RCAR1.

Mechanism of high-affinity abscisic acid binding to PYL9/RCAR1 Masahiro Nakagawa†, Megumi Kagiyama†, Nobuyuki Shibata, Yoshinori Hirano and Toshio Hakoshima* Structural Biology Laboratory, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan

Arabidopsis receptors of abscisic acid (ABA), the key plant hormone for adaptation to water stress, comprise 14 PYR/PYLs/RCARs proteins classified into three subfamilies I, II, and III, which suggests functional differentiation. Although their monomer–dimer equilibria may be correlated with differences in their ABA-binding affinities, how the dimerization decreases the affinity is unclear. Comparative structural and binding studies between PYL9, which is a representative of high-affinity subfamily I, and low-affinity members of subfamily III reveals that the nonpolar triplet (Ile110, Val162, and Leu165) and Pro64 contribute to enhance ABA-binding affinity by inducing a shift of the ABA carboxyl group to form additional direct hydrogen bonds with conserved Asn169. Our mutation studies of PYL1 successfully produced a monomeric mutant PYL1 exhibiting low ABA affinity and also a dimeric mutant PYL1 exhibiting high ABA-binding affinity, suggesting that dimer formation of ABA receptors is not essential for their low ABA-binding affinity. Our study contributes toward establishing the structural basis for the higher ABA-binding affinity of the subfamily receptors and provides a clue for understanding the broad spectrum of hormone actions in plants manifested by the different hormone-binding affinity of multiple receptors.

Introduction Recent advances in plant hormone signaling have focused on understanding the molecular and structural levels of receptors (Santner & Estelle 2009). The first breakthrough was achieved by the crystal structure determination of the Arabidopsis auxin receptor TIR1 in complex with auxin and the effector-protein Aux/IAA peptide (Tan et al. 2007), followed by the structural determination of the Arabidopsis gibberellin receptor GID1 in the ternary complex form with gibberellin (GA) and the effector DELLA protein (Murase et al. 2008). These studies revealed at atomic resolution that plant hormone perception mechanisms comprise unique switching mechanisms, such as the molecular glue mechanism of TIR1 proposed for auxin signaling and the closing lid mechanism of GID1 for GA signaling. The following study has shown that the jasmonate receptor COI1 perceives Communicated by: Masao Tasaka *Correspondence: [email protected] † These authors equally contributed to this work.

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jasmonate (JA) and its effector protein JAZ by a molecular glue mechanism similar to that of TIR1 (Sheard et al. 2010). Recent crystallographic studies have provided structural insight into brassinosteroid perception by BRI1 (Hothorn et al. 2011a; She et al. 2011) and cytokinin recognition by Arabidopsis thaliana histidine kinase 4 (Hothorn et al. 2011b). Abscisic acid (ABA) is another vital plant hormone that plays key regulatory roles in physiological pathways required for the adaptation of vegetative tissues to abiotic stresses such as water stress as well as for plant growth and development (Finkelstein et al. 2002; Yamaguchi-Shinozaki & Shinozaki 2006). Like auxin and GA receptors, identification of the ABAreceptor and delineation of the ABA perception mechanism by the receptor remained elusive for a long time. As the discovery of ABA (Ohkuma et al. 1963; Addicott et al. 1968), although ABA-receptor candidates such as FCA, ABAR/CHLH/GUN5, GCR2, GTG1, and GTG2 have been reported, but there is no evidence that the previously identified and established positive and negative regulators of ABA signaling, such the PP2C protein phosphatases

DOI: 10.1111/gtc.12140 © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

High-affinity ABA binding to PYL9

ABI1, ABI2, and HAB1, are regulated by these proposed receptors (Pennisi 2009). Two groups reported on a family of small soluble proteins that perceive ABA and repress the key negative regulators ABI1, ABI2, and HAB1 (Ma et al. 2009; Park et al. 2009). This family contains 14 members in Arabidopsis, regulatory component of ABA-receptor 1-14 (RCAR114), also referred to as pyrabactin resistance 1 (PYR1) and PYR1-like 1-13 (PYL1-PYL13). These members belong to a family of proteins that contain a START (steroidogenic acute regulatory protein-related lipid transfer) domain (Iyer et al. 2001), but also belong to the Bet v 1 superfamily containing the birch pollen allergen Bet v 1a (Radauer et al. 2008). Unlike other plant hormone receptors that contain only a single or few members, the fact that ABAreceptors contain multiple members implies functional differentiation of ABA-receptor members. Interestingly, the PYR/PYLs/RCARs members are classified into three subfamilies based on amino acid sequence identity: subfamily-I comprises 4 members (PYL7-10/RCAR1-4), subfamily-II 6 members (PYL4-6/RCAR8-10 and PYL11-13/RCAR5-7) and subfamily-III 4 members (PYR1//RCAR11 and PYL1-3/RCAR12-14) (Park et al. 2009). Three members (PYL4-6/RCAR8-10) of subfamily-II may be classified into subfamily-III in the neighbor-joining tree (Ma et al. 2009). These members are hereafter referred to as PYR/PYLs. The presence of that many members in a single family suggests that each subfamily may exert different functions by binding to distinct downstream target proteins such as the PP2C protein phosphatase members. Some members may activate their common downstream targets with different strength. Intriguingly, recent quantitative data suggest that three members of subfamily-I, PYL9 (Ma et al. 2009), PYL5 (Santiago et al. 2009a) and PYL8 (Szostkiewicz et al. 2009), display 50- or 90-fold strong affinity, respectively, to ABA with smaller dissociation constant KD values (~1 lM) compared with subfamily-III members PYR1, PYL1, and PYL2 (Ma et al. 2009; Yin et al. 2009). Indeed, some receptor complexes have been shown to differ in their ABA stereo-selectivity and sensitivity (Santiago et al. 2009a; Szostkiewicz et al. 2009). Thus, it is important to clarify the structural differences between members of the three subfamilies to further our understanding of the functional differences displayed by multiple ABA receptors in plants. Recent crystal structures of PYR1, PYL1, and PYL2 reported by five groups have succeeded in delineating a gate-latch-lock mechanism pertaining to

ABA perception and effector recognition (Melcher et al. 2009; Miyazono et al. 2009; Nishimura et al. 2009; Santiago et al. 2009b; Yin et al. 2009). These structures have clarified precise ABA recognition in the deep binding pocket of the receptors and the mechanisms by which ABA-bound receptors inhibit PP2C protein phosphatases by docking into the PP2C active site and forming a water-mediated hydrogen bond between the key tryptophan residue from a PP2C loop and the receptor-bound ABA molecule (Melcher et al. 2009; Miyazono et al. 2009; Yin et al. 2009). Interestingly, PYR1, PYL1 and PYL2 have been shown to form dimers in solution, yet they bind to PP2Cs as monomers. PYR/PYLs proteins could be separated into two distinct subclasses according to the oligomeric state of their apo forms, high-affinity monomeric and low-affinity dimeric forms (Dupeux et al. 2011; Hao et al. 2011). This observation implies a model in which ABA binding to dimeric PYR/PYLs triggers dissociation of the dimer so that binding to the PP2C can occur (Yin et al. 2009). Thermodynamic switch control of receptor oligomerizatoin involving ABA-bound and unbound states can modulate hormonal responses and more generally, the sensitivity of a ligand-dependent signaling system (Dupeux et al. 2011). In addition to the complexity in the multistate equilibrium, PLY10 has been shown to bind PP2C in the absence of ABA (Hao et al. 2011) and PLY3 contains a trans-homodimer in addition to the common cis-homodimer (Zhang et al. 2012). The monomer/dimer equilibrium found in a subset of PYR1/PYL members seems to be correlated with differences in their ABA-affinities. However, details of the mechanisms by which the dimerized PYR/PYLs reduce their affinity for ABA and the monomeric receptors gain a higher affinity to ABA than the dimeric receptors are still unknown. Unfortunately, no structure of the high-affinity receptor bound to ABA has been reported and provides no insight into the way in which members of the three different subfamilies differ structurally and functionally. Here, we report a biophysical and mutational characterization of PYL9 in comparison with PYL4 and PYL5 (subfamily-II), and PYRl (subfamily-III) with the high-resolution three-dimensional structure of PYL9 (subfamily-I) bound to (+)-ABA and its detailed comparison with previously reported structures of subfamily-III. Our study contributes toward establishing the structural basis for the higher ABA affinities of subfamily-I members and provides a clue

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for understanding the broad spectrum of hormone actions in plants manifested by the different hormone-binding affinity of multiple receptors.

deviation in the flexible N-terminal a1-helix orientation and no significant structural differences in the overall structure (Fig. 1D).

Results

Lid loops of PYL9

Structure determination

PYL9 is a prototype ABA receptor that displays strong affinity to (+)-ABA (hereafter referred to as ABA) with a small KD value of 0.66 lM (Ma et al. 2009). The crystallization of PYL9 in the presence of ABA successfully yield diffraction-quality crystals. The crystal structure was determined and refined at 1.9 A resolution to an Rfree value of 20.4% and Rwork value of 18.8%. The crystal contains one PYL9 molecule in the asymmetric unit. The map obtained showed clear electron density for most of the protein residues and the ABA–bound molecule, whereas no models were built for 12 N-terminal and 5 C-terminal residues, which were not observed in the electron density map. Overall structure of PYL9

The PYL9 structure displays a Bet v 1 a + b fold with an N-terminal extension of 32 residues that contains a1 helix associated with the following b1 strand (Fig. 1A,B). The PYL9 core domain consists of seven b-strands (b1–b7) forming a bent antiparallel b-sheet that produces a central cavity in which the ABA-bound molecule exhibits an extended conformation of 10.4 A with the cyclohexenone ring being oriented toward the cavity entrance and the tail formed by the diene and carboxyl groups being oriented inward. The prominent long C-terminal ahelix (a3) is associated with the b-sheet to form a wall that completes the cavity. Short a- and 310-helices (a2 and g1, respectively) between strands b1 and b2 seal the one side (bottom) of the cavity. The other (top) side of the cavity, representing the entrance to the cavity, is covered by three loops; the loop between b3 and b4 (b3–b4 loop), b5-b6 loop and b7-a3 loop (Fig. 1B,C). These structural characteristics of the overall structure of PYL9 resemble to the previously reported structures of subfamily-III members, PYR1, PYL1, and PYL2 (Melcher et al. 2009; Miyazono et al. 2009; Nishimura et al. 2009; Santiago et al. 2009b; Yin et al. 2009). The overall root mean square (rms) deviation of ABA-bound forms of PYL9 and PYR1 (Nishimura et al. 2009) is 2.2 A for 164 Ca carbon atoms, revealing limited 388

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Three loops, b3–b4, b5–b6, and b7–a3, that form the pocket entrance possess highly conserved sequences and have been shown to execute a large conformational change upon transition from the ABA-free to ABA-bound states (Melcher et al. 2009; Miyazono et al. 2009; Nishimura et al. 2009; Santiago et al. 2009b; Yin et al. 2009). b3-b4 loop contains the conserved motif SGLPA, with the exception of PYL12 and PYL13 of subfamily-II, and is referred to as Pro-cap, gate or CL2 loop in previous reports. b5–b6 loop contains the xHRLxNYxS motif (where x represents any amino acid) and is alternately referred to as Leu-lock, latch or CL3 loop. The ABA-induced conformational change is referred to as a cap-lock (Nishimura et al. 2009) or gate-latch-lock (Melcher et al. 2009) mechanism for hormone signaling. On binding to ABA, b3–b4 loop forms a closed cap or gate, and b5–b6 loop locks or latches the closed cap/gate by making intimate contacts with Pro-cap/gate (b3–b4) loop (Fig. 1B). In addition to these two loops, b7–a3 loop, which is referred to as a Recoil motif (Nishimura et al. 2009), induces a conformational change to interact with Pro-cap/gate loop. Pro-cap/gate and Leu-lock/latch loops of PYL9 essentially display the same conformations as the closed form of subfamily-III members (PYR1, PYL1 and PYL2) bound to ABA (Fig. 1D). Notably, we observed a water molecule that bridges Pro-cap/ gate and Leu-lock/latch loops by hydrogen bonding to both the main-chain carbonyl and amide groups of Pro90 and Arg118, respectively (Fig. 1E). This water molecule is also present in the PYL1-HAB1 and PYL2-ABI1 complexes and plays an essential role in interactions with the target PP2C enzyme by hydrogen bonding to the key Trp residue from PP2C (Melcher et al. 2009; Miyazono et al. 2009; Yin et al. 2009). A similar water molecule was also observed in the ABA-PYR1 structure (Santiago et al. 2009b) (Fig. 1F). In our crystal, one PYL9 molecule exists in the crystallographically asymmetric unit. This molecule makes direct contacts with the adjacent molecule that is related by a crystallographic twofold axis (Fig. 2A). These two molecules make contact with each other through the C-terminal long helices a3 with relatively small interface of 1250 A2, which is in contrast


High-affinity ABA binding to PYL9 (A)

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Figure 1 Overall structure of the PYL9-ABA complex. PYL9 displays a core domain of the Bet v 1-fold consisting of seven b-strands (b1–b7) forming a bent antiparallel b-sheet that produces a central cavity. (A) A view of PYL9 (green) in ribbon representation with the bound abscisic acid (ABA) molecule as a space-filled model (carbon and oxygen atoms in yellow and red, respectively). (B) A view rotated by 180° from that in A. Three loops, b3–b4 (Pro-cap/gate), b5–b6 (Leu-lock/latch) and b7–a3 (Recoil), which possess highly conserved sequences, cover the ABA-binding pocket. (C) Molecular surface representation with the same view as in B with the bound ABA (stick model) located at the mouth of the deep ABA-binding pocket. (D) Structural comparison of PYL9 with PYR1 (subfamily III). PYL9 (gray) is superimposed onto PYR1 (magenta, PDB ID 3K3K). The Bet v 1 domains superimpose well with a small average rms deviation of 2.2 A over the Ca carbon atoms. A similar superposition was obtained for PYL1 and PYL2. (E) Close-up views of the carbonyl group of the ABA cyclohexenone bound to PYL9 in this study. Hydrogen bonds are shown with broken lines. The water molecule and the ABA molecule are shown as a red ball and a stick model (green), respectively. (F) As in E, but for the PYR1-ABA complex (3K3K).


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Figure 2 Results of analytical ultracentrifugation of PYL9 and related abscisic acid (ABA) receptors. (A) The PYL9 pair found in our crystal structure. The second molecule of PYL9 is rotated by 100° from the corresponding position of PYL1 from subfamilyIII shown in B. (B) The PYL1 dimer in the crystal (PDB ID 3JRS). Bound ABA molecules are shown as space-filled models. (C) The distribution of apparent molecular mass obtained from sedimentation velocity analysis of the ABA-free form of PYL9. The single peak was found at 23.1 kDa, suggesting the presence of a monomer in solution. (D) As in C, but for PYL9 bound to ABA. The single peak is found at 23.7 kDa, suggesting the presence of a monomer in solution.

with larger interfaces (1400 A2 for PYR1 or 1766 A2 for PYL2) observed in the dimers of subfamily-III members (Melcher et al. 2009; Miyazono et al. 2009; Nishimura et al. 2009; Santiago et al. 2009b; Yin et al. 2009) (Fig. 2B). The smaller contact area in PYL9 is caused by a large shift of the tilt angle against the dyad axis. Compared with the PYR1 dimer, the second protomer of PLY9 is rotated by ~100° relative to that of PYR1. The dimerized structures of previously reported subfamily-III members are all similar, whereas the PYL9 dimer in our crystal is distinct from any of those previously reported structures of subfamily-III members. Our analytical ultra-centrifugation (AUC) and gelfiltration analysis indicated that PYL9 exists as a monomer in solution in the presence or absence of ABA (Fig. 2C,D), which are consistent with the previous reports, which showed members of subfamily-I (PYL6, PYL8, PYL9) and subfamily-II (PYL4, PYL5, PYL6) exist as monomers, but members of subfamilyIII (PYR1, PYL1, PYL2) as dimers (Nishimura et al. 2009; Santiago et al. 2009b; Szostkiewicz et al. 2009; Yin et al. 2009; Dupeux et al. 2011; Hao et al. 2011). However, apo-PYL1 and PYL3 were determined to 390

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exist in a monomer-dimer equilibrium in solution (Melcher et al. 2009; Hao et al. 2011). In the dimers of these subfamily-III members, the Pro-cap/gate loop of one protomer protrudes toward the other and contacts with three loops, Pro-cap/gate, Leu-lock/ latch and Recoil of the other protomer. These intimate interprotomer interactions involving three loops are modified in our PYL9 structure as described below. ABA perception

PYL9 accommodates one ABA molecule and 13 water molecules inside the hormone-binding cavity between the twisted seven-stranded b-sheet and the C-terminal long a3-helix. The presence of multiple hydrated water molecules inside the cavity is in common with reported structures of subfamily-III members. The ABA-bound molecule is almost buried inside the PYL9 cavity. The cavity is larger than the ABA molecule, while the overall molecular shape and polar and nonpolar surfaces of the ABA-bound molecule match those of the cavity and hydrated water molecules that stabilize the ABA-bound



molecule by multiple hydrogen bonds which bridge the ABA molecule and protein residues inside the cavity (Fig. 3A). The extended ABA conformation is stabilized with two water molecules by forming water-mediated hydrogen bonds between the cyclohexenone ring and the terminal carboxyl group. These water molecules are members of a cluster of four hydrated water molecules forming a hydrogen-bonding network that is linked to polar residues (Lys63, Glu96, Tyr122, Glu143, and Asn169). The tail isoprenoid moiety is found in a plane that is nearly orthogonal to the ring plane of the cyclohexenone, which displays a puckered conformation with the dimethylated ring carbon (at C60 ) being off-plane. The ABA cyclohexenone ring is located at the upper part of the cavity and makes contacts with Pro-cap/gate and Leu-lock/latch loops and a hydrogen bond between the ring carbonyl group and the water molecule that bridges these two loops (Fig. 1E). Importantly, the cyclohexenone ring contributes to stereo-specific binding by direct contacts with nonpolar residues of the upper part of the cavity (Fig. 3B). Pro-cap/gate and Leu-lock/latch loops provide nonpolar residues to participate in these contacts and play essential roles in stereo-specific binding of the ABA hydrocarbon portion. One methyl group of the ABA dimethyl group makes nonpolar contacts with two residues (His117 and Leu119) from Leulock/latch loop and one (Ser94) from b4 strand, and the other methyl group with one residue (Ala91) from the Pro-cap/gate loop and two residue (Val85 and Ser94) from the loop base. The mono-methyl group of the ABA ring also makes contacts four residues, two from the Pro-cap/gate loop and base (Leu89 and Val85, respectively) and two from loop g1-b2 and a3 helix (Phe65 and Leu165, respectively). In addition to the cyclohexenone ring, the methyl group of the ABA isoprenoid moiety makes contacts with two latch residues (Leu119 and Tyr122) and two residues (Val162 (Ala160 in PYR1), Leu165 (Val163 in PYR1)) from a3 helix. Thus, the ABA tail isoprenoid also contributes to stereo-specific binding by direct contacts with protein residues. The most conspicuous contacts are mediated by the ABA terminal carboxyl group, which is anchored to the conserved Lys63 residue from loop g1-b2 at the inward floor by forming a salt bridge with two direct hydrogen bonds (Fig. 3C). Moreover, the carboxyl group forms two direct bifurcated hydrogen-bonding interactions with the conserved Asn169 residue from a3 helix. In our complex between PYL9 and ABA, this carboxyl group is the only ABA

(A)

(B)

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Figure 3 Abscisic acid (ABA) perception by PYL9. (A) Summary of intermolecular interactions between ABA and PYL9 in our complex structure. Van der Waals contacts (dotted lines), hydrogen bonds and salt bridges (blue arrows) are shown. Val162 and Leu165 are highlighted in yellow. (B) ABA recognition by nonpolar contacts. The nonpolar residues of the protein are shown as stick models (brown) with van der Waals surfaces (dots) and the secondary structures shown in green. The stick model of the bound ABA molecule is highlighted using the same color codes as in Fig. 1A. (C) Polar interactions (dashed lines) between ABA (yellow) and PLY9 at the bottom of the binding pocket. The distances between atoms are shown in angstroms.

group that participates in direct polar interactions with protein residues. Thus, the carboxyl group plays a key role in the stabilization of ABA within the


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cavity. At the bottom of the cavity, an additional cluster of eight water molecules occupy the space and form a water-mediated hydrogen-bonding network involving the ABA carboxyl group and inward polar residues (Tyr122, Ser124 and Glu143) in addition to two residues (Lys63 and Asn169) that directly interact with the ABA carboxyl group. Comparison of ABA binding with subfamily-III members

PYL9 and PYL8 displayed a higher affinity to ABA with a reported KD value of 0.66 lM (Ma et al. 2009) and 0.97 lM) (Szostkiewicz et al. 2009), respectively. These KD values are extremely small compared with the reported values of 52 lM for PYL1 (Miyazono et al. 2009) and 59 lM for PYL2 (Yin et al. 2009). Moreover, the KD value for PYR1 was reported to be too low to be measured with conventional techniques such as surface plasmon resonance (SPR) measurements (Nishimura et al. 2009) and has been estimated to be at least larger than 50 lM by isothermal titration calorimetry (ITC) experiments (Dupeux et al. 2011). We compared our PYL9 structure with reported structures of subfamily-III members. As the ABA-binding modes of the reported PYR1, PYL1 and PYL2 structures are essentially the same, we focused on a structural comparison between PYL9 and PYR1 and, when necessary, made comparisons with PYL1 and PYL2. We compared the PYL9 sequence with other members of the Arabodopsis ABA receptors. Partial sequence alignment of the ABA-binding regions is shown in Fig. 4. Compared with PYR1, PYL9 possesses three nonconserved residues, Ile110, Val162, and Leu165, inside the ABA-binding cavity (Fig. 5A,B). These three residues are replaced with Phe108, Ala160, and Val163 in PYR1. Our close inspection of these residues has clarified that Leu165 plays a key role in fine-tuning the position and orientation of the ABA-bound molecule inside the PYL9 cavity by fitting the hydrocarbon side chain of Leu165 to the puckered cyclohexenone moiety of ABA (Fig. 5A,C). This intimate contact enables the ABA terminal carboxyl group to form direct bifurcated hydrogen bonds to Asn169 in addition to Lys63 as described previously. Val162 assists Leu165 in fitting the side chain to the ABA cyclohexenone moiety by contacting the methyl group (C6) of the ABA isoprenoid moiety, while Ile110 makes no direct van der Waals contacts with the ABA 392

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molecule. Three water molecules participate in forming the hydrogen bond network involving ABA and Asn169 by forming water-mediated hydrogen bonds (Fig. 5D). In sharp contrast to Leu165 of PYL9, Val163 of PYR1 contacts with the methyl group (C70 ) protruding from the ABA cyclohexenone ring and prevents the ABA carboxyl group from being oriented toward Asn167 to form direct hydrogen bonds (Fig. 5E). Unlike Ile110 of PYL9, Phe108 makes direct contacts with the ABA carboxyl group, which may stabilize the orientation of the ABA carboxyl group. Compared with the ABA molecule bound to PYL9, the ABA molecule bound to PYR1 exhibits a small rotation (~5°) of ABA around the long molecular axis and a shift (~1 A˚) of ABA away from Asn167. Similar shifts are observed in PYL1. Compared with Val162 of PYL9, the corresponding residue Ala160 of PYR1 loosely packs with the methyl group of the ABA isoprenoid. This small residue may in part facilitate ABA rotation. Like PYR1, three water molecules participate in forming the hydrogen-bonding network involving ABA and the conserved Asn169 residue of PYL9, whereas their networks differ from one another (Fig. 5D,F). In the case of PYL9, both terminal atoms of the side chain of Asn169 participate in water-mediated hydrogen-bonding interactions, whereas in the case of PYR1, the Asn167 side chain is fixed by forming an intramolecular hydrogen bond with the His60 main chain, which prevents fine-tuning of the Asn side chain position to form direct hydrogen bonds to ABA. Thus, in PYR1, only the terminal amide N atom of Asn167 could participate in water-mediated hydrogen bonds to ABA. The intramolecular hydrogen bond between the conserved Asn side chain and the His main chain is conserved in PYL1 and PYL2. This hydrogen bond is absent in PYL9 given replacement of the His residue with a Pro residue (Pro64) which lacks the mainchain amide group as a hydrogen donor for hydrogen bond formation. A Pro residue at this position is conserved in all subfamily-I members and thus contributes to the high ABA-affinity of the members (Fig. 4A). PYL1 and PYL2 also lack direct hydrogen bonds to their conserved Asn residues (Melcher et al. 2009; Miyazono et al. 2009; Yin et al. 2009). Thus, the absence or presence of direct hydrogen-bonding interactions between the conserved Asn residue and the ABA terminal carboxyl group is one prominent difference in the ABA-receptor contacts between



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(C)

Figure 4 Sequence comparison of abscisic acid (ABA) receptors of subfasmilies I, II and III. Partial sequence alignment of the ABA-binding regions of Arabodopsis PYR/PRLs/RACRs. Secondary structures of PYL9 are shown at the top. The 14 members are grouped into the three subfamilies I, II and III according to the reported phylogenetic tree (13). Residues that interact with ABA are in bold and residues conserved throughout the 14 members are highlighted in gray. Residues characteristic for subfamily I members are highlighted in yellow, while residues characteristic for subfamily III members are in blue and other residues in pale yellow. Subfamily II members are divided into two groups. (A) The loop between g1 310-helix and b2-strand (g1-b2 loop) of PYL9 and other subfamily-I members contains KPF (Lys63, Pro64 and Phe65) sequence. Lys63 protrudes its side chain inside the ABA-binding pocket to interact with the ABA carboxyl group (Fig. 3C), whereas Pro64 and Phe65 protrude their side chains outside. The Pro residue is conserved in the subfamily-I, but varied in the other subfamilies. Large residues corresponding to Pro64 and Phe65 play a key role in forming a dimer of the subfamilies III members. (B) As in A, but for the aligned region contains a nonconserved residue, Ile110 of PYL9, that participate in forming the inside wall of the ABA-binding cavities. Ile110 is located in b5 strand. (C) As in A, but for the aligned region contains two non-conserved residues, Va162 and Leu165 of PYL9, that participate in forming the inside wall of the ABA-binding cavities. Va162 and Leu165 are located in a3 helix. The conserved asparagine residues (Asn167 of PYL9) that directly interact with ABA in PYL9 are highlighted in pink.

subfamily-I member PYL9 and subfamily-III members whose structures were determined. Our structural inspection suggests that small differences in the shapes of the ABA-binding cavities induce differences in the ligand-protein interactions and may cause the observed differences in ABA-binding affinity. To test this hypothesis, we attempted to convert PYR1 to a

high-affinity receptor by a triple mutation comprising replacement of Phe108, Ala160 and Val163 with the corresponding residues of PYL9, Ile, Val and Leu, respectively. Our optimized ITC measurements with injections of high concentrations of ABA showed a high KD value (~629 lM) for PYR1 (Fig. 6A). Compared with this high KD value of PYR1, the triple


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Figure 5 Comparison of the nonpolar triplet between PYL9 (subfamily-I) and PYR1 (subfamily III). (A) A top view of the nonpolar triplet residues, I110, V162 and L165, of PYL9 form the binding pocket. The triplet residues are shown as stick models with their van der Waals surfaces (dots). The bound abscisic acid (ABA) molecule (a space-filled model) makes intimate contacts with V162 and L165. (B) As in A, but for PYR1 (subfamily III). F108, A160, and V163 correspond to the nonpolar triplet residues (I110, V162, and L165) of PYL9. In triple mutant PYR (F108I, A160V, V163L), the nonpolar triplet of PYR1 is replaced with that of PYL9. The bound ABA molecule makes loose contacts with A160 and V163. (C) A side view of ABA bound to PYL9. The ABA molecule (green) and three non-conserved nonpolar residues, Ile110, Val162 and Ile165, are shown as stick models with van der Walls surfaces (dots). Direct hydrogen bonds from ABA to conserved Lys63 and Asn169 are shown with broken lines. Water molecules are omitted for clarity. (D) The hydrogen bond network involving conserved Asn169 of PYL9 is highlighted. Water molecules are shown as balls. (E) A side view of ABA bound to PYR1. The ABA molecule (orange) is shifted away from Asn167 by a close contact between the ABA methyl group (C70 ) and Val163 indicated with a red arrow. No direct hydrogen bonds are formed between the ABA carboxyl group and Asn167. Lack of direct hydrogen bonds to the conserved Asn residue is also seen in other subfamily III members, PYL1 and PYL2. (F) The hydrogen bond network involving conserved Asn167 of PYR1 is highlighted.

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mutation (F108I,A160V,V163L) in PYR1 protein exhibited enhanced affinity to ABA with a fivefold smaller KD value (Fig. 6B). Moreover, our quadruple mutation (H60P,F108I,A160V,V163L), which has additional mutation of His60 to a Pro residue found in PYL9. This mutation was expected to enable finetuning of the Asn167 side chain position to form direct hydrogen bonds to ABA by preventing formation of the hydrogen bond between the His60 main chain and the Asn167 side chain. The quadruple mutation successfully convert PYR1 to a high-affinity ABA receptor with a smaller KD value (2.87 lM) (Fig. 6C). In our structure, the carbonyl group of the ABA cyclohexenone ring is perceived through the watermediated hydrogen bond to both the Pro-cap/gate and Leu-lock/latch loops of PYL9 as mentioned above (Fig. 1E). In the ABA-PYR1 structure (Santiago et al. 2009b), the water molecule is hydrogen bonded to the Leu-lock/latch loop alone and not to the Pro-cap/gate loop (Fig. 1F). Moreover, PYR1 makes a weak (3.3 A or 3.4 A) but direct hydrogen bond between the main-chain amide group of Ala89 (corresponding to Ala91 of PYL9) of the Pro-cap/ gate loop and the ring carbonyl group of ABA. These differences seem to be coupled with a small difference in the peptide conformation of the Pro-cap/gate loop, inducing reorientation of the peptide bond between Pro90 and Ala91. Interestingly, the direct hydrogen bond between the ABA carbonyl group and the Ala main chain amide group is also observed in the ABA-PYL1 complex (Melcher et al. 2009), while it is absent in the ABA-PYL2 complex (Yin et al. 2009). Thus, in this case, formation of a direct hydrogen bond may not contribute to the difference in ABA-binding affinity observed between members of subfamilies I and III. Comparison of ABA binding between subfamily-I and II members

It is of interest to determine whether subfamily-II members exhibit strong or weak ABA-binding affinity. A recent report revealed a small KD value of 1.06 lM for PYL5, a member of the subfamily-II (Santiago et al. 2009a). Our ITC measurements showed that two other subfamily-II members, PYL4 and PYL6, exhibit relatively strong affinity to ABA with smaller KD values, 1.32 and 1.48 lM, respectively (Fig. 7A,B). The KD value for PYL6 is consistent with the previous report, in which PYL6 has been shown to exist as a monomer exhibiting a strong binding to ABA

(KD = 1.1 lM) (Dupeux et al. 2011). These subfamily-II members possess an Ile residue at the position of Leu165 of PYL9, suggesting that Ile may function in a manner similar to Leu of PYL9. Our modeling study suggests that, like Leu165 of PYL9, Ile could pack its hydrocarbon side chain with the ABA cyclohexenone ring and stabilize ABA at the position where the ABA carboxyl group could form direct hydrogen bonds to the conserved Asn residue. PYL4 has I182 corresponding to L165 of PYL9. We successfully prepared mutant PYL4(I182V) for its binding assay and found this mutation indeed lowering its affinity by ~4-fold (Fig. 7C). We also tried to perform a mutation study of PYL9 to examine contribution of the key residues to the ABA-binding affinity. However, the mutant PYL9 proteins with a single mutation (L165A) or multiple mutations such as (I110F, V162A, L165V) were poorly expressed, preventing our quantitative binding assay. Monomer/dimer equilibrium and ABA-binding affinity

PYL2 is a member of subfamily-III exhibiting a lowbinding affinity to ABA and forming a dimer in solution. Recently, it has been reported that a single mutation, I88K, converts this low affinity (58.5 lM) to a 7-fold higher affinity (a KD value of 8.2 lM), which exists as a monomer (Hao et al. 2011). Ile88 participates in formation of the dimer interface, suggesting that the I88K mutation modifies the dimer interface. However, Ile88 located in the Pro-Cap/ gate (CL2) loop is important for ABA binding, implying that the enhancement of ABA-binding affinity may be caused by artificial effects on the conformation and/or the physical properties such as flexibility of the Pro-Cap/gate loop. To resolve this difficulty, we tried to convert PYL1, another lowaffinity member of subfamily-III, to a monomer in solution using mutations of other residues located at the dimer interface but not at the Pro-Cap/gate loop. We found that PYL1 (H87A, F88A, K90S) exists as a monomer in solution (Fig. 8A) but exhibits a low affinity (69 lM), which is comparable with that of the wild-type protein (76 lM) (Fig. 8B,C). Moreover, we found that PYL1 (S112R, N117R), which has a double mutation located at the Pro-Cap/gate loop, existed as a dimer but exhibited a ~6-fold higher affinity (13.7 lM) than that of the wild-type protein (Fig. 9A,B). Like PYL2(I88K), PYL1(I111K), which has the mutation corresponding to I88K of PYL2, existed as a monomer and exhibited a higher


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(A)

(B)

(C)

Figure 6 Binding of abscisic acid (ABA) to PYR1 (subfamily III) and its PYL9-like mutant. (A) Binding to the wild-type PYR1. Isothermal titration calorimetry profiles and thermodynamic data for binding experiments at 20 °C in 20 mM HEPES (pH 7.5) and 150 mM NaCl. Injections of 6 lL of 6.0 mM ABA into 295 lM of the wild-type protein were performed over a period of 6 s with an 8-min interval between injections. The final concentration reached was 550 lM. The protein exhibited a KD value of 750 lM with of DH and D S values of 8.0 kcal/mol and 13 cal/mol per K, respectively. (B) Binding to the triple mutant (F108I, A160V, V163L) form of PYR1. Injections of 6 lL of 6.0 mM ABA into 300 lM of the triple mutant protein were performed as in the wild-type protein. The triple mutant protein exhibited a KD value of 159 lM with DH and D S values of 2.08 kcal/mol and 10.4 cal/mol per K, respectively. (C) Binding to the quadruple mutant PYR1(H60P, F108I, A160V, V163L). Injections of 2 lL of 2.5 mM ABA into 150 lM of protein. The injections were performed over a period of 6 s with an 180-s interval between injections. The quadruple mutant protein exhibited a KD value of 2.87 lM with DH and DS values of 6.07 kcal/mol and 4.65 cal/mol per K, respectively.

affinity (12.4 lM) (Fig. 9C). These data strongly suggest that the monomer-dimer equilibrium is nonessential for determining each ABA-binding affinity.

Discussion As previously pointed out (Santiago et al. 2009a; Szostkiewicz et al. 2009; Lumba et al. 2010), highly 396

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redundant Arabidopsis ABA receptors comprising 14 PYR/PYL/RCAR members sharply contrast with the six auxin receptors, three GA receptors, and only one JA receptor (Dharmasiri et al. 2005; Nakajima et al. 2006; Browse 2009; Park et al. 2009). Moreover, six PP2Cs are inhibited by ABA receptors (Gosti et al. 1999; Saez et al. 2004; Nishimura et al. 2007; Browse 2009; Park et al. 2009; Rubio et al.



(C)

(B)

Figure 7 Binding of abscisic acid (ABA) to two subfamily-II members, PYL4 and PYL6. (A) Binding to PYL4 belonging to the subfamily-II. Injections of 0.85 lL of 1.5 mM ABA into 110 lM of protein were performed over a period of 6 s with an 200-s interval between injections. PYL4 exhibited a KD value of 1.32 lM with DH and D S values of 10.59 kcal/mol and 9.22 cal/ mol per K, respectively. (B) As in A, but for binding to PYL6, another subfamily-II member. The titration was performed as in A. PYL6 exhibited a KD value of 1.48 lM with DH and D S values of 0.56 kcal/mol and 33.2 cal/mol per K, respectively. (C) As in A, but for binding to the mutant PYL4 (I182V) protein. I182 of PYL4 is a counterpart of I165 of PYL9, a key residue making direct contacts with the bound ABA. This mutation converts this key residue of PYL4 to a valine found in PYR1 and other subfamily-III members. The injection procedure was the same as that for the wild-type PYL4 in A. The PYL4 mutant exhibited a KD value of 4.90 lM with DH and D S values of 15.27 kcal/mol and 27.80 cal/mol, respectively.

2009), which could provide 84 possible combinations of receptor-phosphatase complexes. In addition to differential regulation brought about by various stresses with overlapping but sometimes specific target PP2Cs, individual combinations exhibiting different ABA affinities could encompass a broad spectrum of ABA responses to allow for fine-tuned physiological responses. The sensitivity of ABA signaling depends on the ABA affinities of the receptors in addition to the affinities of the ABA-bound receptors to target

PP2Cs. An estimation of the endogenous ABA concentration under unstressed conditions corresponds to the micromolar range (Harris et al. 1988; Zhang et al. 2001; Christmann et al. 2005, 2007; McCourt & Creelman 2008; Melcher et al. 2010). In this context, it is surprising that the receptor-PP2C complexes, which may act as co-receptors of ABA exhibit relatively high ABA affinities with KD values ranging from several tens to hundreds nM (Ma et al. 2009; Melcher et al. 2009; Miyazono et al. 2009; Nishimura


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(B)

(C)

Figure 8 Binding of abscisic acid (ABA) to PYL1 (subfamily-III) and its monomeric mutant with substitution located at the dimer interface but not at the Pro-Cap/gate loop. (A) Results of analytical ultracentrifugation of PYL1 and its triple mutant PYL1 (H87A, F88A, K90S). The distribution of apparent molecular mass obtained from sedimentation velocity analyses. The triple mutant PYL1 gave single peaks at 23.1 kDa both in the absence (orange lines) and presence (red lines) of ABA, suggesting a monomer in solution. The wild type (black lines) gave a single peak at 56 kDa, suggesting a dimer in solution as reported previously. (B) Binding to PYL1 belonging to the subfamily-III. Injections of 6 lL of 6.0 mM ABA into 200 lM of protein were performed over a period of 12 s with an 8 min interval between injections, and the final concentration reached was 950 lM. The wild-type PYL1 exhibited a KD value of 76 lM with DH and D S values of 14.6 kcal/mol and 23.8 cal/mol per K, respectively. These results are consistent with the previous reported KD value (52 lM) (24). (C) Binding to the triple mutant PYL1(H87A, F88A, K90S) protein. Injections were performed as in the wild-type PYL1. The triple mutant protein exhibited a KD value of 69 lM with DH and D S values of 16.4 kcal/mol and 13.4 cal/mol per K, respectively.

et al. 2009; Park et al. 2009; Santiago et al. 2009a,b; Szostkiewicz et al. 2009; Yin et al. 2009), which are paradoxically sufficient to bind and activate several different ABA receptor-PP2C complexes under unstressed conditions. Contrary to the relatively high affinities of the ABA receptor-PP2C complexes, individual isolated ABA receptors display relatively low ABA affinities with KD values ranging from micromolar to near millimolar levels. More intriguingly, cytosolic ABA levels can range from the nanomolar to the micromolar range depending on environmental challenge and/or developmental stage (Priest et al. 398

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2006; Christmann et al. 2007). We speculate that, to respond to the given broad dynamic range, some ABA receptors should be colocalized with their target PP2Cs under unstressed conditions and function as co-receptors that sense low-level changes in ABA concentration, while isolated ABA receptors sense high-level changes in ABA concentration. We showed that ABA perception by PYL9, which is representative of subfamily-I members exhibiting high ABA affinities, differs from that by subfamily-III members that display low ABA affinities. Three nonpolar residues, Ile110, Val162, and Leu165 of PYL9,



(B)

(C)

Figure 9 Binding of abscisic acid (ABA) to PYL1 (subfamily-III) and its monomeric mutants with substitution located at the ProCap/gate loop. (A) Elusion profiles of size-exclusion chromatography using a superdex200 HR 10/13 column with size marker proteins (see text). The wild-type PYL1 was eluted at the apparent molecular mass of 70 kDa, which is somewhat larger than the calculated value (51 kDa for a dimer) and that obtained from AUC (56 kDa, see Fig. 8A). PYL1(S112R, N119R) appeared at the same position as that of the wild-type PLY1 dimer, whereas PYL1 (I111K) at the half of the apparent molecular mass, suggesting a monomer in solution. (B) Binding to the double-mutant PYL1(S112R, N119R). Injections of 2 lL of 2.5 mM ABA into 400 lM of protein were performed over a period of 6 s with an 200 s interval between injections. The double-mutatnt protein exhibited a KD value of 13.7 lM with DH and D S values of 2.16 kcal/mol and 2.96 cal/mol per K, respectively. (C) Binding to the single mutant PYL1(I111K). Injections of 2 lL of 2.7 mM ABA into 200 lM of protein. The injections were performed over a period of 6 s with an 200-s interval between injections. The single-mutant protein exhibited a KD value of 12.4 lM with DH and DS values of 2.57 kcal/mol and 13.7 cal/mol per K, respectively.

located inside the ABA-binding cavity are key residues that participate in intimate interactions with the bound ABA molecules and contribute to its high ABA-binding affinity. This ‘nonpolar triplet’ is completely conserved in subfamily-I members, PYL7-10, suggesting that subfamily-I comprises high affinity receptors. Subfamily-III members which display lower ABA affinities have Phe, Ala/Val, or Val at each position of the nonpolar triplet of subfamily-I. We also showed that replacement of these three

residues of PYR1 with Ile, Val and Leu, the nonpolar triplet of subfamily-I, enhances ABA affinity (Fig. 4). In addition to the nonpolar triplet, Pro64 of PYL9 is another key residue contributing to the formation of the direct hydrogen bonds to ABA and is conserved in all subfamily-I members. Recently, a mutant PYR1(H60P) protein has been shown to exhibit a higher ABA-binding affinity with a KD value of 3 lM, and the structure of the complex between ABA-bound PYR(H60P) and HAB1 has


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been determined (Dupeux et al. 2011). We found that PYR1(H60P) binds ABA with a hydrogen bond network similar to that found in our ABA-PYL9 structure (Fig. 5D), suggesting that the observed enhancement of the ABA affinity is induced by the modified hydrogen-bonding interactions by the H60P mutation. Interestingly, PYR1(H60P) has been found to exist as a monomer in solution (Dupeux et al. 2011). This dimer-to-monomer conversion is caused by the mutation, which hinders conformational changes at the peptide backbone required to accommodate Phe61 at the dimer interface. However, no rational explanation was provided for the question how the conversion from a dimer to a monomer enhances the affinity. Our mutation studies indicate that dimer formation is not necessarily prerequisite for low binding affinity receptors (Figs 7,8). Interestingly, PYR1 exhibits an asymmetric state in which one ABA-bound closed-lid protomer may stabilize an open conformation of the other protomer by intimate interactions involving the Pro-cap/gate loop (Park et al. 2009; Santiago et al. 2009a). We speculate that this open conformation in the asymmetric dimer could be formed in solution as a transient state and is in part be responsible for the low affinity of dimerized receptors. It should be noted that subfamily-I members, PYL9 and PYL8, have been shown to display relatively high ABA affinity in the presence of ABI1 and ABI2 with IC50 values that are 5~6-fold smaller than those of PYL1 (Santiago et al. 2009b). Thus, the ABA affinities of the isolated receptors are somehow correlated with the affinities in the presence of PP2Cs, that is, the ABA affinities of co-receptors. Subfamily-II members do not conserve the nonpolar triplet but have a conserved Ile residue at the position of Leu165 of PYL9. Our model building suggests this Ile residue is capable in engaging in intimate interaction with ABA as does Leu165 in our complex. Therefore, these members may exhibit high ABA affinity as is the case with subfamily-I members. This speculation is supported by the observed small KD values of PYL4, PYL5 and PYL6. Recently, I110 of PYR1 (I137 of PYL1, V114 of PYL2) has been shown to play a key role in pyrabactin agonist/antagonist selectivity (Melcher et al. 2010; Peterson et al. 2010; Yuan et al. 2010). It was shown that the I137V mutation converts PYL1 to a pyrabactin-inhibited receptor and also the V114I mutation converts PYL2 to a pyrabactin-sensitive receptor (Melcher et al. 2010; Yuan et al. 2010). The corresponding residue in PYL9 is I112, which is located at 400

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the same position and orientation as those of I110 of PYR1 and I137 of PYL1 and makes a contact with the ABA ring methyl group (C9) (Fig. 3A). An Ile residue appears at this position of other high-affinity members, but also some low-affinity members such as PYR1 and PYL1. Similarly, a Val residue appears at this position of both the high- and low-affinity members. Thus, I110 of PYL9 seems to be not important for its high ABA affinity (Fig. 5B). Among the 14 ABA receptor members, it is anticipated that certain pairs of PYR/PYL/RCAR-PP2C proteins could show stronger or weaker interactions, based on biochemical parameters such as ABA-binding affinity or binding affinity of ABA-bound receptors to each target PP2C, subcellular localization, and organ, tissue and developmental expression, as well as developmental and environmental regulation of ABA biosynthesis. These combinations enable a broad dynamic range of ABA-induced responses in living cells. We have provided the first three-dimensional structure of a high-affinity member of ABA receptors, PYL9, and delineated the structural basis for factors that enhance ABA affinity. We also showed that a subset of high-affinity receptors includes subfamily-III members and, at least, a portion of subfamily-II members, while a subset of low-affinity receptors includes subfamily-I members.

Experimental procedures Protein expression and purification The cDNAs coding for Arabidopsis PYL9 (residues 1–187, NCBI Accession No. AK227623), PYL5 (residues 1–203, AY052251), PYRl (residues 1–191, AY042890), PYL4 (residues 1–207, NCBI Accession No. AY039586), PYL6 (residues 1–215, BT004281) and PYLl (residues 1–221, AY063877) were subcloned into pET49b(+) plasmid (Novagen) using the SmaI and EcoRI restriction-enzyme sites. These receptor proteins were expressed in Escherichia coli Rosetta2 (DE3) cells (Novagen) as fusion proteins with glutathione-S-transferase (GST) and purified as previously described (Shibata et al. 2010). In brief, cells were grown at 16 °C in Luria–Bertani (LB) medium and were then collected by centrifugation (Beckman J-HC). Wet cells were suspended in 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, (Buffer A) and disrupted by sonication at 4 °C. The soluble portion of the cell extract was purified by affinity chromatography on glutathione Sepharose 4B (GE Healthcare) resin and cleaved using HRV3C protease (Novagen). The cleaved proteins were further purified by HiTrap Q HP column (GE Healthcare) chromatography and gel-filtration chromatography on a Superdex 75 prep grade column (GE Healthcare). The identity of the purified protein was confirmed using matrix-assisted laser desorption/ionization time-of-flight


High-affinity ABA binding to PYL9 mass spectroscopy (MALDI–TOF MS; PerSeptive Inc.). Each protein begins after two extra residues (Gly-Pro) that form a part of the HRV3C protease recognition sequence. The mutant proteins, PYR1 (F108I, A160V, V163L), PYL4 (I182V) and PYL1 (H87A, F88A, K90S) were expressed and purified in a similar manner described previously. The protein yields of the recombinant proteins of PYR1 and PYL5 are relatively high, 4.7 and 4.0 mg from one liter culture, respectively. The protein yields of PYL4 and PYL1 are 1.4 and 1.1 mg, respectively, and those of PYL9 and PYL6 are relatively low, ~0.5 mg/L culture. The protein yield of PYL4(I182V) was extremely low (0.08 mg/L).

Crystallization Purified PYL9 (1–187 plus two extra N-terminal residues, Gly-Pro) was concentrated to 15 mg/mL (~0.7 mM) with Buffer A and 2 mM (+)-ABA for crystallization. Crystallization screening was performed as described (Shibata et al. 2010). A microseeding method was employed in an effort to obtain larger crystals suitable for X-ray diffraction. The best crystals were obtained from a solution containing 7.5 mg/mL protein, 1.5 mM (+)-ABA and 10 mM Tris-HCl buffer (pH 8.0) containing 75 mM NaCl with 40 mM malic acid-MES-Tris (MMT) buffer (pH 7.0) and 10% (w/v) PEG 1500 equilibrated against 16.5% (w/v) PEG 1500 in 100 mM MMT buffer (pH 7.0). The crystals were found to belong to space group P3221 with unit cell dimensions, a = b = 111.4 A, c = 40.5 A, and diffracted to a resolution of 1.9 A (Table 1).

X-ray data collection and structure determination Initial diffraction tests were performed using a Rigaku X-ray generator FR-E equipped with a Rigaku R-AXIS VII detector at 173 °C. X-ray diffraction data from PYL9 was collected using an Rayonix MX225HE CCD detector installed on the BL41XU beamline at SPring-8 (Shibata et al. 2010). All data were processed and scaled with HKL-2000 (Otwinowski & Minor 1997). Initial phases were calculated by molecular replacement using different ranges of intensity data and integration radii with the program MOLREP (Vagin & Teplyakov 2010). Following rigid-body refinement of the search model performed with the program CNS (Br€ unger et al. 1998), the resultant initial map showed clear electron densities for most of the protein and the bound ABA. An initial model of the peptide was built into the electron density map using the graphics program Coot (Emsley & Cowtan 2004) and refined by the method of simulated annealing using CNS (Br€ unger et al. 1998) and the translation, rotation, screw-rotation (TLS) refinement using REFMAC (Murshudov et al. 1997). Refinement resulted in a final crystallographic free R value of 20.4% (working R value of 18.8%) for data between 50.0 and 1.9 A. Our electron density at 1.9 A resolution clearly represents the (+)-ABA molecule inside the cavity of PYL9 between the twisted seven-stranded b-sheet and the C-terminal long a3-helix. In the Ramachandran plots using

Table 1 Crystallographic data for the PYL9-ABA complex X-ray data Space group Cell parameters, (A˚) Resolution (A˚)† Mosaicity Reflections, total/unique Redundancy Completeness (%) Rmerge (%) Refinement Number of atoms Protein (residues 1–187) Ligand/ion Water Number of reflections Rwork/Rfree (%)‡ Average B-factor (A˚2) Protein Ligand/ion Water R.m.s. bond length (A˚), angles (º)

P3221 a = b = 111.4, c = 40.5 50–1.9 (1.97–1.90) 0.51–0.80 247 223/23 009 10.7 (10.4) 99.8 (100.0) 60.8 (4.7) 5.0 (57.0)

1352 (residues 13–183) 38 70 21 785 18.8/20.4 40.3 37.4 45.0 0.009, 1.27

ABA, abscisic acid. †Numbers in parentheses refer to statistics for the outer resolution shell. ‡Rwork = Σ | | Fobs | – | Fcalc | |/Σ | Fobs |. Rfree is the same as Rwork except for a 5% subset of all reflections that were never used in the crystallographic refinement.

MolProbity (Davis et al. 2007), no outliers were flagged. In the course of publication of our work, a lower resolution (2.68 A) study has been appeared (Zhang et al. 2013). The overall structure is similar to our structure (rms deviation of 1.3 A), but the N-terminal segments (1–30 residues) were poorly refined and displayed a loop and a short helix with high-temperature factors. This part of our structure forms a long helix associated with the following b-sheet. As the reported resolution, 2.68 A, is insufficient for detailed discussion of the ligand–protein and ligand–water interactions, we discussed these interactions based on our higher resolution structure. The atomic coordinates and structure factors (code 3W9R) of ABA-bound PYL9 have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

Analytical ultracentrifugation (AUC) Sedimentation velocity ultracentrifugation experiments were performed at 20 °C using a Beckman Coulter Optima XLA analytical ultracentrifuge equipped with an An-60 Ti rotor and


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M Nakagawa et al. double sector centerpieces. Purified samples were dissolved in 20 mM Tris buffer (pH 8.0) containing 150 mM NaCl and 1 mM DTT (TSD buffer) at a sample concentration of 1 mg/ mL (~50 lM) and then centrifuged at 3993 g. Radial absorbance scans were measured every 15 min at a wavelength of 280 nm (for the free form) or 5 min at a wavelength of 295 nm (for the ABA-bound form). The resultant data were analyzed using the programs Sedfit and Sednterp. In an effort to glean further insight into the PYL9 conformation in the ABA-bound state, similar experiments were performed for PYL9 in the presence of 100 lM (+)-ABA.

Size-exclusion chromatography The molecular sizes of the ABA receptors were examined using conventional gel-filtration techniques. The receptors chromatographed at 4 °C through a Superdex 200 HR 10/13 column (GE Healthcare Bio-Sciences, USA) with 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl. The molecular mass was determined on the basis of the elution volume from a plot of log (molecular mass) of standard proteins, comprising thyroglobulin (756 kDa), aldolase (182 kDa), carbonic anhydrase (29 kDa) and aprotinin (6.5 kDa) (GE Healthcare Bio-Sciences), versus the elution volume.

ITC-binding assay Binding studies by ITC analysis were conducted using a calorimeter (MicroCal VP-ITC, USA). Purified PYR1 was dialyzed overnight in buffer containing 20 mM Hepes-Na (pH 7.5), 150 mM NaCl and 2 mM TCEP. Ligand (6.0 mM (+)-ABA) in incubation buffer was injected into the protein solution (6 lL each, 8 min pause). Data fitting was performed by using ORIGINTM software supplied with the instrument. Similarly, the sample solutions of PYR1 (F108I, A160V, V163L), PYL6, PYL1, and PYL1 (H87A, F88A, K90S) were dialyzed against the same buffer as in the wild-type PYR1 and served for ITC measurements. Details of each titration were given in the figure legends. We found that PYR1 exhibits a high solubility (higher than 1 mM) and employed a relatively high PYR1 concentration (300 lM) in our titration to PYR1. At this concentration, we observed no precipitation in the sample cell. Measurements with low concentration such as 20 lM yielded only small heat preventing our accurate analysis. The protein yield of PYL4 mutants was very low as mentioned above. To overcome this difficulty, we used an iTC200 calorimeter (MicroCal) for ITC analyses of PYL4 and its mutant protein. Each purified protein was dialyzed overnight in the same buffer as described above. Ligand (1.5 mM (+)-ABA) in incubation buffer was injected into the protein solution (110 lM protein, ~150 lL) with 0.85 lL each in 200-sec interval (total 27 injections). Data fitting was performed by using ORIGINTM software. Details of each titration were given in the figure legends.

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Acknowledgements We would like to thank beamline staffs for data collection at SPring-8 using BL41XU. We also acknowledge J. Tsukamoto, R. Kurata and Y. Tawara for technical support in performing the MALDI-TOF MS analysis. The cDNAs were provided by RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. This work was supported by a Grant-inAid for Scientific Research on Innovative Area “Structural Cell Biology” from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan and by a research grant in the Life Sciences from the Takeda Science Foundation, Japan (to T.H.).

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PYL9 interacts with an R2R3-type MYB transcription factor, AtMYB44.

RCAR1.

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PYL9 interacts with an R2R3-type MYB transcription factor, AtMYB44.

RCAR1.

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