Structural Biology Studies Of Arabidopsis FLU And Its Regulatory Target Protein GluTR

In plants, algae, and most bacteria, tetrapyrrole biosynthesis provides important pigments including chlorophyll and heme. A key regulatory mechanism of tetrapyrrole biosynthesis is the metabolic feedback inhibition of glutamyl-tRNA reductase (GluTR) by heme. GluTR is the rate-limiting enzyme for tetrapyrrole biosynthesis, and in the dark condition, FLU can suppress GluTR activity by negatively regulating and directly interacting with GluTR.Recombinant FLU and GluTR proteins were obtained by prokaryotic expression, followed by Ni-NTA affinity chromatography and size-exclusion chromatography. High purity of FLU and FLU-GluTR complex were obtained, and through crystal screening and optimization, their crystals were grown. Using molecular replacement method, structure of the FLU TPR domain was successfully determined and refined to a resolution of 1.45 A. Structure of the FLU TPR domain in complex with the GluTR dimeric domain was then solved by using the FLU TPR domain as search model, and refined to a resolution of 2.4 A. The FLU TPR domain structure shows that it consists of three non-canonical TPR motifs, and forms dimers through TPR3. Ala262 in TPR2 is one of the few absolutely conserved amino-acid residues, and its mutation will lead to lethal phenotype in Arabidopsis thaliana. The 2.4-A complex structure displays that FLU and GluTR interact at a 2:2 ratio, and FLU binds to the GluTR dimeric domain through TPR1 and TPR3. This interaction occurs through five salt bridges and a hydrogen bond. The ITC experiments evaluate the effects of key amino-acid residues for the FLU-GluTR interaction.For GluTR in higher plants, its C-terminal region is very important for its activity regulation, and the structural integrity of a GluTR dimer is required the interaction with FLU. FLU and GluTR can form a stable complex in vitro. GluTR lacking the C-terminal 15 amino-acid residues can form a dimer, and can interact with FLU. An in vitro activity assay shows that GluTR activity is significantly inhibited in the presence of FLU. In addition, FLU and GluBP, a stimulator protein for GluTR, have antagonizing effects on GluTR activity, and function independently from each other.