期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 112, 期 19, 页码 6230-6235出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1424077112
关键词
auxin signaling; transcription factors; AUX/IAA proteins; PB1 domain; NMR structure
资金
- Deutsche Forschungsgemeinschaft Research Training School [GRK 1026]
- Federal Republic of Germany
- state of Saxony-Anhalt
- European Regional Development Fund
The plant hormone auxin activates primary response genes by facilitating proteolytic removal of AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA)-inducible repressors, which directly bind to transcriptional AUXIN RESPONSE FACTORS (ARF). Most AUX/IAA and ARF proteins share highly conserved C-termini mediating homotypic and heterotypic interactions within and between both protein families. The high-resolution NMR structure of C-terminal domains III and IV of the AUX/IAA protein PsIAA4 from pea (Pisum sativum) revealed a globular ubiquitin-like beta-grasp fold with homologies to the Phox and Bem1p (PB1) domain. The PB1 domain of wildtype PsIAA4 features two distinct surface patches of oppositely charged amino acid residues, mediating front-to-back multimerization via electrostatic interactions. Mutations of conserved basic or acidic residues on either face suppressed PsIAA4 PB1 homo-oligomerization in vitro and confirmed directional interaction of full-length PsIAA4 in vivo (yeast two-hybrid system). Mixing of oppositely mutated PsIAA4 PB1 monomers enabled NMR mapping of the negatively charged interface of the reconstituted PsIAA4 PB1 homodimer variant, whose stoichiometry (1:1) and equilibrium binding constant (K-D similar to 6.4 mu M) were determined by isothermal titration calorimetry. In silico protein-protein docking studies based on NMR and yeast interaction data derived a model of the PsIAA4 PB1 homodimer, which is comparable with other PB1 domain dimers, but indicated considerable differences between the homodimeric interfaces of AUX/IAA and ARF PB1 domains. Our study provides an impetus for elucidating the molecular determinants that confer specificity to complex protein-protein interaction circuits between members of the two central families of transcription factors important to the regulation of auxin-responsive gene expression.
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