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.
Highlights
Three mechanisms have been characterized for plant glutamyl-tRNA reductase (GluTR) activity regulation, which are (i) the end-product feedback inhibition by heme[4], (ii) repression by a membrane protein FLUORESCENT (FLU)[5], and (iii) formation of complex with a soluble GluTR-binding protein (GBP)[6]
We show that GBP has higher affinity to GluTR than FLU’s TPR domain (FLUTPR) when quantified by isothermal titration calorimetry (ITC) experiment
The purified recombinant GluTR, GBP and FLUTPR were mixed at molar ratio of 2:3:3, and the mixture was subjected to size-exclusion chromatography
Summary
Three mechanisms have been characterized for plant GluTR activity regulation, which are (i) the end-product feedback inhibition by heme[4], (ii) repression by a membrane protein FLUORESCENT (FLU)[5], and (iii) formation of complex with a soluble GluTR-binding protein (GBP)[6]. Recent structural studies of the GluTR‒GBP complex[11] and of FLU’s TPR domain (FLUTPR) complexed with GluTR’s dimerization domain[10] have revealed that FLU and GBP bind to different sites on GluTR. These findings indicate that the three post-translational mechanisms of GluTR regulation may function simultaneously. ITC did not detect GSAM binding to GluTR or to the GluTR‒GBP complex These results advance the understanding of plant GluTR regulation at the molecular level and provide a clue to the spatial organization of these proteins
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