Structural basis for Auxin induced transcription regulator interaction and the control of auxin response

Abstract

The plant hormone auxin is involved in all stages of plant development. Aux/IAAs are the transcriptional repressors that bind to the Auxin Response Factors (ARFs) to regulate the gene expression upon auxin release. Aux/IAA have highly conserved C-terminal domains (domains III-IV) that mediate both homotypic and heterotypic interactions between Aux/IAA and ARF family proteins. It has been known that Aux/IAA and ARF form homo- and hetero-oligomers for the transcriptional regulation, but what determines their association states is poorly understood. Here I report, to our knowledge, the first solution structure of domain III−IV of Aux/IAA17 (IAA17), and characterize molecular interactions underlying the homotypic and heterotypic oligomerization. The structure exhibits a compact β-grasp fold with a highly dynamic insert helix that is unique in Aux/IAA family proteins. The insert helix exhibited fast motions in the ps-ns time scale from 15N relaxation data, but the amplitude of the motion is likely limited to the local neighborhood. IAA17 associates to form a heterogeneous ensemble of front-to-back oligomers in a concentration-dependent manner. IAA17 and ARF5 associate to form homo- or hetero-oligomers using a common scaffold and binding interfaces, but their affinities vary significantly. The equilibrium dissociation constants (KD) for homo-oligomerization are 6.6 μM and 0.87 μM for IAA17 and ARF5, respectively, whereas hetero-oligomerization reveals a ∼10- to ∼100-fold greater affinity (KD = 73 nM). Thus, individual homo-oligomers of IAA17 and ARF5 spontaneously exchange their subunits to form alternating hetero-oligomers for transcriptional repression. Oligomerization is mainly driven by electrostatic interactions, so that charge complementarity at the interface determines the binding affinity. The heterotypic association between ARF1 repressor and Aux/IAA17 was weaker than individual homotypic associations, in contrast to the preferred heterotypic association between ARF5 and Aux/IAA17. This finding suggests that ARF repressors do not respond to auxin, simply competing with ARF activators for binding to the auxin response element. Selective binding might be a general strategy of auxin response transcriptional regulation in plants.