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Supplementary Figure S1
Dupeux et al., 2011
PYL8: 21.5kDa
NO (+)ABA
PYL6: 24kDa
ΔNHAB1: 37kDa
(+)ABA
PYL1
PYL1-HAB1
50±1
1500
59±2
33±1
49±1
37±2
33±3
ΔNHAB1
1200
PYL1
ΔNHAB1 10
900
ΔNHAB1
600
PYL1
PYL1
300
1
0
16
18
14
20
16
PYL6
600
18
20
PYL6-HAB1
62±2
23±1
400
43±4
43±3
52±1
23±1
ΔNHAB1
ΔNHAB1
PYL6
300
10
PYL6
ΔNHAB1
200
PYL6
100
0
14
16
18
20
14
PYL8
16
59±2
22±2
22±1
1
20
18
PYL8-HAB1
1000
800
100
32±1
40±2
38±1
ΔNHAB1
PYL8
600
ΔNHAB1
400
200
10
PYL8
ΔNHAB1
PYL8
1
0
14
16
Molecular mass (kDa)
100
500
Excess refractive index (x108)
Excess refractive index (x108)
Molecular mass (kDa)
100
1800
14
Excess refractive index (x108)
PYL1: 25.5kDa
18
Elution volume (ml)
20
14
16
18
Elution volume (ml)
20
Molecular mass (kDa)
Excess refractive index (x108)
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).
Supplementary Figure S2
Dupeux et al., 2011
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=847M) 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=9736M.
aA
D53
110
115
115
- 15 N (ppm)
w1-15N (ppm)
110
1
T125
120
120
125
10.2
125
T124
9.8
9.8
10.2
9.4
9.4
10.2
9.4
9.8
10.2
w2- 1H (ppm)
2-
bB
1
H (ppm)
1.0
T125
0.6
bound
p
pbound
0.8
0.4
0.2
0.0
0.0
0.5
1.0
1.5
2.0
[ABA] / [PYR1]
[ABA]/[PYR1]
2.5
3.0
9.8
9.4
10.2
9.8
9.4
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)
Phe61
Lys51
His60
(wt)
Pro60
Supplementary Figure S4
Dupeux et al., 2011
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
60
70
80
90
150
PYR1
PYL1
PYL2
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.