ 3.1 Nζ Thr86 (α) Oγ1 3.3 Gln79 Oε1 Ser43 (α) Oγ 2.6 Asp81 Oδ2 Arg42 (α) Nη1 3.0 Oδ2 Arg42 (α) Nη2 3.2 Oδ2 Lys45 (α) Nζ 2.9 Glu99 Oε1 Thr86 (α) Oγ1 3.4 Oε1 Tyr88 (α) OH 2.6 Lys104 Nζ Asp93 (β) Oδ2 3.3 Asn106 N Thr46 (α) O 3.2 Asn106 Nδ2 Lys45 (α) O 3.1 Asn129 Oδ1 Leu48 (α) N 3.2 Oδ1 Val49 (α) N 3.0 Asp150 Oδ2 Lys91 (α) Nζ 2.8 Gln152 Oε1 Lys91 (α) Nζ 2.9 Asp153 Oδ1 Lys51 (α) Nζ 3.1 Asp153 Oδ2 Lys51 (α) Nζ 3.2 Lys179 Nζ Ser92 (α) Oγ 3.3 Nζ Ser89 (β) Oγ 2.6 Nζ Asp90 (β) Oδ1 3.4 Lys243 Nζ Ala43 (β) O 3.2 Hydrogen bonds through water molecule 1 Wat-33 O Lys74 (FSHR) Nζ 2.7 Wat-33 O Glu99 (FSHR) Oε1 3.3 Wat-33 O Ser85 (FSHα) Oγ 3.2 Wat-33 O Tyr88 (FSHα) OH 3.3 Non-hydrogen bonding polar interactions 2 FSHR Gln79 Asp81 Glu99 Lys104 Ala105 Asn129 Asp150 Lys179 Aps202 Hydrophobic contacts 3 FSHR Leu55 Gln79 Asp81 Arg101 Lys104 Asn106 Tyr124 Asn129 Thr130 Gly131 Leu148 Trp176 Lys179 Val221 Ile222 Lys243 FSH Ser43 (α), Thr46 (a) Arg42 (α), Lys45 (a), Thr46 (α) Thr86 (α) Asp93 (β) Thr46 (α) Val49 (α) Lys91 (α) Ser89 (β) Asp90 (β) FSH Arg42 (α), Leu99 (β), Y103 (β) Thr46 (α), Leu99 (β) Thr46 (α) Val96 (β) Thr46 (α) Thr46 (a) Tyr88 (α) Met47 (α) Leu48 (α) Leu48 (α) Tyr88 (α) Tyr89 (α) Ser89 (β), Asp90 (β) Ala43 (β) Pro42 (β), Pro45 (β) Pro45 (β) 1 Hydrogen bond distances are in the range of Å. 2 Polar interactions have contact distances between 3.4 and 4.0 Å. 3 Carbon-carbon contacts are within 4.0 Å.

8 Supplementary Methods Protein expression and purification Human FSHR and FSH were coexpressed using the baculovirus system. The cdna for FSHR and FSH α- and β-chains were modified by PCR and inserted into the same pfastbac Dual plasmid (Invitrogen). The construct encodes a covalently linked FSH αβ heterodimer under the control of the polyhedrin promoter and a truncated ectodomain of FSHR under the control of the p10 promoter. The covalently linked FSH comprises the signal peptide and entire mature protein of FSH β (residues 1-111), followed by a 15- residue linker (GGGSGGGSGGGSGGG) and the mature protein sequence of FSH α (residues 1-92). Secretion of the FSHR ectodomain was facilitated by its native signal sequence. A FLAG tag (DYKDDDDK) was engineered at the C-terminus of FSHR for affinity purification. Constructs with different C-terminal truncations of the FSHR ectodomain were tested for expression and proper folding. All expressed poorly with a majority of the secreted protein forming large soluble aggregates. The most stable FSHR construct contains residues (FSHR HB ) where residues 1-17 correspond to its putative signal peptide. N-terminal sequencing analysis of the secreted FSHR HB indicates that signal peptide cleavage from the mature protein occurs at one of two sites, Gly17 or Cys18. Hi5 cells were grown in monolayer or suspension culture to 2.0x10 6 cells/ml, which were then infected at a 10-fold multiplicity of infection with recombinant baculovirus carrying the FSHR HB and FSH genes. The cell culture supernatant was harvested 72 hours after infection and applied to an anti-flag antibody (M2) affinity column. FSH-FSHR HB complex bound to the M2 column and was eluted with 100 µg/ml FLAG peptide in 50 mm Tris, ph7.5, 150 mm NaCl. The protein complex was further purified by size exclusion chromatography (Superdex200, Pharmacia) in 20 mm Tris, ph8.0, 150 mm NaCl to remove aggregates. The yield of properly folded FSH-FSHR HB protein was about 0.1 mg per liter of insect cell culture. Partial deglycosylation of the FSH-FSHR HB complex using a combination of endoglycosidase F2 and F3 was carried out at 20ºC for 5 days in 50 mm sodium acetate, ph4.5. FSH-FSHR HB protein was separated from the deglycosylation enzymes by ion exchange chromatography (MonoQ, Pharmacia) in 20 mm Tris, ph8.0 using a linear salt gradient from 0 to 500 mm NaCl. Crystallization and data collection Crystals of fully glycosylated FSH-FSHR HB complex were grown at 4ºC from 18% tertbutanol, 100 mm Na Citrate, ph6.0. Diffraction data were measured at National Synchrotron Light Source (NSLS) beamline X4A from crystals frozen directly from the vapor-diffusion drop. These crystals have the space group R32 (a = Å, α = 80.3 ) with an estimated 5-10 complexes per asymmetric unit and diffract only to 9 Å spacings. Crystals of the partially deglycosylated FSH-FSHR HB complex were grown at 20ºC by vapor diffusion method. The crystallization drops contained 0.5 µl of 10% PEG3350, 0.1 M Li 2 SO 4 as the precipitant and 0.5 µl of the protein complex at 9.8 mg/ml in 20 mm Tris, ph8.0. Plate crystals with a thickness of only 5-10 µm grew as clusters; single crystals were broken off the clusters for data collection. 1

9 The crystals were soaked briefly in a cryoprotecting solution containing 10% PEG3350, 0.1 M Li 2 SO 4, 25% ethylene glycol, and flash-cooled with liquid nitrogen. Native data were collected to 2.9 Å spacings at SGX-CAT beamline of the Advanced Photon Source (APS). These crystals belong to the space group C2 (a=121.3 Å, b=66.9 Å, c=148.6 Å, and β=99.1º), and have two FSH-FSHR HB complexes per asymmetric unit. Data were integrated and scaled using Denzo and Scalepack 1. Most of the subsequent processing used CCP4 programs 2. Structure determination and refinement The structure of FSH-FSHR HB complex was solved by molecular replacement 3. The presence of a two-fold non-crystallographic symmetry (NCS) axis was identified by analysis of the self-rotation function, and the known structure of FSH 4 (PDB code: 1FL7) was used as the search model in Amore 3. One FSH molecule was readily located as the top rotation and translation solution, and the second FSH was found by fixing the position of the first hormone molecule. The final two-fsh solution had a correlation coefficient of 28.1% and R-value of 54.3% for data from 20 to 4 Å. After rigid body refinement of each hormone molecule in CNS 5 with all data ( Å), this model (R free =52.1%; R work =53.1%) was used to obtain partial phases, which were improved by two-fold NCS-averaging, histogram matching and solvent flattening in DM 2. The resulting electron density map gave additional density for the bound receptor. Furthermore, it clearly revealed a β-sheet in the receptor region which is characteristic of the LRR motif predicted for FSHR ectodomain 6. A model of the FSH-FSHR HB complex structure was gradually constructed as the density was improved through manual building and rebuilding in O 7 followed by refinement. Refinement was carried out using CNS 5 initially and at the final stages using REFMAC 2 with TLS refinement 8. All data ( F >0) were included, with 5% of randomly selected reflections used for R free monitoring. Tight non-crystallographic symmetry restraints were gradually relaxed during the refinement process. The optimal NCSrestraints were determined by systematically monitoring the variation of R free as a function of the restraint weights in REFMAC (data shown elsewhere). The model was refined to a final R free of 25.9 % and R work of 21.9 %. Continuous electron density was observed for carbohydrate moieties at one N- linked glycosylation site on each receptor molecule (a disaccharide at N191 of FSHR HB and a trisaccharide at it symmetry mate N191 ), all four sites on one of the hormone molecules (monosaccharides at αn52, αn78, βn7 and βn24), and two sites on the other hormone (monosaccharides at αn78 and βn24 ). Broken density was observed for glycosylation at αn52 and βn7, but no carbohydrate residues were modeled. The final model contained two FSH molecules (α3-92 and α6-92 ; β3-107 and β3-107 ), two FSHR HB molecules (residues and ), 10 N- acetylglucosamine and 1 mannose residue, 50 water molecules and 3 sulfate ions. A Ramachandran analysis ( places 92.1% of all residues in favored regions and 0.9% in outlier regions. Residues of the FLAG-tag peptide and the linker between FSH β- and α-chains were not visible in the electron density maps, possibly due to disordering. The linker peptide used to stabilize the FSH αβ heterodimer does not appear to affect the mode of association between FSH α- and β-subunits or the receptor binding 2

10 mode of FSH. First of all, the entire linker together with four C-terminal residues of FSHβ and two N-terminal residues of FSHα (five in the second protomer) are disordered, indicating that they are highly flexible. In addition, a covalent linker connecting the C- terminus of FSHβ and N-terminus of FSHα would span a region on the FSH surface opposite to the receptor-binding site, and therefore would not hinder receptor binding. Chemical crosslinking The FSH-FSHR HB complex was dialyzed extensively against 20 mm HEPES, ph8.0, 150 mm NaCl, and then reacted with glutaraldehyde at 20 C to completion (overnight) at increasing concentrations of FSH-FSHR HB (30, 60, 120, 180, 240, 300 and 360 µm) and constant molar ratios of protein to glutaraldehyde (3:1, 3:2 or 1:1). The low glutaraldehyde concentration was chosen to prevent adventitious crosslinking at higher concentrations of FSH-FSHR HB. Equivalent amounts of crosslinked protein (2 µg) were loaded on 8-25% native gels or under non-reducing conditions on SDS gels. Because glycosylated versions of single-chain FSH and FSHR HB had similar apparent molecular weight, the dimer band on SDS gels could result from the crosslinking of two receptor molecules located at the dimer interface or from the 1:1 receptor-hormone complex. A novel band on native gels, which contained both FSH and FSHR HB by N-terminal sequencing, represented a true dimer of the complex and was therefore chosen for quantification. Gels were stained by Coomassie blue, and quantified with a densitometer. Crosslinking was not observed for BSA under the same conditions. Given the abundance of lysine residues present in FSH-FSHR HB (30 lysines per FSH-FSHR HB complex, including 2 from the FLAG tag) and the low glutaraldehyde concentration, the amount of crosslinked species is a subpopulation of the total amount of dimer in solution at equilibrium, but it is proportional to the dimer fraction. Based on a monomer-dimer equilibrium model of A+A A 2, the fraction of dimers at a specific total protein concentration (C T ) is given by ( 1+ 8CT K d 1) y = ( 1+ 8CT K d + 1) where K d is the dissociation constant. The dimer band density (D) is proportional to the dimer fraction, D=S y, where S is a scaling factor. A least-squares fitting of the D(C T ) data give estimates of S and K d. Analytical ultracentrifugation Sedimentation equilibrium experiments were carried out in a Beckman/Coulter XL-I analytical centrifuge using absorbance optics at 280 nm. Purified fully glycosylated FSH- FSHR HB complex was dialyzed extensively against a buffer containing 20 mm HEPES, ph 8.0, 150 mm NaCl and loaded into a six-channel equilibrium cell at 0.33, 0.66 and 1.05 mg/ml. Samples were centrifuged at 7,000, 9,000 and 12,000 r.p.m. in an An50Ti rotor at 4 C. Equilibrium was reached when no further change was observed in protein distribution spectra acquired at 1-h intervals. Data were analyzed with the WinNONLIN fitting program 9 ( Because the exact molecular mass of the FSH-FSHR HB complex was not known due to heavy glycosylation, the monomer molecular mass was allowed to float as a variable in the fitting to a monomer-dimer equilibrium model. The data clearly demonstrated the 3

11 presence of a thermodynamic monomer-dimer equilibrium because the dimer dissociation constant obtained separately from the three different concentrations and speeds converged within their 95% confidence intervals. The molecular mass of the FSH- FSHR HB complex was determined to be 78 kd, 32% of which corresponded to carbohydrates. Dynamic light scattering Hydrodynamic radii of FSH-FSHR HB at various concentrations were obtained from lightscattering measurements using a DynaPro Molecular Sizing Instrument (Protein Solutions, Inc). BSA control showed less than 5% variation over the same concentration range. Illustration Figures were generated using Ribbons 10, Grasp 11, Adobe Illustrator and Photoshop. References 1. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, (1997). 2. Collaborative Computational Project, Number 4. The CCP4 Suite: Programs for protein crystallography. Acta Crystallogr. D. 50, (1994). 3. Navaza, J. Implementation of molecular replacement in AMoRe. Acta Crystallogr. D. 57, (2001). 4. Fox, K. M., Dias, J. A. & Van Roey, P. Three-dimensional structure of human follicle-stimulating hormone. Mol. Endocrinol. 15, (2001). 5. Brünger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D. 54, (1998). 6. Jiang, X. et al. Structural predictions for the ligand-binding region of glycoprotein hormone receptors and the nature of hormone-receptor interactions. Structure 3, (1995). 7. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in the models. Acta Crystallogr. A. 47, (1991). 8. Winn, M. D., Isupov, M. N. & Murshudov, G. N. Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr. D. 57, (2001). 9. Laue, T. M., Shah, B. D., Ridgeway, T. M. & Pelletier, S. L. in Analytical ultracentrifugation in biochemistry and polymer science (eds. Harding, S. E., Rowe, A. J. & Horton, J. C.) (Royal Society of Chemistry, Cambridge, UK, 1992). 10. Carson, M. Ribbons. Methods Enzymol. 277, (1997). 11. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, (1991). 4

Supplemental Information. The structural basis of R Spondin recognition by LGR5 and RNF43

Supplemental Information. The structural basis of R Spondin recognition by LGR5 and RNF43 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