Inter- and intra-molecular interactions of Arabidopsis thaliana DELLA protein RGL1

Abstract

The phytohormone gibberellin and the DELLA proteins act together to control key aspects of plant development. Gibberellin induces degradation of DELLA proteins by recruitment of an F-box protein using a molecular switch: a gibberellin-bound nuclear receptor interacts with the N-terminal domain of DELLA proteins, and this event primes the DELLA C-terminal domain for interaction with the F-box protein. However, the mechanism of signalling between the N- and C-terminal domains of DELLA proteins is unresolved. In the present study, we used in vivo and in vitro approaches to characterize di- and tri-partite interactions of the DELLA protein RGL1 (REPRESSOR OF GA1-3-LIKE 1) of Arabidopsis thaliana with the gibberellin receptor GID1A (GIBBERELLIC ACID-INSENSITIVE DWARF-1A) and the F-box protein SLY1 (SLEEPY1). Deuterium-exchange MS unequivocally showed that the entire N-terminal domain of RGL1 is disordered prior to interaction with the GID1A; furthermore, association/dissociation kinetics, determined by surface plasmon resonance, predicts a two-state conformational change of the RGL1 N-terminal domain upon interaction with GID1A. Additionally, competition assays with monoclonal antibodies revealed that contacts mediated by the short helix Asp-Glu-Leu-Leu of the hallmark DELLA motif are not essential for the GID1A–RGL1 N-terminal domain interaction. Finally, yeast two- and three-hybrid experiments determined that unabated communication between N- and C-terminal domains of RGL1 is required for recruitment of the F-box protein SLY1.

Multifarious Roles of Intrinsic Disorder in Proteins Illustrate Its Broad Impact on Plant Biology

“…Murase et al (2008); Sun et al (2010); Sheerin et al (2011) and disordered domains for interaction with partners in signaling processes ( Figure 3C, III). These potential modes of binding have some experimental support (Table 2).…”

Applications of Bioinformatics and Experimental Methods to Intrinsic Disorder-Based Protein-Protein Interactions

“…The MBP moiety of a well folded structure exhibited inaccessibility to solvent for most of the protein except the two terminal regions. In contrast, AtRGL1n showed instantly high exchange rate for the whole polypeptide chain, implying that free AtRGL1n without interacting partners is totally unfolded (Sheerin et al, 2011). …”

Functional analysis of synthetic DELLA domain peptides and bioactive gibberellin assay using surface plasmon resonance technology

“…All DELLA proteins consist of an N-terminal DELLA domain and a C-terminal GRAS domain [2,10]. The N-terminal DELLA domain includes two conserved motifs (DELLA and VHYNP) [2,3,6,11,12]. In Arabidopsis, DELLA proteins comprise five homologs (GAI, RGA, RGL1, RGL2 and RGL3) [1,11,[13][14][15].…”

“…Most of the studies focused on understanding the mechanisms of hormone metabolic pathways and signal transductions. Glutathione S-transgerase (GST)-fused SLR1 (the unique DELLA protein in rice) N-terminal fragment (SLR1 4-125 ) and maltosebinding protein (MBP)-tagged RGL1 N-terminal fragment (RGL1 1-137 ) were expressed in Escherichia coli and purified as ligands, which were respectively immobilized on the sensor chip surface using anti-GST and anti-BMP antibodies, and the SPR biosensor assay demonstrated the binding of SLR1 4-125 and RGL1 to GID1s in the presence of bioactive GAs [12,43,44]. However, the recombinant expression and purification of N-terminal fragment of the DELLA protein with high quality is a laborious and costly process, and the protein is prone to lose activity during the in vitro assay.…”