title: 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.
authors.affiliation: Institute of Molecular Biosciences, Massey University, Private Bag 11 222, Palmerston North, New Zealand.
authors.forename: David J
authors.initials: DJ
authors.lastname: Sheerin
authors.forename: Jeremy
authors.initials: J
authors.lastname: Buchanan
authors.forename: Chris
authors.initials: C
authors.lastname: Kirk
authors.forename: Dawn
authors.initials: D
authors.lastname: Harvey
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Sun
authors.forename: Julian
authors.initials: J
authors.lastname: Spagnuolo
authors.forename: Sheng
authors.initials: S
authors.lastname: Li
authors.forename: Tong
authors.initials: T
authors.lastname: Liu
authors.forename: Virgil A
authors.initials: VA
authors.lastname: Woods
authors.forename: Toshi
authors.initials: T
authors.lastname: Foster
authors.forename: William T
authors.initials: WT
authors.lastname: Jones
authors.forename: Jasna
authors.initials: J
authors.lastname: Rakonjac
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D005875:Gibberellins / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins
chemical_list: D029681:Arabidopsis Proteins; C512225:GID1a protein, Arabidopsis; D005875:Gibberellins; C450824:RGL1 protein, Arabidopsis; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C108186:gibberellin 3beta-hydroxylase