Molecular basis for AUXIN RESPONSE FACTOR protein interaction and the control of auxin response repression

Abstract

In plants, the AUXIN RESPONSE FACTOR (ARF) transcription factor family regulates gene expression in response to auxin. In the absence of auxin, ARF transcription factors are repressed by interaction with AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) proteins. Although the C termini of ARF and Aux/IAA proteins facilitate their homo- and heterooligomerization, the molecular basis for this interaction remained undefined. The crystal structure of the C-terminal interaction domain of Arabidopsis ARF7 reveals a Phox and Bem1p (PB1) domain that provides both positive and negative electrostatic interfaces for directional protein interaction. Mutation of interface residues in the ARF7 PB1 domain yields monomeric protein and abolishes interaction with both itself and IAA17. Expression of a stabilized Aux/IAA protein (i.e., IAA16) bearing PB1 mutations in Arabidopsis suggests a multimerization requirement for ARF protein repression, leading to a refined auxin-signaling model.

Fig. 1.

ARF7 contains a C-terminal type I/II PB1 domain. (A) In the current auxin-signaling model, Aux/IAAs dimerize with and repress ARF transcription factors in the absence of auxin. In the presence of auxin, Aux/IAAs interact with SCF^TIR1 resulting in repressor degradation, freeing ARFs for auxin-responsive gene transcription. (B) Sequence alignment of ARF and Aux/IAA proteins identify canonical PB1 domain features, including a conserved lysine (blue) and the OPCA-like motif (red) that align with type I (p67phox and Cdc24p) and type II (p40phox) PB1 domains. The ARF and Aux/IAA PB1 domain variable region (var.) is also indicated. (C) The ARF7PB1 crystal structure reveals that the ARF7 C terminus is a PB1 domain. Secondary structure features are labeled and colored as in B with modeled lysine (blue) and OPCA (red) residues shown as stick representations. (D) ARF7PB1 structural features define ARF and Aux/IAA sequence motifs III and IV, which are colored as in B.

Fig. 2.

ARF and Aux/IAA PB1 domain interactions are driven by charge–charge interactions. (A) The modeled ARF7PB1 electrostatic surface potential reveals positive (blue) and negative (red) interaction interface, containing the invariant lysine and OPCA motif, respectively. (B) PISA predictions of residue contributions to interactions identified the ARF7PB1 interaction interface containing the invariant lysine (blue), OPCA motif (red), and residues important for hydrogen bonding (orange) and charge–charge interactions (green). (C) Size-exclusion chromatography reveals that wild-type ARF7Cterm exists as soluble aggregate in solution (black), whereas mutations in the invariant lysine (blue), the OPCA motif (red), or both (yellow) afford monomeric protein. (D) Size-exclusion chromatography reveals that mixing ARF7Cterm^K1042A (blue) and ARF7Cterm^opca (red), which exist as monomeric protein in solution, results in the formation of a dimer (ARF7Cterm^K1042A and ARF7Cterm^opca mixed in a 1:1 ratio, purple). Inset, protein molecular weight standards. (E) Yeast two-hybrid assays reveal that introduction of single unlike PB1 mutations in ARF7 and IAA17 does not affect ARF–ARF or ARF–Aux/IAA interactions. Combining both PB1 mutations (lysine and opca) or like mutations—e.g., both proteins contain lysine or OPCA mutations—abrogates protein–protein interactions. BD, binding domain; AD, activation domain.

Fig. 3.

ARF7 PB1 domains formed an extended directional pentamer within the crystal. (A) The ARF7PB1 crystal contains an oriented ARF7PB1 domain pentamer of chains A, B, L, O, and P. (B) Stereo view of ARF7PB1 pentamer topology suggests formation of a curved helix multimer. The N-terminal residue of each chain is shown as a space-filling model.

Fig. 4.

Aux/IAA PB1 domain dimerization is insufficient for ARF repression in Arabidopsis. (A) Models representing stabilized iaa16-1 with a single-interaction interface. (B) Histograms and (C) representative photographs of rosette diameters from wild-type plants overexpressing IAA16 (gray; n = 117), iaa16-1 (black; n = 47), iaa16-1^K122A (blue; n = 117), or iaa16-1^opca (red; n = 80) reveal that mutation of the PB1 invariant lysine or OPCA motif abrogates iaa16-1 low-auxin phenotypes.

Fig. 5.

A refined auxin-signaling model. Higher-order oligomerization of Aux/IAA proteins may be required for ARF repression. In the presence of auxin, Aux/IAA proteins are targeted for degradation by the SCF^TIR1 complex, freeing ARF proteins for auxin-responsive gene transcription. ARF–ARF interactions can occur through the DBD (28) and/or through the PB1 domain.