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Publikation

Winkler, M., Niemeyer, M., Hellmuth, A., Janitza, P., Christ, G., Samodelov, S. L., Wilde, V., Majovsky, P., Trujillo, M., Zurbriggen, M. D., Hoehenwarter, W., Quint, M. & Calderón Villalobos, L. I. A. Variation in auxin sensing guides AUX/IAA transcriptional repressor ubiquitylation and destruction. Nature Commun. 8, 15706, (2017) DOI: 10.1038/ncomms15706

Auxin is a small molecule morphogen that bridges SCFTIR1/AFB-AUX/IAA co-receptor interactions leading to ubiquitylation and proteasome-dependent degradation of AUX/IAA transcriptional repressors. Here, we systematically dissect auxin sensing by SCFTIR1-IAA6 and SCFTIR1-IAA19 co-receptor complexes, and assess IAA6/IAA19 ubiquitylation in vitro and IAA6/IAA19 degradation in vivo. We show that TIR1-IAA19 and TIR1-IAA6 have distinct auxin affinities that correlate with ubiquitylation and turnover dynamics of the AUX/IAA. We establish a system to track AUX/IAA ubiquitylation in IAA6 and IAA19 in vitro and show that it occurs in flexible hotspots in degron-flanking regions adorned with specific Lys residues. We propose that this signature is exploited during auxin-mediated SCFTIR1-AUX/IAA interactions. We present evidence for an evolving AUX/IAA repertoire, typified by the IAA6/IAA19 ohnologues, that discriminates the range of auxin concentrations found in plants. We postulate that the intrinsic flexibility of AUX/IAAs might bias their ubiquitylation and destruction kinetics enabling specific auxin responses.
Bücher und Buchkapitel

Hellmuth, A. & Calderón Villalobos, L. I. A. Radioligand Binding Assays for Determining Dissociation Constants of Phytohormone Receptors. In: Plant Proteostasis   (Lois, L. M.; Matthiesen, R. ). Meth. Mol. Biol 1450, 23-34, (2016) ISBN: 978-1-4939-3757-8 DOI: 10.1007/978-1-4939-3759-2_3

In receptor–ligand interactions, dissociation constants provide a key parameter for characterizing binding. Here, we describe filter-based radioligand binding assays at equilibrium, either varying ligand concentrations up to receptor saturation or outcompeting ligand from its receptor with increasing concentrations of ligand analogue. Using the auxin coreceptor system, we illustrate how to use a saturation binding assay to determine the apparent dissociation constant (K D ′ ) for the formation of a ternary TIR1–auxin–AUX/IAA complex. Also, we show how to determine the inhibitory constant (K i) for auxin binding by the coreceptor complex via a competition binding assay. These assays can be applied broadly to characterize a one-site binding reaction of a hormone to its receptor.

Publikationen in Druck

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

Moss, B. L., Mao, H., Guseman, J. M., Hinds, T. R., Hellmuth, A., Kovenock, M., Noorassa, A., Lanctot, A., Calderón Villalobos, L. I. A., Zheng, N. & Nemhauser, J. Rate motifs tune Auxin/Indole-3-Acetic acid degradation dynamics. Plant Physiol. 169, 803-813, (2015) DOI: 10.1104/pp.15.00587

Ubiquitin-mediated protein degradation is a common feature in diverse plant cell signaling pathways; however, the factors that control the dynamics of regulated protein turnover are largely unknown. One of the best-characterized families of E3 ubiquitin ligases, SCFTIR1/AFBs, facilitates ubiquitination of Aux/IAA repressor proteins in the presence of auxin. Rates of auxin-induced degradation vary widely within the Aux/IAA family, and sequences outside of the characterized degron (the minimum region required for auxin-induced degradation) can accelerate or decelerate degradation. We have used synthetic auxin degradation assays in yeast and in plants to characterize motifs flanking the degron that contribute to tuning the dynamics of Aux/IAA degradation. The presence of these "rate motifs" is conserved in phylogenetically-distant members of the Arabidopsis thaliana Aux/IAA family, as well as in their putative Brassica rapa orthologs. We found that rate motifs can act by enhancing interaction between repressors and the E3, but that this is not the only mechanism of action. Phenotypes of transgenic plants expressing a deletion in a rate motif in IAA28 resembled plants expressing degron mutations, underscoring the functional relevance of Aux/IAA degradation dynamics in regulating auxin responses

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 PNAS 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

Guseman, J. M., Hellmuth, A., Lanctot, A., Feldman, T. P., Moss, B. L., Klavins, E., Calderón Villalobos, L. I. A. & Nemhauser, J. L. Auxin-induced degradation dynamics set the pace for lateral root development Development 142, 1-5, (2015) DOI: 10.1242/dev.117234


Auxin elicits diverse cell behaviors through a simple nuclear signaling pathway initiated by degradation of Aux/IAA co-repressors. Our previous work revealed that members of the large Arabidopsis Aux/IAA family exhibit a range of degradation rates in synthetic contexts. However, it remained an unresolved issue whether differences in Aux/IAA turnover rates played a significant role in plant responses to auxin. Here, we use the well-established model of lateral root development to directly test the hypothesis that the rate of auxin-induced Aux/IAA turnover sets the pace for auxin-regulated developmental events. We did this by generating transgenic plants expressing degradation rate variants of IAA14, a crucial determinant of lateral root initiation. Progression through the well-established stages of lateral root development was strongly correlated with the engineered rates of IAA14 turnover, leading to the conclusion that Aux/IAAs are auxin-initiated timers that synchronize developmental transitions

Publikationen in Druck

Bagchi, R., Melnyk, C. W., Christ, G., Winkler, M., Kirchsteiner, K., Salehin, M., Mergner, J., Niemeyer, M., Schwechheimer, C., Calderón Villalobos, L. I. A. & Estelle, M. . In:  The Arabidopsis ALF4 protein is a regulator of SCF E3 ligases.  EMBO J e201797159, DOI: 10.15252/embj.201797159

The cullin-RING E3 ligases (CRLs) regulate diverse cellular processes in all eukaryotes. CRL activity is controlled by several proteins or protein complexes, including NEDD8, CAND1, and the CSN. Recently, a mammalian protein called Glomulin (GLMN) was shown to inhibit CRLs by binding to the RING BOX (RBX1) subunit and preventing binding to the ubiquitin-conjugating enzyme. Here, we show that Arabidopsis ABERRANT LATERAL ROOT FORMATION4 (ALF4) is an ortholog of GLMN. The alf4 mutant exhibits a phenotype that suggests defects in plant hormone response. We show that ALF4 binds to RBX1 and inhibits the activity of SCFTIR1, an E3 ligase responsible for degradation of the Aux/IAA transcriptional repressors. In vivo, the alf4 mutation destabilizes the CUL1 subunit of the SCF. Reduced CUL1 levels are associated with increased levels of the Aux/IAA proteins as well as the DELLA repressors, substrate of SCFSLY1. We propose that the alf4 phenotype is partly due to increased levels of the Aux/IAA and DELLA proteins.
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