Abstract
Gibberellins (GAs) are phytohormones that regulate various developmental processes in plants. The initial GA signalling events involve the binding of a GA to the soluble GA receptor protein GID1, followed by the binding of the complex to the negative transcriptional regulator of GA signaling, the DELLA protein. Although X-ray structures for certain Arabidopsis GID1/GA/DELLA protein complexes have previously been determined, examination of these complexes did not fully clarify how a DELLA protein recognizes and binds to a GID1/GA complex. Herein, we present a study aimed at physically defining, via a combination of gel chromatography, isothermal titration calorimetry (ITC), small-angle X-ray scattering experiments (SAXS), NMR spectroscopy and mutagenesis, how the rice DELLA protein (SLR1) binds to the rice GID1/GA complex. We have identified the shortest SLR1 sequence (M28-A112) that binds the rice GID/GA complex tightly. The binding constant for the ternary complex that includes SLR1(M28-A112) is 2.9 × 107 M−1; the binding is enthalpically driven and does not depend on the chemical nature of the bound GA. Furthermore, the results of SAXS, ITC, and gel filtration experiments indicate that when free in solution, SLR1(M28-A112) is a natively unfolded protein. The NMR experiments expand this observation to show that the unfolded mutant also contains a small amount of marginally stable secondary structure. Conversely, the protein has a highly ordered structure when bound one-to-one to GID1/GA.
Highlights
The gibberellins (GAs) are a large family of tetracyclic diterpenoid plant hormones that induce a wide range of plant growth responses, including seed germination, stem elongation, leaf expansion, induction of flowering, and pollen maturation[1,2]
Using a combination of gel chromatography, isothermal titration calorimetry (ITC), small-angle X-ray scattering (SAXS) measurements, and NMR spectroscopy, we report an examination of various solution OsGID1/GA/SLR1 complexes that differ according to the SLR1 variant present and the isolated components in solution
It appears from the chromatograms that, with the exception of Trx-SLR1(M1-E104), all of the Trx-SLR1 mutants bind Trx-OsGID1/GA3; as the results of the isothermal titration calorimetry experiment show, SLR1(M1-E104) does bind the OsGID1/GA3 complex, albeit with a binding constant that is only approximately 0.4% of that found for SLR1(M28-A112)
Summary
The gibberellins (GAs) are a large family of tetracyclic diterpenoid plant hormones that induce a wide range of plant growth responses, including seed germination, stem elongation, leaf expansion, induction of flowering, and pollen maturation[1,2]. A number of rice proteins involved in GA signalling pathways have been identified by screening for rice (Oryza sativa) variants with aberrant growth characteristics owing to genetic mutations (reviewed in[4,5,6]). The GA receptor protein (OsGID1)[7], the DELLA protein (SLR1)[8], and the F-box protein (GID2)[9,10] have been shown to be essential for GA signalling in rice. Much remains to be learned about the molecular recognition mechanisms that occur during the initial steps of GA-mediated plant growth pathways. The rice proteins OsGID1 and SLR1 are suitable models for such studies because they can be isolated as recombinant proteins in quantities large enough for in vitro structural studies
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