Supplementary Figure S1 (+)ABA NO (+)ABA Excess refractive index (x10 8 ) Molecular mass (kDa) 50±1...

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upplementary Figure S1 (+)ABA NO (+)ABA Excess refractive index (x10 8 ) Excess refractive index (x10 8 ) Molecular mass (kDa) 14 16 18 20 0 300 600 900 1200 1500 1800 50±1 33±1 Excess refractive index (x10 8 ) 14 16 18 20 1 10 100 Molecular mass (kDa) 37±2 49±1 59±2 33±3 PYL1 ΔNHAB1 Molecular mass (kDa) PYL1 PYL1-HAB1 14 16 18 20 0 100 200 300 400 500 600 23±1 23±1 Excess refractive index 14 16 18 20 0 200 400 600 800 1000 22±2 22±1 14 16 18 20 1 10 100 43±4 52±1 62±2 43±3 PYL6 ΔNHAB1 14 16 18 20 1 10 100 40±2 32±1 59±2 38±1 PYL8 ΔNHAB1 PYL6 PYL6-HAB1 PYL8 PYL8-HAB1 Dupeux et al., 2011 PYL6: 24kDa PYL1: 25.5kDa Elution volume (ml) Elution volume (ml) PYL1 ΔNHAB1 PYL1 ΔNHAB1 PYL8 ΔNHAB1 PYL8 ΔNHAB1 PYL6 ΔNHAB1 PYL6 ΔNHAB1 ABA receptors exist in dimeric and monomeric forms. SEC-MALLS analysis of PYL1, PYL6 and PYL8 alone (left panels) and in the presence of ΔNHAB1 (right panels). The experiments were done in the absence (blue) and presence (red) of 1mM (PYL6 & PYL8) or 5mM ( PYL1) (+)ABA. The apparent size of PYL6 and PYL8 indicates that they are monomeric both in the presence and absence of (+)ABA. PYL1 is dimeric in the absence of ABA and addition of 5mM ABA produces partial dissociation. All receptor proteins tested in this study form 1:1 complexes when combined with ΔNHAB1 in the presence of (+)ABA (right panels). However, while dimeric proteins including PYL1 and PYR1 (see figure 2 in main text) do not interact with ΔNHAB1 in the absence of (+)ABA the formation of less stable complexes between monomeric receptors PYL6, PYL8 and ΔNHAB1 in the same conditions is revealed by a decrease in the height of the peaks corresponding to monomeric ΔNHAB1 and the appearance of new peaks containing both receptor proteins and ΔNHAB1 (figure 2 in main text shows similar experiments for PYR1 and PYL5). PYL8: 21.5kDa ΔNHAB1: 37kDa

Transcript of Supplementary Figure S1 (+)ABA NO (+)ABA Excess refractive index (x10 8 ) Molecular mass (kDa) 50±1...

Page 1: Supplementary Figure S1 (+)ABA NO (+)ABA Excess refractive index (x10 8 ) Molecular mass (kDa) 50±1 33±1 Excess refractive index (x10 8 ) Molecular mass.

Supplementary Figure S1

(+)ABA

NO (+)ABA

Exc

ess

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(x1

08)

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50±133±1

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ess

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14 16 18 20

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lecu

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37±249±159±2

33±3

PYL1

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Mo

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PYL1 PYL1-HAB1

14 16 18 20

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23±123±1

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43±452±162±243±3

PYL6

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14 16 18 20

1

10

100

40±232±159±238±1

PYL8

ΔNHAB1

PYL6 PYL6-HAB1

PYL8 PYL8-HAB1

Dupeux et al., 2011

PYL6: 24kDa

PYL1: 25.5kDa

Elution volume (ml) Elution volume (ml)

PYL1

ΔNHAB1

PYL1

ΔNHAB1

PYL8

ΔNHAB1

PYL8

ΔNHAB1

PYL6

ΔNHAB1

PYL6

ΔNHAB1

ABA receptors exist in dimeric and monomeric forms. SEC-MALLS analysis of PYL1, PYL6 and PYL8 alone (left panels) and in the presence of ΔNHAB1 (right panels). The experiments were done in the absence (blue) and presence (red) of 1mM (PYL6 & PYL8) or 5mM ( PYL1) (+)ABA. The apparent size of PYL6 and PYL8 indicates that they are monomeric both in the presence and absence of (+)ABA. PYL1 is dimeric in the absence of ABA and addition of 5mM ABA produces partial dissociation. All receptor proteins tested in this study form 1:1 complexes when combined with ΔNHAB1 in the presence of (+)ABA (right panels). However, while dimeric proteins including PYL1 and PYR1 (see figure 2 in main text) do not interact with ΔNHAB1 in the absence of (+)ABA the formation of less stable complexes between monomeric receptors PYL6, PYL8 and ΔNHAB1 in the same conditions is revealed by a decrease in the height of the peaks corresponding to monomeric ΔNHAB1 and the appearance of new peaks containing both receptor proteins and ΔNHAB1 (figure 2 in main text shows similar experiments for PYR1 and PYL5).

PYL8: 21.5kDa

ΔNHAB1: 37kDa

Page 2: Supplementary Figure S1 (+)ABA NO (+)ABA Excess refractive index (x10 8 ) Molecular mass (kDa) 50±1 33±1 Excess refractive index (x10 8 ) Molecular mass.

Supplementary Figure S2 Dupeux et al., 2011

10.2 9.8 9.4

125

120

115

110

1

- 15

N (

pp

m)

10.2 9.8 9.4 10.2 9.8 9.4

2 - 1 H (ppm)

10.2 9.8 9.4 10.2 9.8 9.4

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120

115

110D 5 3

T 1 2 5

T 1 2 4

a

b

[A B A ]/[P Y R 1 ]

p boun

d

0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 3 .00 .0

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1 .0T 1 2 5

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B

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15N

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m)

2- 1H (ppm)

p boun

d

[ABA] / [PYR1]

Determination of the dissociation constant, Kd, of the PYR1:ABA complex by solution NMR. (a) Part of 1H-15N HSQC spectra of PYR1 with increasing amounts of ABA. The spectra correspond to the following [ABA]/[PYR1] ratios: 0.00 (red), 0.23 (blue), 0.47 (magenta), 0.70 (green), 2.59 (black). As the concentration of ABA increases, two resonances of D53, T124 and T125 are visible corresponding to free and ABA-bound PYR1, respectively. The assignment of the resonances was taken from Melcher et al. 2010. (b) Determination of the Kd value from the intensities of the double resonances of T125 (Kd=847M) as described in Materials and Methods. Filled circles indicate experimental points, while the solid line corresponds to the least squares fit of Eq. 1. A weighted average over all three residues yields Kd=9736M.

Page 3: Supplementary Figure S1 (+)ABA NO (+)ABA Excess refractive index (x10 8 ) Molecular mass (kDa) 50±1 33±1 Excess refractive index (x10 8 ) Molecular mass.

Pro60

His60(wt)

Phe61

Lys51

Supplementary Figure S3 Dupeux et al., 2011

Omit electron density map around proline 60 of the PYRH60P-ABA-HAB1 complex structure. The electron density confirms the substitution of histidine by proline at position 60. The structure of wt PYR has been superimposed on that of PYR1H60P to help in the interpretation)

Page 4: Supplementary Figure S1 (+)ABA NO (+)ABA Excess refractive index (x10 8 ) Molecular mass (kDa) 50±1 33±1 Excess refractive index (x10 8 ) Molecular mass.

Supplementary Figure S4 Dupeux et al., 2011

B. Sequence variation in regions determining the oligomeric structure of PYR/PYL proteins. Multiple sequence and secondary structure alignment of selected regions containing amino acids involved in ABA binding (red squares) and dimer formation (for PYR1,PYL1 and PYL2 , green squares). PYR1, PYL1 and PYL2 are dimeric proteins while PYL5,PYL6 and PYL8 are monomeric. The structural and biochemical analysis of the PYRH60P mutant indicates that other proteins containing proline at the equivalent position, like PYL7, PYL9 and PYL10 are likely to be monomeric and show high intrinsic affinity for ABA.

A. Structural proximity of amino acids involved in ABA binding and dimerization . Multiple sequence and secondary structure alignment of selected regions of dimeric PYR/PYL proteins. Residues involved in ABA binding (red squares) and dimer formation (green squares) are indicated.

PYR1

PYR1PYL1

PYL2

60 70 80 90 150