THOUSAND-GRAIN WEIGHT 6, which is an IAA-glucose hydrolase, preferentially recognizes the structure of the indole ring

Abstract

An indole-3-acetic acid (IAA)-glucose hydrolase, THOUSAND-GRAIN WEIGHT 6 (TGW6), negatively regulates the grain weight in rice. TGW6 has been used as a target for breeding increased rice yield. Moreover, the activity of TGW6 has been thought to involve auxin homeostasis, yet the details of this putative TGW6 activity remain unclear. Here, we show the three-dimensional structure and substrate preference of TGW6 using X-ray crystallography, thermal shift assays and fluorine nuclear magnetic resonance (¹⁹F NMR). The crystal structure of TGW6 was determined at 2.6 Å resolution and exhibited a six-bladed β-propeller structure. Thermal shift assays revealed that TGW6 preferably interacted with indole compounds among the tested substrates, enzyme products and their analogs. Further analysis using ¹⁹F NMR with 1,134 fluorinated fragments emphasized the importance of indole fragments in recognition by TGW6. Finally, docking simulation analyses of the substrate and related fragments in the presence of TGW6 supported the interaction specificity for indole compounds. Herein, we describe the structure and substrate preference of TGW6 for interacting with indole fragments during substrate recognition. Uncovering the molecular details of TGW6 activity will stimulate the use of this enzyme for increasing crop yields and contributes to functional studies of IAA glycoconjugate hydrolases in auxin homeostasis.

Figure 1

Overview of TGW6 structure. (a) Topology models of TGW6 structure (PDB: 8KG3). (b) A schematic model of the crystal structure of TGW6 (PDB: 8KG3). The numbers 1–6 indicate the blade structure number. The letters A to D show the location of each β-strand within a blade structure.

Figure 2

Structural comparison of TGW6 and structurally similar proteins. (a) Schematic models of proteins structurally similar to TGW6 (PDB: 8KG3). STR1; strictosidine synthase (PDB: 2FP8), DFPase; diisopropylfluorophosphatase (PDB: 1PJX), XC5397; gluconolactonase (PDB: 3DR2), Drp35; drug resistance protein 35 (PDB: 2DG1), NHL domain of TRIM71; the NHL domain of E3 ubiquitin-protein ligase tripartite motif-containing protein 71 (PDB: 6FPT). (b) Conserved putative Ca²⁺-binding sites in TGW6 and similar proteins. The gray spheres indicate the locations of Ca²⁺. The side chains of conserved Ca²⁺-binding residues are shown as stick models with colors denoting oxygen (red), carbon (green), and nitrogen (blue).

Figure 3

Thermal stability of TGW6 with various small compounds. The white bar is the T_m point of TGW6 in the control condition (no ligand). Black bars are the T_m points of TGW6 in the presence of a substrate, enzyme products or their analogs. The gray and hatched bars are T_m points of TGW6 in the presence of the six hit fragments. The horizontal solid line is the T_m point in the control condition, and that of dashed line is the T_m shifted temperature. This figure shows the standard deviations of T_m points for three independent experiments as error bars, and the plot indicates the T_m value for each experiment. For the measurements, the final concentration of recombinant TGW6 was adjusted to 27.6 μM with a 55.0 μM ligand solution.

Figure 4

Docking pose of IAA-Glc in the presence of TGW6. (a) A portion of TGW6 is represented as a drawing and the surface model is shown in gray. IAA-Glc is shown as a stick model with atom colors denoting oxygen (red), carbon (purple), nitrogen (blue) and hydrogen (gray). The side chains of key residues interacting with IAA-Glc are shown as stick models with colors denoting oxygen (red), carbon (green), nitrogen (blue) and hydrogen (gray). (b) The binding position of IAA-Glc in the TGW6 crystal structure (PDB: 8KG3). TGW6 is schematically shown as a gray surface model. The simulated binding of IAA-Glc is represented as magenta spherical models. (c) The interaction map of IAA-Glc in the presence of TGW6. Interacting residues are represented as colored circles. The dotted lines indicate the kind of interaction and the position of interaction: hydrophobic interaction (gray), aromatic-aromatic contacts (purple), hydrogen bonds and weak hydrogen bonds (green and light blue), respectively. The colored circle in cyan indicates subpocket A and that in magenta denotes subpocket B.