www.sciencemag.org/cgi/content/full/science.1242468/DC1

Supplementary Materials for

Molecular Mechanism for Plant Steroid Receptor Activation by Somatic

Embryogenesis Co-Receptor Kinases

Julia Santiago, Christine Henzler, Michael Hothorn*

*To whom correspondence should be addressed. E-mail: mhothorn@tuebingen.mpg.de

Published 8 August 2013 on Science Express

DOI: 10.1126/science.1242468

This PDF file includes:

Materials and Methods

Figs. S1 to S6

Table S1

References

μΜ

Μ

μ

μ

°

Fig. S1.The wild-type BRI1 and SERK1 ectodomains interact upon brassinolide binding. (A) UVabsorbance traces from analytical size-exclusion chromatography experiments. BL-boundBRI1 wild-type elutes as a monomer (black dotted line), as does the isolated SERK1LRR domain (blue dotted line). The BRI1 wild-type – BL – SERK1 complex elutes as anapparent heterodimer (red line), while a mixture of BRI1 wild-type and SERK1 in theabsence of BL yields two isolated peaks that correspond to the monomeric BRI1 andSERK1 ectodomains, respectively (black line). Void (V0) and total volume (Vt) areshown, together with elution volumes for molecular mass standards (A, Thyroglobulin,669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). The calculatedmolecular mass for the BRI1 wild-type and SERK1 elution peaks are ~125 and ~35 kDa,respectively. Purified BRI1 wild-type and SERK1 are ~110 and ~30 kDa. (B)SDS-PAGE analysis of elution fractions from the size-exclusion chromatographyexperiments shown in (A).

7

A

0 5 10 15 20 25

050

100

150

200

elution volume (ml)

A 280

(a.u

.)

V0 VtA B C D E F

BRI1 wild-typeSERK1BRI1 wild-type - SERK1 -BL BRI1 wild-type - SERK1 +BL

peak1

peak2

B

marker

comple

x

BRI1 wild

-type

250

130100

70

55

3525

15

SERK1

peak

1pe

ak2

BRI1 wild-type

SERK1

Fig. S2Crystal lattice interactions of the bri1sud1 and SERK1 ectodomains. (A) Orientation of 12bri1sud1 – BL – SERK1 complexes in the unit cell of the P65 crystal structure (a=b=~70 Åand c=874 Å). Cα-traces of BRI1 molecules A and B are shown in dark- and light-blue,respectively, the corresponding SERK1 ectodomains are depicted in yellow and red(chains C, D). Two bri1sud1 – BL – SERK1 complexes form a crystallographic dimer inthe asymmetric unit. (B) Crystal packing brings two BRI1 ectodomain superhelices into ahead-to-head arrangement. The lattice propagates by establishing contacts between theC-termini of two SERK1 LRR domains and the corresponding C-termini of the BRI1ectodomains. This figure has been prepared with the supercell.py script as implementedin the program PYMOL (http://pymol.sourceforge.org).

8

70 70

874

CC C

C

N

N

N

N

A B

Fig. S3SERK1 ectodomain residues participate in the specific recognition of the steroidhormone. (A) The LIGPLOT (41) diagram summarizes key interactions betweenbrassinolide (yellow lines) and hormone binding pocket residues that originate from theBRI1 LRR core (shown in blue), from the BRI1 island domain (in green) and from theSERK1 N-terminal capping domain (shown in orange). His62SERK1 establishes hydrogenbonds with both the 2α and 3α hydroxyl groups of BL, which are known to be critical forbioactivity. Semicircles with radiating lines indicate non-polar interactions. Chemicalstructures of (B) brassinolide and (C) BL 2,3-acetonide are included for comparison.

9

3.072.41

3.30

3.00

2

3

N

ND1

H62

Y597

N

S647

Y642N705

Y599

K601

M657

T729

I563T646

P648

F681

I540

I706

F61

A

B

C

D

A

H

H

CH3

CH3

CH3

OH

OH

OH

OH

CH3

CH3

H3C

HH

H O

O

3

2

B

H

H

CH3

CH3

CH3

OH

OH

CH3

CH3

H3C

HH

H O

O

O

O 3

2

C

Fig. S4BRI1 – SERK1 complex interface residues are conserved among known SERK-family members and in other LRR receptor kinases. Structure-basedsequence alignment of the known BRI1 interacting SERK-family members Arabidopsis thaliana SERK1 (Uniprot (http://www.uniprot.org) identifier:Q94AG2), A. thaliana SERK2 (Uniprot identifier: Q9XIC7), A. thaliana SERK3/BAK1 (Uniprot identifier: Q94F62), A. thaliana SERK4 (Uniprotidentifier: Q9SKG5) and Oryza sativa subsp. japonica BAK1 (Uniprot identifier: Q7XV05) (9–11, 42, 43). Based on the SERK1 interface residues inthe bri1sud1 – BL – SERK1 complex, we identified other, putative, BRI1-interacting receptor kinases (in grey): Selaginella moellendorffii SERKx(Uniprot identifier: D8RKF6), Capsella rubella SERKx (44) (GenBank identifier: EOA30137.1), Vitis vinifera SERKx (Uniprot identifier: D7STF5),Glycine max (Uniprot identifier: I1KR51), Nicotiana benthamiana SERKx (Uniprot identifier: E3VXE7), Medicago truncatula SERKx (Uniprotidentifier: G7ILB9). The alignment includes a secondary structure assignment calculated with the program DSSP (45) and colored according to Fig. 1C.The N- and C-terminal caps and the five LRRs in SERK1 are indicated in red and blue, respectively. Cysteine residues in the N- and C-terminal cappingdomains are highlighted in green. Note that most SERK proteins have the C-terminal disulfide bond replaced by a proline-rich region. N-glycosylationsites observed in SERK1 crystals are marked with a yellow star. The position of the serk3 elg point-mutation (corresponds to Asp123 in SERK1) isindicated in cyan, SERK-residues interacting with BRI1 in the bri1sud1 – BL – SERK1 complex are highlighted in orange.

10

310αN-terminal cap

signal peptide

*AtSERK1 MESSYV----V-FILL--SLILLPNHSLWLASANLEGDALHTLRVTLV--DP-NNVLQSWDPTLVNPCTWFHVTCNNENSVIRVDLGNAELSGHLVPELGVLKNLQYLELYSNNAtSERK2 MGRKKFEAFGF-VCLI--SLLLLFN-SLWLASSNMEGDALHSLRANLV--DP-NNVLQSWDPTLVNPCTWFHVTCNNENSVIRVDLGNADLSGQLVPQLGQLKNLQYLELYSNNAtSERK3 MERR-----LMIPCFF--WLILVLD-LVLRVSGNAEGDALSALKNSLA--DP-NKVLQSWDATLVTPCTWFHVTCNSDNSVTRVDLGNANLSGQLVMQLGQLPNLQYLELYSNNAtSERK4 MTSSKMEQRSL-LCFL--YLLLLFN-FTLRVAGNAEGDALTQLKNSLSSGDPANNVLQSWDATLVTPCTWFHVTCNPENKVTRVDLGNAKLSGKLVPELGQLLNLQYLELYSNNOsBAK1 MAEARLLRRRR-LCLAVPFVWVVAV-AVSRVGANTEGDALYSLRQSLK--DA-NNVLQSWDPTLVNPCTWFHVTCNPDNSVIRVDLGNAQLSGALVPQLGQLKNLQYLELYSNNSmSERKx MEQDAAA--VL-LLLL--CLFCLLG-VQPSLVCVSPVSALFAFKQSLV--DP-QNAMSGWDKNAVDPCSWIHVSCS-EQNVSRVELPGLQLSGQLSPRLADLANLQYLMLQNNNCrSERKx MEQR-----SL-LCFV--WLILLLV-FTLRAAGNTEGDALIVLKNNLSPADPANNVLQSWDATLVTPCTWFHVTCNNENKVTRVDLGNAELSGKLVPELGQLLNLQYLELYSNNVvSERKx MEAI------F-LC--------LIS-LVLRVSGISEGDALYALKSSLV--DP-KDVLQSWDTSSGNPCIWFHVTCNGDGNVIRVDLGNGSLSGQLDSRVGQLTKLEYLGLYNNNGmSERKx MERMISSFMSL-FFIL--WIFVVLD-LVLKVYGHAEGDALIVLKNSMI--DP-NNALHNWDASLVSPCTWFHVTCS-ENSVIRVELGNANLSGKLVPELGQLPNLQYLELYSNNNbSERKx MDQSVLL---I-CVFL--CLTGLLL-SSSPVAGNAEGDALYAQKTNLG--DP-NTVLQSWDQTLVNPCTWFHVTCNNENSVTRVDLGNANLTGQLVPQLGQLQKLQYLELYSNNMtSERKx MERVSSAS-KV-SFLF--WAILVLH-LLLNASSNVESDTLIALKSNLN--DP-NSVFQSWNATNVNPCEWFHVTCNDDKSVILIDLENANLSGTLISKFGDLSNLQYLELSSNN

Asn104

C-terminal cap

AtSERK1 ITGPIPSNLGNLTNLVSLDLYLNSFSGPIPESLGKLSKLRFLRLNNNSLTGSIPMSLTNITTLQVLDLSNNRLSGSVPDNGSFSLFTPISFANNLDLCG-PVT---S-HPCPGSAtSERK2 ITGPVPSDLGNLTNLVSLDLYLNSFTGPIPDSLGKLFKLRFLRLNNNSLTGPIPMSLTNIMTLQVLDLSNNRLSGSVPDNGSFSLFTPISFANNLDLCG-PVT---S-RPCPGSAtSERK3 ITGTIPEQLGNLTELVSLDLYLNNLSGPIPSTLGRLKKLRFLRLNNNSLSGEIPRSLTAVLTLQVLDLSNNPLTGDIPVNGSFSLFTPISFANTKLTPL-PAS---PPPPISPTAtSERK4 ITGEIPEELGDLVELVSLDLYANSISGPIPSSLGKLGKLRFLRLNNNSLSGEIPMTLTSV-QLQVLDISNNRLSGDIPVNGSFSLFTPISFANNSLTDL-PEP---PPTSTSPTOsBAK1 ISGTIPNELGNLTNLVSLDLYLNNFTGFIPETLGQLYKLRFLRLNNNSLSGSIPKSLTNITTLQVLDLSNNNLSGEVPSTGSFSLFTPISFANNKDLCG-PGT---T-KPCPGASmSERKx LSGPIPPEFGNWSRIISVDLSNNNLSDPIPSTLGKLQTLQYLRLNNNSLSGAFPVSVATIRALDFLDVSFNNLSGNVPNATTANL----NVKGNPLLCG-SKT--SRI--CPGDCrSERKx ITGEIPEELGGLRELVSLDLYANNINGPIPSSLGQLEKLRFLRLNNNSLSGGIPMELTAV-QLQVLDISNNRLSGDIPVNGSFSLFTPISFKNNKLTSL-P---------EPPPVvSERKx ISGKIPEELGNLENLMSLDLYFNNLSGPIPGTLGKLRKLHFLRLNNNILMGTIPMSLTAVSSLEILDLSNNKLTGDIPVNGSFSLFTPISFGNNRLSNNSPKRTLDSPSPISPNGmSERKx ITGEIPVELGNLTNLVSLDLYMNKITGPIPDELANLNQLQSLRLNDNSLLGNIPVGLTTINSLQVLDLSNNNLTGDVPVNGSFSIFTPISFNNNPFLNK-T-------IPVTPANbSERKx ISGRIPNELGNLTELVSLDLYLNNLNGPIPDTLGKLQKLRFLRLNNNSLIGLIPMSLTTILALQVLDLSSNHLTGPVPVNGSFSLFTPISFANNQLEVP-PA--------SPPPMtSERKx ITGKIPEELGNLTNLVSLDLYLNHLSGTILNTLGNLHKLCFLRLNNNSLTGVIPISLSNVATLQVLDLSNNNLEGDIPVNGSFLLFTSSSYQNNPRLKQ-P----------KII

310 310 310 310

D123NSERK3 elg

* *

Asn150 Asn184

310

LRR1 LRR2

LRR2 LRR3 LRR4 LRR5

Fig. S5Details of the BRI1 – SERK1 complex interface. (A) Side and (B) front view of the BRI1– SERK1 interface with the BRI1 LRR domain in blue (in surface representation), theSERK1 ectodomain in orange (ribbon diagram) and BL in yellow (in bondsrepresentation). Interface residues are highlighted as sticks. Arg73SERK1 contacts Thr750in the BRI1 C-terminal cap (bri1102), Asp75SERK1 establishes a hydrogen bond withThr729, as does the main-chain oxygen of Gly77 with Thr726. Non-polar contacts aremediated by Tyr97SERK1, Tyr101SERK1, Tyr125SERK1 and Phe145SERK1 and by Met727 andMet745 in BRI1. Asp123SERK1 is centrally located in the complex interface and takes partin a hydrogen-bonding network that involves Glu99SERK1, Ser121SERK1, Tyr125SERK1,Arg147SERK1 and Glu749 in the BRI1 C-terminal cap.

11

LRR4

R73D75 G77

T726

T729 M727

Q747

E99Y97

Y101

Y125

M745

E749

F145R147

S121T750

D123

LRR3

LRR2

N-cap

LRR1

N-cap

R73

D75

G77

T726

T750

T729

Y97

E99Y101

E749

M745

Y125D123

S121

R147

F145

A

B

Fig. S6Conserved surface patches in the SERK1 ectodomain may mediate interaction with otherreceptor kinases. (A) Ribbon diagram of the SERK1 ectodomain colored as in Fig. 1Cand shown in the same orientation as the molecular surfaces in (B) Surface diagram ofthe SERK1 ectodomain with the bri1sud1 – interacting residues shown in blue (left panel),and with the surface colored according to SERK-family sequence conservation (rightpanel, compare fig. S4).

12

N

C

A B

100 %0

B

100 %0

N

C

α, β, γ °

σ

°

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