Abstract
The tetratricopeptide repeat (TPR)-containing protein FLU is a negative regulator of chlorophyll biosynthesis in plants. It directly interacts through its TPR domain with glutamyl-tRNA reductase (GluTR), the rate-limiting enzyme in the formation of δ-aminolevulinic acid (ALA). Delineation of how FLU binds to GluTR is important for understanding the molecular basis for FLU-mediated repression of synthesis of ALA, the universal tetrapyrrole precursor. Here, we characterize the FLU-GluTR interaction by solving the crystal structures of the uncomplexed TPR domain of FLU (FLU(TPR)) at 1.45-Å resolution and the complex of the dimeric domain of GluTR bound to FLU(TPR) at 2.4-Å resolution. Three non-canonical TPR motifs of each FLU(TPR) form a concave surface and clamp the helix bundle in the C-terminal dimeric domain of GluTR. We demonstrate that a 2:2 FLU(TPR)-GluTR complex is the functional unit for FLU-mediated GluTR regulation and suggest that the formation of the FLU-GluTR complex prevents glutamyl-tRNA, the GluTR substrate, from binding with this enzyme. These results also provide insights into the spatial regulation of ALA synthesis by the membrane-located FLU protein.
Highlights
FLU negatively regulates glutamyl-tRNA reductase (GluTR) during chlorophyll biosynthesis
The structure of the FLUTPR-GluTRDD complex was determined by molecular replacement using the FLUTPR structure as the search model, and the dimeric domain of GluTR was fitted into the density manually and refined as described above
FLUTPR consists of three tetratricopeptide repeat (TPR) motifs (TPR1-TPR3) and forms dimer through TPR3 (Fig. 1A)
Summary
FLU negatively regulates glutamyl-tRNA reductase (GluTR) during chlorophyll biosynthesis. The tetratricopeptide repeat (TPR)-containing protein FLU is a negative regulator of chlorophyll biosynthesis in plants. It directly interacts through its TPR domain with glutamyl-tRNA reductase (GluTR), the rate-limiting enzyme in the formation of ␦-aminolevulinic acid (ALA). We demonstrate that a 2:2 FLUTPR-GluTR complex is the functional unit for FLU-mediated GluTR regulation and suggest that the formation of the FLU-GluTR complex prevents glutamyl-tRNA, the GluTR substrate, from binding with this enzyme These results provide insights into the spatial regulation of ALA synthesis by the membrane-located FLU protein. An immunoprecipitation/mass spectrometry study has identified a chloroplast membrane complex containing FLU and several key enzymes devoted to chlorophyll synthesis [14] This complex is proposed to interact with GluTR upon accumulation of fluorescent. Together with an enzymatic ALA synthesis assay, these findings allow an elucidation of the molecular basis for FLU-mediated GluTR regulation
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