The Arabidopsis glutamyl-tRNA reductase (GluTR) forms a ternary complex with FLU and GluTR-binding protein

Abstract

Tetrapyrrole biosynthesis is an essential and tightly regulated process, and glutamyl-tRNA reductase (GluTR) is a key target for multiple regulatory factors at the post-translational level. By binding to the thylakoid membrane protein FLUORESCENT (FLU) or the soluble stromal GluTR-binding protein (GBP), the activity of GluTR is down- or up-regulated. Here, we reconstructed a ternary complex composed of the C-terminal tetratricopepetide-repeat domain of FLU, GBP, and GluTR, crystallized and solved the structure of the complex at 3.2 Å. The overall structure resembles the shape of merged two binary complexes as previously reported, and shows a large conformational change within GluTR. We also demonstrated that GluTR binds tightly with GBP but does not bind to GSAM under the same condition. These findings allow us to suggest a biological role of the ternary complex for the regulation of plant GluTR.

Figure 1. Reconstruction and crystallization of the FLU^TPR-GluTR-GBP ternary complex.

(A) Elution profiles of FLU^TPR, GluTR, GBP, and their mixture. Y-axis: mAU, milli-absorbance units; x-axis: volume in ml. (B) Crystal packing of the ternary complex (left panel) and its comparison with the GluTR-GBP complex (right panel). Color scheme: FLU^TPR, purple; GluTR, green; GBP, orange. The outline and the local 2-fold axis of a protein complex are in black lines. The ternary complex (left panel) is viewed from a direction perpendicular to the crystallographic a–c plane; the GluTR-GBP complex (right panel), to the b,c plane.

Figure 2. Structure of the FLU^TPR-GluTR-GBP ternary complex.

(A) Overall structure of the ternary complex and a superimposition of the two binary complexes. For the ternary complex (left panel), FLU^TPR, GluTR and GBP are colored coded as per each monomer. The catalytic domain (CD), NADPH-binding domain (NBD) and dimerization domain (DD) of GluTR are indicated on one GluTR monomer. For the two binary complexes (right panel), FLU^TPR, GluTR^DD, GluTR and GBP are colored coded as per each dimer. (B) Structural comparison of the GluTR-GBP part in the ternary complex with the GluTR-GBP complex. Structures are colored as in (A), and only halves are shown. The dashed arrow indicates the missing region in the ternary complex; the solid arrow indicates the region observed in the ternary complex but not in the GluTR-GBP complex. The structures are rotated 90 degrees along the y-axis to show the difference in the dimerization domain of GluTR. (C) Structural comparison of the ternary complex with the FLU^TPR-GluTR^DD complex. The inset shows the details of the ionic bond between the catalytic domain of GluTR and FLU^TPR. The abbreviations used are as follows: CD, the catalytic domain of GluTR; NBD, the NADPH-binding domain of GluTR; DD, the dimerization domain of GluTR; FLU^TPR, the recombinant TPR domain of FLU; GluTR^DD, the recombinant dimerization domain of GluTR.

Figure 3. Superimposition of GluTR monomers.

(A) Superimposed backbones of the four GluTR monomers. The color schemes are green for chain (A) of 5CHE, light green for chain (B) of 5CHE, prussian blue for chain (A) of 4N7R, and cyan for chain (B) of 4N7R. Orange arrows denote structural difference in the dimerization domain. (B) Superimposed backbones of the spinal helix and the dimerization domain of the two GluTR dimers. The two arms of the Y-shaped GluTR dimer in the ternary complex are denoted by solid orange lines; the two arms in the GluTR-GBP binary complex are denoted by dashed orange lines.

Figure 4. ITC analysis of GluTR’s interaction with GSAM and GBP.

The top panel shows the heat response upon each injection, and the bottom panel shows the integrated heat value (▪) and the fit (−) to a single-site binding model. N.D., not determined.