Corey S. Westfall

Molecular basis for AUXIN RESPONSE FACTOR protein interaction and the control of auxin response repression

Significance Auxin is a critical plant hormone that regulates every aspect of plant growth and development. AUXIN RESPONSE FACTOR (ARF) transcription factors control auxin-regulated gene transcription, and their activity is regulated by AUXIN/INDOLE 3-ACETIC ACID repressor proteins. This work identifies that dimerization of the repressor with the transcription factor is insufficient to repress activity, suggesting that multimerization is the mechanism of repressing ARF transcriptional activity and further raising the possibility that multimerization in other systems may play roles in transcriptional repression. In plants, the AUXIN RESPONSE FACTOR (ARF) transcription factor family regulates gene expression in response to auxin. In the absence of auxin, ARF transcription factors are repressed by interaction with AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) proteins. Although the C termini of ARF and Aux/IAA proteins facilitate their homo- and heterooligomerization, the molecular basis for this interaction remained undefined. The crystal structure of the C-terminal interaction domain of Arabidopsis ARF7 reveals a Phox and Bem1p (PB1) domain that provides both positive and negative electrostatic interfaces for directional protein interaction. Mutation of interface residues in the ARF7 PB1 domain yields monomeric protein and abolishes interaction with both itself and IAA17. Expression of a stabilized Aux/IAA protein (i.e., IAA16) bearing PB1 mutations in Arabidopsis suggests a multimerization requirement for ARF protein repression, leading to a refined auxin-signaling model.

Structural Basis for Prereceptor Modulation of Plant Hormones by GH3 Proteins

Plant Hormone Modulators The activity and stability of several plant hormones is modulated by conjugation with various amino acids and their derivatives. Westfall et al. (p. 1708, published online 24 May) solved the crystal structures for two acyl acid amido synthetases from Arabidopsis. The findings suggest how the enzymes might discriminate between apolar and acidic amino acids and lend insight into the reaction chemistries that add functional diversity to hormone signaling pathways. Crystal structures of plant GH3 proteins reveal how these enzymes accommodate jasmonates, auxins, and benzoates. Acyl acid amido synthetases of the GH3 family act as critical prereceptor modulators of plant hormone action; however, the molecular basis for their hormone selectivity is unclear. Here, we report the crystal structures of benzoate-specific Arabidopsis thaliana AtGH3.12/PBS3 and jasmonic acid–specific AtGH3.11/JAR1. These structures, combined with biochemical analysis, define features for the conjugation of amino acids to diverse acyl acid substrates and highlight the importance of conformational changes in the carboxyl-terminal domain for catalysis. We also identify residues forming the acyl acid binding site across the GH3 family and residues critical for amino acid recognition. Our results demonstrate how a highly adaptable three-dimensional scaffold is used for the evolution of promiscuous activity across an enzyme family for modulation of plant signaling molecules.

Modulating plant hormones by enzyme action: the GH3 family of acyl acid amido synthetases.

Plants respond to developmental cues and environmental stresses by controlling both the level and activity of various hormones. One mechanism of modulating hormone action involves amino acid conjugation. In plants, the GH3 family of enzymes conjugates various amino acids to jasmonates, auxins, and benzoates. The effect of conjugation can lead to activation, inactivation, or degradation of these molecules. Although the acyl acid and amino acid specificities of a few GH3 enzymes have been examined qualitatively, further indepth analysis of the structure and function of these proteins is needed to reveal the molecular basis for how GH3 proteins modulate plant hormone action.

Kinetic Basis for the Conjugation of Auxin by a GH3 Family Indole-acetic Acid-Amido Synthetase

The GH3 family of acyl-acid-amido synthetases catalyze the ATP-dependent formation of amino acid conjugates to modulate levels of active plant hormones, including auxins and jasmonates. Initial biochemical studies of various GH3s show that these enzymes group into three families based on sequence relationships and acyl-acid substrate preference (I, jasmonate-conjugating; II, auxin- and salicylic acid-conjugating; III, benzoate-conjugating); however, little is known about the kinetic and chemical mechanisms of these enzymes. Here we use GH3-8 from Oryza sativa (rice; OsGH3-8), which functions as an indole-acetic acid (IAA)-amido synthetase, for detailed mechanistic studies. Steady-state kinetic analysis shows that the OsGH3-8 requires either Mg2+ or Mn2+ for maximal activity and is specific for aspartate but accepts asparagine as a substrate with a 45-fold decrease in catalytic efficiency and accepts other auxin analogs, including phenyl-acetic acid, indole butyric acid, and naphthalene-acetic acid, as acyl-acid substrates with 1.4–9-fold reductions in kcat/Km relative to IAA. Initial velocity and product inhibition studies indicate that the enzyme uses a Bi Uni Uni Bi Ping Pong reaction sequence. In the first half-reaction, ATP binds first followed by IAA. Next, formation of an adenylated IAA intermediate results in release of pyrophosphate. The second half-reaction begins with binding of aspartate, which reacts with the adenylated intermediate to release IAA-Asp and AMP. Formation of a catalytically competent adenylated-IAA reaction intermediate was confirmed by mass spectrometry. These mechanistic studies provide insight on the reaction catalyzed by the GH3 family of enzymes to modulate plant hormone action.

A chemical inhibitor of jasmonate signaling targets JAR1 in Arabidopsis thaliana

Jasmonates are lipid-derived plant hormones that regulate plant defenses and numerous developmental processes. Although the biosynthesis and molecular function of the most active form of the hormone, (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), have been unraveled, it remains poorly understood how the diversity of bioactive jasmonates regulates such a multitude of plant responses. Bioactive analogs have been used as chemical tools to interrogate the diverse and dynamic processes of jasmonate action. By contrast, small molecules impairing jasmonate functions are currently unknown. Here, we report on jarin-1 as what is to our knowledge the first small-molecule inhibitor of jasmonate responses that was identified in a chemical screen using Arabidopsis thaliana. Jarin-1 impairs the activity of JA-Ile synthetase, thereby preventing the synthesis of the active hormone, JA-Ile, whereas closely related enzymes are not affected. Thus, jarin-1 may serve as a useful chemical tool in search for missing regulatory components and further dissection of the complex jasmonate signaling networks.

Enzyme Action in the Regulation of Plant Hormone Responses

Plants synthesize a chemically diverse range of hormones that regulate growth, development, and responses to environmental stresses. The major classes of plant hormones are specialized metabolites with exquisitely tailored perception and signaling systems, but equally important are the enzymes that control the dose and exposure to the bioactive forms of these molecules. Here, we review new insights into the role of enzyme families, including the SABATH methyltransferases, the methylesterases, the GH3 acyl acid-amido synthetases, and the hormone peptidyl hydrolases, in controlling the biosynthesis and modifications of plant hormones and how these enzymes contribute to the network of chemical signals responsible for plant growth, development, and environmental adaptation.

Structure and Mechanism of Soybean ATP Sulfurylase and the Committed Step in Plant Sulfur Assimilation

Background: ATP sulfurylase catalyzes the energetically unfavorable formation of adenosine 5′-phosphosulfate in plant sulfur assimilation. Results: Structural and kinetic analyses identifies key active site residues. Conclusion: A reaction mechanism involving distortion of nucleotide conformation and stabilizing interactions is proposed. Significance: These results provide the first molecular insights on a plant ATP sulfurylase and the committed step of plant sulfur assimilation. Enzymes of the sulfur assimilation pathway are potential targets for improving nutrient content and environmental stress responses in plants. The committed step in this pathway is catalyzed by ATP sulfurylase, which synthesizes adenosine 5′-phosphosulfate (APS) from sulfate and ATP. To better understand the molecular basis of this energetically unfavorable reaction, the x-ray crystal structure of ATP sulfurylase isoform 1 from soybean (Glycine max ATP sulfurylase) in complex with APS was determined. This structure revealed several highly conserved substrate-binding motifs in the active site and a distinct dimerization interface compared with other ATP sulfurylases but was similar to mammalian 3′-phosphoadenosine 5′-phosphosulfate synthetase. Steady-state kinetic analysis of 20 G. max ATP sulfurylase point mutants suggests a reaction mechanism in which nucleophilic attack by sulfate on the α-phosphate of ATP involves transition state stabilization by Arg-248, Asn-249, His-255, and Arg-349. The structure and kinetic analysis suggest that ATP sulfurylase overcomes the energetic barrier of APS synthesis by distorting nucleotide structure and identifies critical residues for catalysis. Mutations that alter sulfate assimilation in Arabidopsis were mapped to the structure, which provides a molecular basis for understanding their effects on the sulfur assimilation pathway.

Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle

Background: The soybean genome encodes the most expanded plant heterotrimeric G-protein network reported to date. Results: Each Gα has distinct biochemical properties, and the RGS proteins have different GTPase-activating effects on each Gα. Conclusion: The core G-protein components, their interactions, and biochemical properties are conserved across phyla, but important mechanistic differences exist. Significance: This study provides insight into the complexity of plant G-protein networks. Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1–4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1–4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks.

Quantitation of the Effect of ErbB2 on Epidermal Growth Factor Receptor Binding and Dimerization

Background: ErbB2 is the preferred dimerization partner for the epidermal growth factor (EGF) receptor. Results: Heterodimerization with ErbB2 increases the affinity of the EGFR for EGF and increases the level of dimers maintained at any given concentration of EGF. Conclusion: ErbB2 modulates EGF receptor affinity and dimer stability. Significance: This study elucidates the molecular basis for the enhanced binding of EGF to EGFR/ErbB2 heterodimers. The epidermal growth factor (EGF) receptor is a member of the ErbB family of receptors that also includes ErbB2, ErbB3, and ErbB4. These receptors form homo- and heterodimers in response to ligand with ErbB2 being the preferred dimerization partner. Here we use 125I-EGF binding to quantitate the interaction of the EGF receptor with ErbB2. We show that the EGFR/ErbB2 heterodimer binds EGF with a 7-fold higher affinity than the EGFR homodimer. Because it cannot bind a second ligand, the EGFR/ErbB2 heterodimer is not subject to ligand-induced dissociation caused by the negatively cooperative binding of EGF to the second site on the EGFR homodimer. This increases the stability of the heterodimer relative to the homodimer and is associated with enhanced and prolonged EGF receptor autophosphorylation. These effects are independent of the kinase activity of ErbB2 but require back-to-back dimerization of the EGF receptor with ErbB2. Back-to-back dimerization is also required for phosphorylation of ErbB2. These findings provide a molecular explanation for the apparent preference of the EGF receptor for dimerizing with ErbB2 and suggest that the phosphorylation of ErbB2 occurs largely in the context of the EGFR/ErbB2 heterodimer, rather than through lateral phosphorylation of isolated ErbB2 subunits.

Arabidopsis thaliana GH3.5 acyl acid amido synthetase mediates metabolic crosstalk in auxin and salicylic acid homeostasis.

Significance Plants require precise control over growth regulators during development and in their responses to biotic and abiotic stresses. One strategy for modulating levels of bioactive phytohormones is to conjugate these molecules to amino acids using acyl acid amido synthetases of the Gretchen Hagen 3 (GH3) protein family. Typically, GH3 proteins modify one type of phytohormone. Structural studies, along with in vitro and in planta biochemical analyses, reveal that the GH3.5 protein from the model plant Arabidopsis thaliana conjugates multiple molecules from various phytohormone pathways. This activity mediates crosstalk between auxin developmental and pathogen response pathways. In Arabidopsis thaliana, the acyl acid amido synthetase Gretchen Hagen 3.5 (AtGH3.5) conjugates both indole-3-acetic acid (IAA) and salicylic acid (SA) to modulate auxin and pathogen response pathways. To understand the molecular basis for the activity of AtGH3.5, we determined the X-ray crystal structure of the enzyme in complex with IAA and AMP. Biochemical analysis demonstrates that the substrate preference of AtGH3.5 is wider than originally described and includes the natural auxin phenylacetic acid (PAA) and the potential SA precursor benzoic acid (BA). Residues that determine IAA versus BA substrate preference were identified. The dual functionality of AtGH3.5 is unique to this enzyme although multiple IAA-conjugating GH3 proteins share nearly identical acyl acid binding sites. In planta analysis of IAA, PAA, SA, and BA and their respective aspartyl conjugates were determined in wild-type and overexpressing lines of A. thaliana. This study suggests that AtGH3.5 conjugates auxins (i.e., IAA and PAA) and benzoates (i.e., SA and BA) to mediate crosstalk between different metabolic pathways, broadening the potential roles for GH3 acyl acid amido synthetases in plants.