Structure-based chemical design of abscisic acid antagonists that

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Structure-based chemical design of abscisic acid antagonists that block PYL-PP2C receptor interactions Jun Takeuchi, Naoki Mimura, Masanori Okamoto, Shunsuke Yajima, Masayuki Sue, Tomonori Akiyama, Keina Monda, Koh Iba, Toshiyuki Ohnishi, and Yasushi Todoroki ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00105 • Publication Date (Web): 05 Apr 2018 Downloaded from http://pubs.acs.org on April 7, 2018

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ACS Chemical Biology

Structure-based chemical design of abscisic acid antagonists that block PYL-PP2C receptor interactions Jun Takeuchi1,a*, Naoki Mimura2,a, Masanori Okamoto3,4,a, Shunsuke Yajima5, Masayuki Sue6, Tomonori Akiyama6, Keina Monda7, Koh Iba7, Toshiyuki Ohnishi1,8 &Yasushi Todoroki1,8* 1

Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan.

2

Graduate School of Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan.

3

Center for Bioscience Research and Education, Utsunomiya University, Utsunomiya 321-8505, Japan.

4

PRESTO, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan.

5

Department of Bioscience, Tokyo University of Agriculture, Tokyo 243-0034, Japan.

6

Department of Applied Biology and Chemistry, Tokyo University of Agriculture, Tokyo 243-0034, Japan.

7

Department of Biology, Faculty of Science, Kyushu University, Fukuoka 819-0395, Japan,

8

Research Institute of Green Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan.

a

These authors contributed equally to this work.

*E-mail: [email protected]; [email protected]

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Abstract In Arabidopsis, signaling of the stress hormone abscisic acid (ABA) is mediated by PYR/PYL/RCAR receptors (PYLs), which bind to and inhibit group-A protein phosphatases 2C (PP2Cs), the negative regulators of ABA. X-ray structures of several PYL-ABA and PYL-ABA-PP2C complexes have revealed that a conserved tryptophan in PP2Cs is inserted into a small tunnel adjacent to the C4′ of ABA in the PYL-ABA complex and plays a crucial role in the formation and stabilization of the PYL-ABA-PP2C complex. Here, 4′-modified ABA analogs were designed to prevent the insertion of the tryptophan into the tunnel adjacent to the C4′ of ABA in these complexes. These analogs were predicted to block PYL–PP2C receptor interactions and thus block ABA signaling. To test this, 4′-O-phenylpropynyl ABA analogs were synthesized as novel PYL antagonists (PANs). Structural, thermodynamic, biochemical, and physiological studies demonstrated that PANs completely abolished ABA-induced PYL-PP2C interactions in vitro and suppressed stress-induced ABA responses in vivo more strongly than did 3′-hexylsulfanyl-ABA (AS6), a PYL antagonist we developed previously. The PANs and AS6 antagonized the effects of ABA to different degrees in different plants, suggesting that these PANs can function as chemical scalpels to dissect the complicated regulatory mechanism of ABA signaling in plants.

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Introduction Abscisic acid (ABA) is a plant hormone that plays critical roles in many physiological processes, such as seed dormancy, stomatal closure, and adaptive responses to abiotic stress1. A chemical tool to modulate ABA signaling would be valuable for investigating the function of ABA and its related components in plant physiological processes. Such a tool could be used alone or in combination with genetic analyses. Additionally, an ABA-signaling modulator would be of potential agrichemical value, because the ability to control seed germination rates, fruiting, and water use is important for effective and stable agricultural production. The ABA signaling mechanism is triggered by the activation of the cytosolic ABA receptors PYR/PYL/RCAR (PYR1 and PYLs 1–13 or RCARs 1–14 in Arabidopsis; hereafter referred to as PYLs), which inhibit group-A protein phosphatases 2C (PP2Cs), including HAB1, ABI1, and ABI22,3. ABA stabilizes a gate-closed conformation of PYLs by mediating a hydrophobic interaction between two conserved regions, a mobile gating loop and an α helix, which compose the ABA-binding pocket3. The gate-closed PYLs interact with PP2Cs mainly on the surfaces of these regions to form a ternary PYL-ABA-PP2C complex, in which a conserved Trp residue of PP2Cs (Trp385 in HAB1) plays a crucial role in complex formation and stabilization4,5. ABA-HYPERSENSITIVE GERMINATION 1 (AHG1), which lacks the Trp residue, is unable to form the ternary complex6. An indole ring of the Trp of PP2Cs is inserted into a small solvent-exposed tunnel (4'-tunnel) adjacent to the C4' of ABA in association with gate-closing, resulting in a π- π interaction with the benzene ring of the Phe of PYLs and a water-mediated hydrogen bond with the C4' carbonyl oxygen of ABA (Supplementary Figure S1). The docking site is adjacent to the active site of PP2Cs; the Ser side chain on the gate loop of PYLs is inserted into the chelating site of the magnesium ions of PP2Cs, resulting in their inhibition. The inactivation of PP2Cs leads to the activation of

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SNF1-related protein kinases (SnRK2s), which elicit ABA actions by phosphorylating S-type anion channels and transcription factors by autophosphorylation5. Based on the structural mechanism of the PYL-PP2C co-receptor system for ABA, we previously generated an ABA analog, AS6, as an antagonist of PYL-PP2C interactions7. AS6 bound to the ABA-binding pocket in PYLs and induced a conformational change to the gate-closed form in a manner similar to the formation of PYL-ABA complexes. The 3'-S-hexyl chain of AS6 was accommodated by a small solvent-exposed tunnel (3'-tunnel), which emerged adjacent to AS6’s C3' during gate-closing and protruded onto the PP2C-interacting surface of PYL (Supplementary Figure S2). AS6 inhibited ABA-induced PYL-PP2C interactions through direct steric hindrance of the S-hexyl chain. However, AS6 only weakly suppressed the activity of PP2Cs in a PYL-dependent manner in our in vitro assay system. Because of its basal partial agonist activity, AS6 did not completely abolish PYL-PP2C interactions. Nevertheless, this partial agonist activity in vitro did not correspond to the partial activation of ABA responses in vivo: AS6 did not inhibit Arabidopsis seed germination, upregulate ABA-responsive genes, or reduce transpiration in radish seedlings at the tested range of concentrations. A conformationally restricted analog of AS6, PAO4, which was developed to improve the affinity for PYLs by reducing the entropic penalty for binding events, showed similar partial agonist activity in vitro8. Although the reason for the differences between in vitro and in vivo behaviors remains obscure, the in vitro partial agonist activity suggests that the steric hindrance of the alkyl chain protruding from the 3'-tunnel is not sufficient to completely abolish PYL-PP2C interactions. The insufficient activity of AS6 and PAO4 as PYL antagonists may explain the poor antagonistic activity against exogenous ABA in vivo, as AS6 and PAO4 could not completely restore the germination time course of ABA-treated Arabidopsis seeds to that of untreated seeds, even though all seeds germinated (Supplementary Figure S3).

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In the present work, we focused on the 4'-tunnel to develop a complete PYL antagonist without partial agonist activity in vitro. Because the 4'-tunnel plays a crucial role in the formation and stabilization of the PYL-ABA-PP2C complex as described above, a ligand impairing this role was expected to completely block PYL–PP2C receptor interactions. Here, we describe the structure-based design, synthesis, and in vitro and in vivo activities of 4'-modified ABA analogs as antagonists capable of completely blocking PYL-PP2C binding.

RESULTS AND DISCUSSION Design and synthesis

The insertion of the Trp of PP2Cs into the 4'-tunnel is required for the formation of a stable PYL-ABA-PP2C ternary complex. Therefore, a ligand that blocks the insertion of the indole moiety of the Trp of PP2Cs into the 4'-tunnel would prevent the formation of the ternary complex. A simple design strategy for the ligand is to introduce an alkyl chain at the C4' of ABA. As a first trial, we synthesized 4'-O-butyl/pentyl-ABAs (Supplementary Figure S4A) and evaluated the in vivo activities. 4′-O-Butyl-ABA slightly inhibited Arabidopsis seed germination, whereas 4′-O-pentyl-ABA slightly weakened the effect of exogenous ABA (Supplementary Figure S4B). Because the distance between the Trp-indole nitrogen and the 4'-carbonyl oxygen of ABA in the ternary complex is approximately 4.7 Å, the O-butyl/pentyl chain (4.9/6.2 Å) was considered to be long enough to block the insertion of the Trp-indole. The slight agonist/antagonist effect suggested that the introduction of a simple linear alkyl chain at C4' reduced the affinity for PYLs. The alkyl chain may not fit into the tunnels because of the steric hindrance of the zigzag conformation (Supplementary Figure S5), although further structural characterization of PYL-4′-O-alkyl-ABA complexes is necessary to confirm this hypothesis.

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Based on the results of the first trial, we designed five 4'-O-phenylpropynylated ABA analogs as novel complete PYL antagonists (PANs). In these five ABA analogs, an alkyne and an aromatic ring were integrated into the 4'-O-substituent (Figure 1A). The propynyl moiety was introduced to confer rigid linearity without zigzagging or cranking associated with bond rotations, whereas the phenyl ring was expected to mimic the Trp-indole of PP2Cs, which interacts with the Phe-phenyl ring of PYLs (Figure 1B). The objective was to increase the affinity for PYLs and prevent the insertion of the Trp-indole into the 4'-tunnel. The PANs were synthesized from a natural ABA, S-(+)-ABA, via 1',4'-trans-diol-ABA as shown in Supplementary Figure S6. Thus, all the PANs had the same configuration at C1' as that of natural ABA. Biochemical characterization of PANs

We conducted PP2C phosphatase assays to determine whether the PANs showed partial agonist activity in vitro, similar to that of AS6. In these assays, we used HAB1 as PP2C and PYR1 and PYLs 1–6 and 8–10 as the PYLs (10 PYLs for PANH and PANMe; PYR1 and PYL5 for other PANs). Whereas AS6 at 50 µM inhibited PP2C activity, the tested PANs at 50 µM did not significantly inhibit PP2C activity in the presence of all the tested PYLs (Figure 2A). Moreover, even at 100 µM, the PANs did not inhibit PP2C activity in the presence of PYR1 and PYL5 (Supplementary Figure S7A). This concentration was close to the solubility limit of PANs (other PYLs were not tested). Next, we evaluated the antagonistic effect of PANs on ABA-dependent inhibition of PP2C activity. At 5 µM, ABA strongly inhibited PP2C activity (to 130). Different letters indicate significant differences between treatments (P-value < 0.05, Tukey’s test). (D) Effects of AS6 and PANMe on expression levels of Arabidopsis ABA-responsive genes. AS6 and PANMe were administered at 0, 2.5, 5, and 25 µM in presence of 2.5 µM ABA. Six-day-old Arabidopsis wild-type (Columbia accession) seedlings were incubated in a solution containing chemicals in 0.5× MS and 0.5% sucrose for 6 h (n = 4, error bars represent sd). (E) Spatial expression pattern of MAPKKK18

after co-treatment with

ABA and

PANMe.

Six-day-old

plants

of

promoter-MAPKKK18::GUS transgenic Arabidopsis were incubated in chemical solution for 6 h at 22°C in light conditions. Scale bars = 1 mm (leaf), 50 µm (epidermis), or 0.5 mm (root).

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Table 1 Apparent PANH and PANMe binding affinity for PYL5

a

b

c

d

Compounds

Kda (nM)

n

∆Ha (kcal/mol)

−T∆Sb (kcal/mol)

∆Gc (kcal/mol)

PANH

163 ± 43

1.10

−7.6 ± 0.1

−1.5

−9.1 ± 0.1

PANMe

47 ± 22

0.90

−6.4 ± 0.1

−3.5

−9.8 ± 0.3

ABAd

890 ± 100

0.53

−7.8 ± 0.1

−0.3

−8.1 ± 0.1

AS6d

480 ± 100

0.55

−9.7 ± 0.2

1.2

−8.5 ± 0.1

Kd, n, ∆H obtained from single-set-of-sites fit to data. T∆S = ∆H ｰ∆G ∆G =ｰ RTln(1/Kd). Uncertainties for Kd, ∆H, and ∆G calculated by curve fitting program of MicroCal Origin 7.0. Data from reference 7.

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A

PANH (1): R = H PANMe (2): R = CH3 PANOMe (3): R = OCH3 PANAc (4): R = COCH3 PANCF3 (5): R = CF3

B HAB1

PYR1

W385 F159



DMSO

ABA

AS6

PANH

PANMe

C

AS6

PANH

PANMe

PYR1 120

HAB1 relative activity (%)


A

100 80 60 40 20

100 80 60 40 20 0

0

0

No PYR1 PYL1 PYL2 PYL3 PYL4 PYL5 PYL6 PYL8 PYL9 PYL10 PYL

20

40

60

80 100

PYL5

B 120



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100 80 60 40 20 0 No PYR1 PYL1 PYL2 PYL3 PYL4 PYL5 PYL6 PYL8 PYL9 PYL10 PYL


100 80 60 40 20 0 0

20

40

60

80 100

Chemical concentration (µM)

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ACS Chemical Biology A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

B complex B

complex A

complex A complex B

gate loop

PYR1-ABA (3K90)

C complex A

complex B

F159

F159

D complex A

complex B

3' 4'

3' V393 (HAB1)

4'

PYR1-ABA-HAB1 (3QN1)


W385 (HAB1)

PYR1-ABA-HAB1 (3QN1)


Temperature (C) 20

25

30

35

-4

ΔH / kcal mol−1

-6 -8 PANMe -10 -12

ABA

-14 -16 10 ΔS / kcal mol−1 K−1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

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PANMe

5 0 -5 -10 -15

ABA

-20 -25


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Figure 5. Physiological effects of PANMe compared with those of AS6. (A) Arabidopsis seed germination rate in the presence of 0.3 µM ABA and 30 µM AS6 or 1 µM PANMe (n = 3, error bars represent sd). (B) Effects of AS6 and PANMe on Arabidopsis seedling growth. Ten-day-old plants were sprayed with 1 mL 5 µM ABA and 50 µM PYL antagonists daily for 3 weeks. (C) Inhibitory effects of AS6 and PANMe on ABA-dependent changes in stomatal aperture. Data are mean ± SE (n > 130). Different letters indicate significant differences between treatments (P-value < 0.05, Tukey’s test). (D) Effects of AS6 and PANMe on expression levels of Arabidopsis ABA-responsive genes. AS6 and PANMe were administered at 0, 2.5, 5, and 25 µM in presence of 2.5 µM ABA. Six-day-old Arabidopsis wild-type (Columbia accession) seedlings were incubated in a solution containing chemicals in 0.5× MS and 0.5% sucrose for 6 h (n = 4, error bars represent sd). (E) Spatial expression pattern of MAPKKK18 after co-treatment with ABA and PANMe. Six-day-old plants of promoter-MAPKKK18::GUS transgenic Arabidopsis were incubated in chemical solution for 6 h at 22°C in light conditions. Scale bars = 1 mm (leaf), 50 µm (epidermis), or 0.5 mm (root). 140x131mm (300 x 300 DPI)



Table of contents graphic 75x39mm (300 x 300 DPI)


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Structure-based chemical design of abscisic acid antagonists that

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