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Science 336 (6089): 1708-1711

Copyright © 2012 by the American Association for the Advancement of Science

Structural Basis for Prereceptor Modulation of Plant Hormones by GH3 Proteins

Corey S. Westfall1,*, Chloe Zubieta2,*, Jonathan Herrmann1, Ulrike Kapp2, Max H. Nanao3,4, and Joseph M. Jez1,{dagger}

1 Department of Biology, Washington University, St. Louis, MO 63130, USA.
2 European Synchrotron Radiation Facility, 38000 Grenoble, France.
3 European Molecular Biology Laboratory, Grenoble, France.
4 Unit of Virus Host-Cell Interactions, Université Joseph Fourier–European Molecular Biology Laboratory–CNRS, Grenoble, France.

Figure 1
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Fig. 1. Overall structure of AtGH3.12. Ribbon diagram showing the N- and C-terminal domains with α helices and β strands colored gold and blue, respectively. AMP in the active site is shown as a space-filling model. The hinge loop that switches conformation during the reaction sequence is also indicated.


Figure 2
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Fig. 2. Nucleotide binding site and C-terminal domain movement in AtGH3.12. (A) Active site residues in AtGH3.12bulletAMP. Residues from the P-loop, β-turn-β, and ribose interaction motifs are shown. (B) C-terminal domain pivot. Positions of the hinge loop (residues 420 to 432) and the α18/α19 loop (residues 543 to 554) in the AMP complex (blue) and the AMP-CPP (ATP analog) complex (gold) are shown. AMP-CPP is shown for reference. (C) Conformational change of C-terminal domain opens (ATP form) and closes (AMP form) access to the active site. Surface views of the open and closed forms are shown. In each view, the bound nucleotide is shown as a stick model. Residues in the hinge and α18/α19 loops are colored blue and gold, respectively. Positions of Lys428 and Lys550 in each conformation are indicated for reference.


Figure 3
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Fig. 3. Structural diversity of the acyl acid binding site in GH3 proteins. (A) Residues in the SA binding site of the AtGH3.12bulletAMPbulletSA complex are shown. (B) Residues of the JA-Ile binding site of AtGH3.11 are shown. The position of AMP from AtGH3.12 is shown for reference. (C) Surface view of the JA-Ile binding site in AtGH3.11, looking down the amino acid binding site toward the acyl acid substrate cavity. For clarity, the C-terminal domain has been removed. Surfaces associated with acyl acid substrate binding, Trp336, Phe125, and Ser101 (part of the P loop), are shown in yellow, blue, and red, respectively. The position of ATP in the nucleotide binding site is modeled on the AtGH3.12 structures. (D) Sequence comparison of residues in the acyl binding site of GH3 proteins from Arabidopsis. Numbering at the top and bottom correspond to AtGH3.12 and AtGH3.11, respectively. Stars across the top of the alignment indicate residues with side chains in the pocket; shading indicates conservation. Asterisks near the GH3 protein names indicate known acyl acid specificity as follows: light blue, benzoate; yellow, IAA; red, JA.


Figure 4
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Fig. 4. Amino acid specificity and binding site. (A) Amino acid specificity of AtGH3.11 and AtGH3.12. Relative catalytic efficiency (Vmax/Km) for each amino acid was determined using JA and ATP for AtGH3.11/JAR1 and using 4HBA and ATP for AtGH3.12/PBS3. C, Cys; F, Phe; H, His; I, Ile; P, Pro; V, Val; W, Trp. (B) Targeted sequence comparison of Arabidopsis (At) and rice (Os) GH3 proteins with known amino acid preferences. Numbering at the top and bottom corresponds to AtGH3.12 and AtGH3.11, respectively. (C) Structural comparison of the AtGH3.12bulletAMPbulletSA (green) and AtGH3.11bulletJA-Ile (gold) complexes, showing the positions of the highly conserved lysine residues (Lys428/Lys435) proposed to interact with amino acid substrates and the second lysine (Lys146) proposed to interact with the R group of acidic amino acids.


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