SUPPLEMENTARY INFORMATION

Transcription

1 Supplementary Discussion The HGGG sequence motif forms an oxyanion hole in HLSs. In the case of GID1, the third Gly residue of the sequence motif is replaced with Ser (Ser123 in OsGID1), i.e., the sequence of the motif is HGGS (Supplementary Fig. 9). This replacement enables the side chain of Ser123 as well as Ser198 to interact with the carboxylate group at the C6 position of GAs (Fig. 1c). The importance of Ser123 was confirmed by little GA binding activity of mutated OsGID1 protein, S123G (Supplementary Fig.5). The substitution of the third Gly in the HGGG motif likely caused another replacement in which the Ala next to the catalytic serine in HSL is replaced with Ser, i.e., Ser199 in OsGID1. By this substitution, the side chain of Ser199 is coordinated to the main-chain NH of Ser123. Indeed, the distance and angle between the NH of Ser123 and OG of Ser199 are not the most favorable but still allowed to make hydrogen bond. This interaction makes it possible for the HGGS loop structure to adopt the hydrogen bonding distance between Ser123 and the carboxyl group in GAs. These two amino acid exchanges are conserved in all GID1 proteins from rice (Oryza sativa) 1, Arabidopsis (Arabidopsis thaliana) 2, cotton (Gossypium hirsutum) 3, and lycophytes (Selaginella moellendorffii) 4, as indicated in Supplementary Fig

2 Supplementary Table S1 Data collection and refinement statistics Data collection GID1-GA4 Hg SAD GID1-GA3 Space group P 21 P 21 P 21 Cell dimensions a, b, c (Å) 82.8, 133.9, , 133.3, , 134.1, 118.9,, ( ) 90, 105.0, 90 90, 104.9, 90 90, 105.2, 90 Wavelength (Å) Resolution (Å) ( ) * ( ) ( ) No. of reflections No. of unique reflections Rmerge (0.597) (0.298) (0.365) I/ I 21.8 (1.7) 21.0 (5.6) 27.6 (3.2) Completeness (%) 98.8 (92.8) 99.8 (99.9) 97.6 (87.5) Redundancy 3.6 (3.4) 3.8 (3.8) 3.7 (3.3) Figure of Merit 0.66 Refinement Resolution (Å) No. reflections Rwork / Rfree / / No. atoms Protein Ligand/ion Water B-factors Protein Ligand/ion Water R.m.s deviations Bond lengths (Å) Bond angles (º) *Highest resolution shell is shown in parentheses. R merge = I i -<I i > / I i, where I i = observed intensit y, <I i > = average intensity of multiple 2

3 Supplementary Figures Supplementary Fig. 1: Topology of rice GID1 structure: the central -sheet is shown in pink ( 1, Val63 Ile69; 2, Leu76 Ala83; 3, Val115 Phe118; 4, Val148 Val152; 5, Val192 Asp197; 6, Asn221 Leu224; 7, Ser288 Ser293; 8, Val316 Cys321) and the central helices ( - and helices ( )) in blue ( 1, Thr132 Ser145; 2, Cys164 Ser178; 1, Pro180 Met182; 3, Ser199 Glu214; 6, Cys299 Asp312; 2, Phe328 Leu330; 7, Val335 Asn351). An additional region containing the N-terminal lid ( b, Leu18 Leu34; c, Arg43 Leu49) and insertion helices between 6 and 7 ( 4, Glu235 Leu240; 5, Leu248 Tyr258) are shown in green and orange, respectively. A segment from Ala85 to Thr105 that is invisible in the electron density map is shown as a broken line. Functional loops are shown as bold lines (L120, link: 3 and 1; L240, link: 4 and 5; L290, link 7 and 6; L320, link: 8 and 2). Supplementary Fig. 2: Electron density map around the GA binding site of rice GID1. a, GA 4 GID1 complex; b, GA 3 GID1 complex; c, their superimposed 3

4 structural model, shown in orange (GA 4 ) and in green (GA 3 ); d, chemical structure of GA 4 and GA 3. The GA molecules are shown as a ball-and-stick model, with its positive F o -F c electron density calculated and contoured at 3.5 before it was built into the OsGID1 model. Supplementary Fig. 3: Superimposed structure of rice GID1 (green) and AeCXE1 (grey) around the GA binding site, including their corresponding residues for the HGGG motif and catalytic triad of HSL. The residues of GID1 are indicated, with the corresponding residues of AeCXE1 in parentheses. Structural changes are indicated by arrows. Supplementary Fig. 4: Stereo view of Fig. 2a. Supplementary Fig. 5: GA binding activities of mutated rice GID1s proteins with conserved amino acids replaced with Ala or the corresponding amino acid residues of SmGID1s. Ala exchange, mutated GID1 proteins with amino acids related to polar 4

5 and hydrophobic interactions with GA 4 (described in the text) replaced with Ala. Miscellaneous exchange, mutated GID1 proteins with amino acids related to other functions replaced with Ala or Gly. SmGID1 type, mutated GID1 proteins with amino acids, that are not conserved in S. moellendorffii replaced with the corresponding SmGID1 amino acid residue. mlid, mutated GID1 proteins with 6 amino acids (Leu18, Trp21, Leu29, Ile33, Leu45 and Tyr48) replaced with Ala. Values are means s.d.; n = 3. Supplementary Fig. 6: Schematic rice GID1 dimer interaction in the crystalline state. GID1 molecules are likely packed into a crystalline lattice as dimers; nevertheless, the interface area is narrower than typical protein dimers. The functional relevance of the dimer is unclear, as the gel filtration profile indicates that GID1 functions as a monomer. The N-terminal lid region including b and c in each monomer is colored (blue-green in one monomer and green in the other). The six hydrophobic amino acid residues that appear to participate in the interaction in each monomer are shown by a stick model. These residues are marked by blue in Supplementary Fig

6 Supplementary Fig. 7: Gel filtration elution profile of rice GID1 complexed with GA 4. Molecular mass calibration is shown at the top of the figure. Supplementary Fig. 8: Hydrophobic amino acids that lie at the upper side of N-terminal lid region are essential for interaction between rice GID1 and SLR1. Interactions established by yeast two-hybrid assay using mutated GID1s as bait and the full-length SLR1 as prey with or without 10-4 M GA 4. mlid, mutated GID1 proteins with 6 amino acids (Leu18, Trp21, Leu29, Ile33, Leu45 and Tyr48), replaced with Ala. Values are means SD, n = 3. Supplementary Fig. 9: Sequence alignment of GID1s and its homologues based on the crystal structure of rice GID1. GID1 from rice, OsGID1; Arabidopsis, AtGID1abc; cotton, GhGID1ab; lycophyte, SmGID1ab; bryophyte (putative; GID1 function not detected), PpGID1L12; plant carboxylesterase AeCXE1 5, 2o7r; thermostable carboxylesterase Este1 (unpublished), 2C7B; Archaeon carboxylesterase 6

7 AFEST 6, 1jji; thermophilic esterase Est2 7, 1EVQ. The last four HSL members are designated by their PDB codes; their similarity to OsGID1 was found by SSM analysis. The - and helices of GID1 are indicated by cylinders and the -strands with arrows. Unmodeled regions are indicated by a broken line. The HGG motif and catalytic triad (Ser, Asp, His; Ser198, Asp296, Val326 in OsGID1) of HSL are marked in green. The GA recognition residues are marked in yellow, functional residues in the lid closure are light-blue and other protein interactions indicated in Supplementary Fig. 6 are blue. Fully conserved residues are filled in whereas partially conserved ones are open boxes. Supplementary References 1. Ueguchi-Tanaka, M. et al. GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin. Nature 437, (2005). 2. Nakajima, M. et al. Identification and characterization of Arabidopsis gibberellin receptors. Plant J 46, (2006). 7

8 3. Aleman, L. et al. Functional analysis of cotton orthologs of GA signal transduction factors GID1 and SLR1. Plant Mol Biol 68, 1-16 (2008). 4. Hirano, K. et al. The GID1-mediated gibberellin perception mechanism is conserved in the Lycophyte Selaginella moellendorffii but not in the Bryophyte Physcomitrella patens. Plant Cell 19, (2007). 5. Ileperuma, N. R. et al. High-resolution crystal structure of plant carboxylesterase AeCXE1, from Actinidia eriantha, and its complex with a high-affinity inhibitor paraoxon. Biochemistry 46, (2007). 6. De Simone, G. et al. The crystal structure of a hyper-thermophilic carboxylesterase from the archaeon Archaeoglobus fulgidus. J Mol Biol 314, (2001). 7. De Simone, G. et al. A snapshot of a transition state analogue of a novel thermophilic esterase belonging to the subfamily of mammalian hormone-sensitive lipase. J Mol Biol 303, (2000). 8

9 N HGGS Ser198 Asp Val C Supplementary Fig. 1 M.Matsuoka et al. 9

10 a b Phe27 Ile24 Tyr31 Phe27 Ile24 Tyr31 Ser127 Asp250 Ser127 Asp250 Val264 Val264 Tyr134 GA4 Ser123 Tyr134 GA3 Ser123 Ser198 Ser198 c GA3 Asp250 d HO 2 OC O COOH GA O OH 2 OC 13 HO GA3 COOH Supplementary Fig. 2 M.Matsuoka et al. 10

11 GA4 Val326 Gly121 (91) Ser198 (169) (His306) Asp296 (276) His120 (90) Gly122 (92) Ser123 (Gly93) Supplementary Fig. 3 M.Matsuoka et al. 11

12 (carboxyl-end of β6) Asn225 (η2) (L320) Tyr329 Gly327m Asp296 (L290) Arg251 (α5) (carboxyl-end of β6) Asn225 (η2) (L320) Tyr329 Gly327m Asp296 (L290) Arg251 (α5) (α1) Tyr134 GA4 C3 Ser198 (nucleophilic elbow) C6 Ser123 (L120) Gly122m (L120) Asp250 (α5) (α1) Tyr134 GA4 C3 Ser198 (nucleophilic elbow) C6 Ser123 (L120) Gly122m (L120) Asp250 (α5) Ser127 (L120) Tyr31 (αb) Ser127 (L120) Tyr31 (αb) Supplementary Fig. 4 M.Matsuoka et al. 12

13 GA binding activity (dpm) Wild I24A F27A Y31A S123A S127A I133A Y134A S198A N225A F245A V246A D250A R251A Y254A V326A Y329A L330A H126A W253A S123G mlid F27L S127M I133L I133V N225I N225M Y329F L330I Ala exchange I Miscellaneous exchange SmGID1 type Supplementary Fig. 5 M.Matsuoka et al. 13

14 doi: /nature07546 Trp21 Leu29 Leu45 Tyr48 Ile33 Leu29 Leu18 Leu18 Ile33 Leu45 Tyr48 Trp21 Supplementary Fig. 6 M.Matsuoka et al. 14

15 kda 158 kda 44 kda 17 kda elution volume (ml) Supplementary Fig. 7 M.Matsuoka et al. 15

16 GA bait prey 4 Wild- GID1 SLR1 - + mlid- GID1 SLR β-galactosidase activity (Miller unit) Supplementary Fig. 8 M.Matsuoka et al. 16

17 20 40 OsGID1 MA-----GSDEVNRNECKTVVPLHTWVLISNFKLSYNILRRADGTFERDLGEYLDRRVPA 55 AtGID1a MA-----ASDEVNLIESRTVVPLNTWVLISNFKVAYNILRRPDGTFNRHLAEYLDRKVTA 55 AtGID1b MA-----GGNEVNLNECKRIVPLNTWVLISNFKLAYKVLRRPDGSFNRDLAEFLDRKVPA 55 AtGID1c MA-----GSEEVNLIESKTVVPLNTWVLISNFKLAYNLLRRPDGTFNRHLAEFLDRKVPA 55 GhGID1a MA-----GSNEVNLNESKRVVPLNTWVLISNFKLAYNLQRRPDGTFNRDLSEFLDRRVPA 55 GhGID1b MA-----GSNEVNLNESKRVVPLNTWVLISNFKLSYNLQRRPDGTFNRDLSEFLDRRVPA 55 SmGID1a MN-----SCSKAILKPSKDVVPLSTWILISKLKVEYMLTRGADGSFNRNLAEFHDRKASA 55 SmGID1b MEPEEDSSAEELQLKESKEVVPLNTWVLISNLKLAYNLTRNSDGSFNRNLDEFLDRKVPV 60 PpGID1L MASLVQLRILSEFVVRANRVTRRRDGTINRWLADTLEKKVPA 42 PpGID1L MLYDLQMASMMQLRLLCKVVVKANDLARRKDGTINRWLADVCERKVPA 48 2O7R MSNDHLETTGSSDPNTNLLKYLPIVLNPDRTITRPIQIPSTAA 43 2C7B MPLDPQIKPILERIRALSIAASPQELRRQVEEQSRL 36 1JJI MLDMPIDPVYYQLAEYFDSLPKFDQF------SSAREYREAINRIYEE 42 1EVQ MPLDPVIQQVLDQLNRMPAPDYKHLSAQQFRSQQS OsGID1 NARP----LEGVSSFDHIIDQ-SVGLEVRIYRAAAEGDAEEGAAAVTRPILEFLTDAPAA 110 AtGID1a NANP----VDGVFSFDVLIDR-RINLLSRVYRPAYAD------QEQPPSILDLEKPVDG- 103 AtGID1b NSFP----LDGVFSFD-HVDS-TTNLLTRIYQPASLL------HQTRHGTLELTKPLSTT 103 AtGID1c NANP----VNGVFSFDVIIDR-QTNLLSRVYRPADAG TSPSITDLQNPVDG- 101 GhGID1a NINP----VDGVFSFD-HVDG-ATGLLNRVYQPSSL NEAQWGMVDLEKPLSTT 102 GhGID1b NINP----VDGVFSFD-HVDG-ATGLLNRVYQPSPK NEAQWGIVDLEKPLSTT 102 SmGID1a SLAP----HDGVASMDVTIDR-SSGLWSRIFLPAIAY AQEEQANRD 96 SmGID1b SSVERE -DDPVTFMDVTIDR-TSGIWSRIFIPRASH NNNASSTTH 104 PpGID1L1 NPIP----VKGVSSADVTIDA-EAGIWARVFSLTEEI------EETSLPTATDGNQR-LF 90 PpGID1L2 NPKP----IKGVHTVDVTIDP-EAGVWVRLFIPTEET------VETPSKSASNDTQIESN 97 2O7R SPDPTSS -SPVLTKDLALNPL-HNTFVRLFLPRHALYN S 80 2C7B LTAAVQEP--IAETRDVHIPVSGGSIRARVYFPKKAAG JJI RNRQLSQHERVERVEDRTIKGRNGDIRVRVYQQKPD EVQ LFPPVKKEPVAEVREFDMDLPG-RTLKVRMYRPEGVEP OsGID1 EPFPVIIFFHGGSFVHSSASSTIYDSLCRRFVKLSKGVVVSVNYRRAPEHRYPCAYDDGW 170 AtGID1a DIVPVILFFHGGSFAHSSANSAIYDTLCRRLVGLCKCVVVSVNYRRAPENPYPCAYDDGW 163 AtGID1b EIVPVLIFFHGGSFTHSSANSAIYDTFCRRLVTICGVVVVSVDYRRSPEHRYPCAYDDGW 163 AtGID1c EIVPVIVFFHGGSFAHSSANSAIYDTLCRRLVGLCGAVVVSVNYRRAPENRYPCAYDDGW 161 GhGID1a EIVPVIVFFHGGSFTHSSANSAIYDTFCRRLVSLCKAVVVSVNYRRSPEHRYPCAYDDGW 162 GhGID1b EVVPVIVFFHGGSFTHSSANSAIYDTFCRRLVNICKAVVVSVNYRRSPEHRYPCAYDDGW 162 SmGID1a DKVPIIFYFHGGSYAHSSANTALYDMVCRQLCRTCRAVVISVNYRRAPEHRCPAAYRDGL 156 SmGID1b G-TPIFFYFHGGSFVHMSANSAVYHTVCQQLARLCQAVVISVNYRRAPEHKYPAAYNDCY 162 PpGID1L1 KTMPIILYYHGGGFAVLCPNFYLYDIFCRRLARKCNAIVISVHYRRAPEFKFPTAYDDSY 150 PpGID1L2 KTMPIVYYYHGGGFTILCPDFYLYDVFCRRLAKCCKSVVISLHYRRAPEFKFPTAYDDSF 157 2O7R AKLPLVVYFHGGGFILFSAASTIFHDFCCEMAVHAGVVIASVDYRLAPEHRLPAAYDDAM 140 2C7B --LPAVLYYHGGGFVFGSIET--HDHICRRLSRLSDSVVVSVDYRLAPEYKFPTAVEDAY 128 1JJI --SPVLVYYHGGGFVICSIES--HDALCRRIARLSNSTVVSVDYRLAPEHKFPAAVYDCY 134 1EVQ -PYPALVYYHGGGWVVGDLET--HDPVCRVLAKDGRAVVFSVDYRLAPEHKFPAAVEDAY OsGID1 TALKWVMS QPFM-RSGGDAQARVFLSGDSSGGNIAHHVAVRAADEG-----VK 217 AtGID1a IALNWVNS RSWL-KSKKDSKVHIFLAGDSSGGNIAHNVALRAGESG-----ID 210 AtGID1b NALNWVKS RVWL-QSGKDSNVYVYLAGDSSGGNIAHNVAVRATNEG-----VK 210 AtGID1c AVLKWVNS SSWL-RSKKDSKVRIFLAGDSSGGNIVHNVAVRAVESR-----ID 208 GhGID1a AALKWVKS RTWL-QSGKDSNVHVYLAGDSSGGNIAHHVAVRAAEAD-----VE 209 GhGID1b AALKWVKS RTWL-QSGKDSKVHVYLAGDSSGGNIAHHVAVRAAEAD-----VE 209 SmGID1a AALRWLRLQAARHVAATWL-PPGAD-LSRCFLAGDSSGGNMVHHVGVAAATARHELWPVR 214 SmGID1b AALTWLKVQVLRGVAHAWL-PRTAD-LGRCFLVGDSNGGNIVHHVGVRAAESGAELGPLR 220 PpGID1L1 KAMEWLQSKE----ATVSL-PPNVD-FSRVFLSGDSAGGNIAHHVALRAAGKDLG--RLS 202 PpGID1L2 KGLEWLQSEK----ATASL-PLNVD-FSRVFLCGDSAGANIAYHMALQSARKDLG--RVS 209 2O7R EALQWIKD SRDEWLTNFADFSNCFIMGESAGGNIAYHAGLRAAAVADELLPLK 193 2C7B AALKWVAD RA---DELGVDPDRIAVAGDSAGGNLAAVVSILDRNSG----EKL 174 1JJI DATKWVAE NA---EELRIDPSKIFVGGDSAGGNLAAAVSIMARDSG----EDF 180 1EVQ DALQWIAE R---AADFHLDPARIAVGGDSAGGNLAAVTSILAKERG----GPA Supplementary Fig. 9-1 M.Matsuoka et al.

18 OsGID1 VCGNILLNAMFG--GTERTESERRLDG--KYFVTLQDRDWYWKAYLPEDADRDHPACN-P 272 AtGID1a VLGNILLNPMFG--GNERTESEKSLDG--KYFVTVRDRDWYWKAFLPEGEDREHPACN-P 265 AtGID1b VLGNILLHPMFG--GQERTQSEKTLDG--KYFVTIQDRDWYWRAYLPEGEDRDHPACN-P 265 AtGID1c VLGNILLNPMFG--GTERTESEKRLDG--KYFVTVRDRDWYWRAFLPEGEDREHPACS-P 263 GhGID1a VLGDILLHPMFG--GQKRTESEKRLDG--KYFVTLHDRDWYWRAYLPEGEDRDHPACN-P 264 GhGID1b VLGNILLHPMFG--GQMRTESEKRLDG--KYFVTLHDRDWYWRAYLPEGEDRDHPACN-P 264 SmGID1a VVGHVLLMPMFG--GVERTASERRLDG--QYFVTVKDRDYYWKLFLPEGADRDHPACNV- 269 SmGID1b VAGHILIIPMFG--GNRRTQSELRFDG--QYFVTIKDRDFYWQSFLPAGADRDHPACNI- 275 PpGID1L1 LKGLVLIQPFFG--GEERTSAELRLKN--VPIVSVESLDWHWKAYLPEGANRDHPSCNI- 257 PpGID1L2 LKGVVIIQGFFG--GEERTPAELRLKN--VPLVSVESLDWYWKSYLPKGSNRDHPACNI O7R IKGLVLDEPGFG--GSKRTGSELRLAN--DSRLPTFVLDLIWELSLPMGADRDHEYCN P 248 2C7B VKKQVLIYPVVNM-TGVPTASLVEFGVAETTSLPIELMVWFGRQYLKRPEEAYDFKAS-P 232 1JJI IKHQILIYPVVN--FVAPTPSLLEFG-EGLWILDQKIMSWFSEQYFSREEDKFNPLAS-V 236 1EVQ LAFQLLIYPSTGYDPAHPPASIEENAEGY--LLTGGMMLWFRDQYLNSLEELTHPWFSPV OsGID1 FGP--NGRRLGGLP----FAKSLIIVSGLDLTCDRQLAYADALREDGHHVKVVQCENATV 326 AtGID1a FSPRGKSLEGVSFP------KSLVVVAGLDLIRDWQLAYAEGLKKAGQEVKLMHLEKATV 319 AtGID1b FGPRGQSLKGVNFP------KSLVVVAGLDLVQDWQLAYVDGLKKTGLEVNLLYLKQATI 319 AtGID1c FGPRSKSLEGLSFP------KSLVVVAGLDLIQDWQLKYAEGLKKAGQEVKLLYLEQATI 317 GhGID1a FGPRGRSLEGLKFP------KSLVVVAGLDLIQDWQLAYVEGLKKSGQEVNLLFLEKATI 318 GhGID1b FGPRGRTLEGLKFP------KSLVVVAGLDLIQDWQLAYVEGLKKSGQEVKLLFLEKATI 326 SmGID1a FGPGSDAERVLGEIPVP---KSLVVVAGLDLTQDWQLRYARGMERSGKSVEVLVLEDTPV 322 SmGID1b FGPSSRSLEGVVLP------PSLVAVAGLDMIKDWQLQYVEGMRNAGKDVELLFLEEATV 329 PpGID1L1 FGPNSPDLSDVPLP------PILNIVGGLDILQDWEMRYSEGMKKAGKEVQTIFYEEGIH 311 PpGID1L2 FGPNSSDLSDVSLP------PFLNIVGGLDILQDWEMRFAEGLQKAGKQVQTIFYEEGIH 318 2O7R TAE--SEPLYSFDKIRSLGWRVMVVGCHGDPMIDRQMELAERLEKKGVDVVAQFDVGGYH 306 2C7B LLA--DLGG LPPALVVTAEYDPLRDEGELYAYKMKASGSRAVAVRFAGMVH 281 1JJI I-----FADLEN------LPPALIITAEYDPLRDEGEVFGQMLRRAGVEASIVRYRGVLH 285 1EVQ LYP--DLSG LPPAYIATAQYDPLRDVGKLYAEALNKAGVKVEIENFEDLIH OsGID1 GFYLLP-NTVHYHEVMEEISDFLNANLYY AtGID1a GFYLLP-NNNHFHNVMDEISAFVNAEC AtGID1b GFYFLP-NNDHFHCLMEELNKFVHSIEDSQSKSSPVLLTP AtGID1c GFYLLP-NNNHFHTVMDEIAAFVNAECQ GhGID1a GFYFLP-NNNHFYCLMEEIKNFVNPNC GhGID1b GFYFLP-NNDHFYRLMEEMNNFVHSNC SmGID1a GFFIFP-NTEQYYRVMDKIRGFV-RDEQEPMDSST SmGID1b GFFIFP-NTGHFHRLMDKITAFIDRDGDQHLTRRTGITRRTTT 371 PpGID1L1 TFALLN-QAKLASQMLLDVAAFINSH PpGID1L2 TFALLN-QAKVGPKMFLDVAAFINSH O7R AVKLE-DPEKAKQFFVILKKFVVDSCTTKLKLN C7B GFVSFYPFVDAGREALDLAAASIRSGLQPS JJI GFINYYPVLKAARDAINQIAALLVFD EVQ GFAQFYSLSPGATKALVRIAEKLRDALA Supplementary Fig. 9-2 M.Matsuoka et al.