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Publikation

Dinesh, D. C.; Calderón Villalobos, L. I. A.; Abel, S.; Structural Biology of Nuclear Auxin Action Trends Plant Sci. 21, 302-316, (2016) DOI: 10.1016/j.tplants.2015.10.019

Auxin coordinates plant development largely via hierarchical control of gene expression. During the past decades, the study of early auxin genes paired with the power of Arabidopsis genetics have unraveled key nuclear components and molecular interactions that perceive the hormone and activate primary response genes. Recent research in the realm of structural biology allowed unprecedented insight into: (i) the recognition of auxin-responsive DNA elements by auxin transcription factors; (ii) the inactivation of those auxin response factors by early auxin-inducible repressors; and (iii) the activation of target genes by auxin-triggered repressor degradation. The biophysical studies reviewed here provide an impetus for elucidating the molecular determinants of the intricate interactions between core components of the nuclear auxin response module.
Publikation

Dinesh, D. C.; Kovermann, M.; Gopalswamy, M.; Hellmuth, A.; Calderón Villalobos, L. I. A.; Lilie, H.; Balbach, J.; Abel, S.; Solution structure of the PsIAA4 oligomerization domain reveals interaction modes for transcription factors in early auxin response Proc. Natl. Acad. Sci. U.S.A. 112, 6230-6235, (2015) DOI: 10.1073/pnas.1424077112

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 β-grasp fold with homologies to the Phox and Bem1p (PB1) domain. The PB1 domain of wild-type 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 (KD ∼6.4 μM) 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.
Publikation

Bürstenbinder, K.; Savchenko, T.; Müller, J.; Adamson, A. W.; Stamm, G.; Kwong, R.; Zipp, B. J.; Dinesh, D. C.; Abel, S.; Arabidopsis Calmodulin-binding Protein IQ67-Domain 1 Localizes to Microtubules and Interacts with Kinesin Light Chain-related Protein-1 J. Biol. Chem. 288, 1871-1882, (2013) DOI: 10.1074/jbc.M112.396200

Calcium (Ca2+) is a key second messenger in eukaryotes and regulates diverse cellular processes, most notably via calmodulin (CaM). In Arabidopsis thaliana, IQD1 (IQ67 domain 1) is the founding member of the IQD family of putative CaM targets. The 33 predicted IQD proteins share a conserved domain of 67 amino acids that is characterized by a unique arrangement of multiple CaM recruitment motifs, including so-called IQ motifs. Whereas IQD1 has been implicated in the regulation of defense metabolism, the biochemical functions of IQD proteins remain to be elucidated. In this study we show that IQD1 binds to multiple Arabidopsis CaM and CaM-like (CML) proteins in vitro and in yeast two-hybrid interaction assays. CaM overlay assays revealed moderate affinity of IQD1 to CaM2 (Kd ∼ 0.6 μm). Deletion mapping of IQD1 demonstrated the importance of the IQ67 domain for CaM2 binding in vitro, which is corroborated by interaction of the shortest IQD member, IQD20, with Arabidopsis CaM/CMLs in yeast. A genetic screen of a cDNA library identified Arabidopsis kinesin light chain-related protein-1 (KLCR1) as an IQD1 interactor. The subcellular localization of GFP-tagged IQD1 proteins to microtubules and the cell nucleus in transiently and stably transformed plant tissues (tobacco leaves and Arabidopsis seedlings) suggests direct interaction of IQD1 and KLCR1 in planta that is supported by GFP∼IQD1-dependent recruitment of RFP∼KLCR1 and RFP∼CaM2 to microtubules. Collectively, the prospect arises that IQD1 and related proteins provide Ca2+/CaM-regulated scaffolds for facilitating cellular transport of specific cargo along microtubular tracks via kinesin motor proteins.
Publikation

Schumann, N.; Navarro-Quezada, A.; Ullrich, K.; Kuhl, C.; Quint, M.; Molecular Evolution and Selection Patterns of Plant F-Box Proteins with C-Terminal Kelch Repeats Plant Physiol. 155, 835-850, (2011) DOI: 10.1104/pp.110.166579

The F-box protein superfamily represents one of the largest families in the plant kingdom. F-box proteins phylogenetically organize into numerous subfamilies characterized by their carboxyl (C)-terminal protein-protein interaction domain. Among the largest F-box protein subfamilies in plant genomes are those with C-terminal kelch repeats. In this study, we analyzed the phylogeny and evolution of F-box kelch proteins/genes (FBKs) in seven completely sequenced land plant genomes including a bryophyte, a lycophyte, monocots, and eudicots. While absent in prokaryotes, F-box kelch proteins are widespread in eukaryotes. Nonplant eukaryotes usually contain only a single FBK gene. In land plant genomes, however, FBKs expanded dramatically. Arabidopsis thaliana, for example, contains at least 103 F-box genes with well-conserved C-terminal kelch repeats. The construction of a phylogenetic tree based on the full-length amino acid sequences of the FBKs that we identified in the seven species enabled us to classify FBK genes into unstable/stable/superstable categories. In contrast to superstable genes, which are conserved across all seven species, kelch domains of unstable genes, which are defined as lineage specific, showed strong signatures of positive selection, indicating adaptational potential. We found evidence for conserved protein features such as binding affinities toward A. thaliana SKP1-like adaptor proteins and subcellular localization among closely related FBKs. Pseudogenization seems to occur only rarely, but differential transcriptional regulation of close relatives may result in subfunctionalization.
Publikation

Delker, C.; Pöschl, Y.; Raschke, A.; Ullrich, K.; Ettingshausen, S.; Hauptmann, V.; Grosse, I.; Quint, M.; Natural Variation of Transcriptional Auxin Response Networks in Arabidopsis thaliana Plant Cell 22, 2184-2200, (2010) DOI: 10.1105/tpc.110.073957

Natural variation has been observed for various traits in Arabidopsis thaliana. Here, we investigated natural variation in the context of physiological and transcriptional responses to the phytohormone auxin, a key regulator of plant development. A survey of the general extent of natural variation to auxin stimuli revealed significant physiological variation among 20 genetically diverse natural accessions. Moreover, we observed dramatic variation on the global transcriptome level after induction of auxin responses in seven accessions. Although we detect isolated cases of major-effect polymorphisms, sequencing of signaling genes revealed sequence conservation, making selective pressures that favor functionally different protein variants among accessions unlikely. However, coexpression analyses of a priori defined auxin signaling networks identified variations in the transcriptional equilibrium of signaling components. In agreement with this, cluster analyses of genome-wide expression profiles followed by analyses of a posteriori defined gene networks revealed accession-specific auxin responses. We hypothesize that quantitative distortions in the ratios of interacting signaling components contribute to the detected transcriptional variation, resulting in physiological variation of auxin responses among accessions.
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