Volume 20, Issue 5, Pages 780-790 (May 2012) Complex Structures of the Abscisic Acid Receptor PYL3/RCAR13 Reveal a Unique Regulatory Mechanism  Xingliang Zhang, Qi Zhang, Qi Xin, Lin Yu, Zheng Wang, Wei Wu, Lun Jiang, Guoqiang Wang, Wenli Tian, Zengqin Deng, Yang Wang, Zhao Liu, Jiafu Long, Zhizhong Gong, Zhongzhou Chen  Structure  Volume 20, Issue 5, Pages 780-790 (May 2012) DOI: 10.1016/j.str.2012.02.019 Copyright © 2012 Elsevier Ltd Terms and Conditions

Structure 2012 20, 780-790DOI: (10.1016/j.str.2012.02.019) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 1 Formation of the PYL3 trans-Dimers with the Addition of Two Ligands (A) Apo-PYL3 cis-homodimer, two protomers are related by a 2-fold rotation axis, parallel to the plane of the page. (B) Superposition of (+)-ABA-bound PYL3 trans-homodimer (green) with apo-PYL3 cis-homodimer (purple) in Cα trace mode. (+)-ABA is labeled in blue. (C) The stereo view of the PYL3-(+)-ABA complex structure. The ABA (color blue) binding pocket in ligand-bound PYL3 is exposed to the solvent and cycled (magenta). Two protomers are related by a 2-fold rotation axis, perpendicular to the plane of the page. See also Figures S1, S2, and S3 and Table S1. Structure 2012 20, 780-790DOI: (10.1016/j.str.2012.02.019) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 2 Conformation Change of PYL3 Protomer upon the Addition of Two Ligands and the Dimeric Interface in the trans-Homodimer (A) Superposition of two ligand-bound PYL3 protomers with apo-PYL3 (apo-PYL3, yellow; S-(+)-ABA complex, green; pyrabactin complex, purple). (B) Interaction of two PYL3 molecules in PYL3-(+)-ABA structure (cyan and green). Two hydrophobic zones are shown as brown ellipse and two hydrogen bonds are labeled. Asn180 and Asp184 of one protomer are close to Thr209 and Pro208 of another protomer in the trans-homodimer. See also Figures S3, S5, and S8. Structure 2012 20, 780-790DOI: (10.1016/j.str.2012.02.019) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 3 Formation of trans-Homodimer in Solution (A) Elution profile of PYL3 or mutants with or without the addition of (+)-ABA by size-exclusion column chromatography. (B) Crosslinking of PYL3 or mutants by EGS in different concentration. (C) SDS-PAGE of PYL3 mutants under different reducing conditions. It shows that N180C T209C mutant forms a trans-dimer with the addition of ABA under nonreducing conditions, whereas S195L block the formation of trans-dimer. (D) Mass spectra of the N180C T209C mutant under different conditions. See also Figures S4 and S5. Structure 2012 20, 780-790DOI: (10.1016/j.str.2012.02.019) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 4 Inhibition of HAB1 by PYL3 Mutants in the Presence of (+)-ABA (A) The relative phosphatase activity of each reaction was normalized to that of the reaction containing phosphopeptide substrate and HAB1 (100%). Each reaction was repeated at least three times. (B) GST pulldown experiments of PYL3 mutants by GST-HAB1 in the presence of (+)-ABA. GST-HAB1 and PYL3 are highlighted by arrows. See also Figure S3. Structure 2012 20, 780-790DOI: (10.1016/j.str.2012.02.019) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 5 Analytical Ultracentrifugation Data of PYL3 Mutants and Complexes c(s) distribution from sedimentation velocity analytical ultracentrifugation experiments (SV) performed at 0.2 mM protein with or without 0.6 mM (+)-ABA. The change due to the adding of (+)-ABA observed in the c(s) distribution confirms the presence of different kinds of dimers and monomer. PYL3 S195L with (+)-ABA in the solution resembles PYL3 WT, overwhelmingly exists in cis-dimer conformation. Nonetheless, the triple mutant F81A V202A I203A almost exists as a monomer in the absence of (+)-ABA. See also Figure S6. Structure 2012 20, 780-790DOI: (10.1016/j.str.2012.02.019) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 6 Diagrammatic Representation of the Relationships of Ions Detected by MALDI-TOF-MS of Nonreduced and Reduced Samples The N and C termini of each sequence are shown in single-letter code with their numerical locations in the protein sequence. The trypsin cleavage sites are indicated by arrow. The m/z values correspond to the combinations of the ions from the reduced spectrum or theoretical values. An example of the computational process would be a summation of the masses of V164-R186 (m/z 2616.9) plus S195-C209 (m/z 1518.7), less 2 u to form one interchain disulfide, to give the nonreduced values of m/z 4132.5, which loses two N-terminal residues VY to give a mass of m/z 3871.0. In addition, the above two m/z values can not be formed for cis-dimer or monomer theoretically. All measurements were made with external mass calibration. Structure 2012 20, 780-790DOI: (10.1016/j.str.2012.02.019) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 7 PYL3–ABA–HAB1 Complex Structure (A) Overall structure of PYL3–ABA–HAB1 complex. (B) The interface between PYL3 and HAB1 in the PYL3–ABA–HAB1 complex. S195 is in the hydrophobic zone (magenta ellipse). W385 of HAB1 inserts the binding pocket. ABA, yellow; PYL3 main chain, blue; HAB1 main chain, red; side chain, cyan. See also Figures S3 and S7. Structure 2012 20, 780-790DOI: (10.1016/j.str.2012.02.019) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 8 The Mechanism for the Inhibition of PP2Cs by PYL3 with Ligands A characteristic change that one PYL3 in the apo-PYL3 cis-homodimer rotates and forms a trans-homodimer occurs after apo-PYL3 recognizes and binds to ligands. In turn, the trans-homodimer dissociates to monomer more easily and binds to PP2C more conveniently than the cis-homodimer. Noteworthily, only the appropriate gate closure can induce PP2C binding. See also Figures S7 and S8 and Table S1. Structure 2012 20, 780-790DOI: (10.1016/j.str.2012.02.019) Copyright © 2012 Elsevier Ltd Terms and Conditions