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Publikation

Niemeyer, M.; Moreno Castillo, E.; Ihling, C. H.; Iacobucci, C.; Wilde, V.; Hellmuth, A.; Hoehenwarter, W.; Samodelov, S. L.; Zurbriggen, M. D.; Kastritis, P. L.; Sinz, A.; Calderón Villalobos, L. I. A.; Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin co-receptor assemblies Nat. Commun. 11, 2277, (2020) DOI: 10.1038/s41467-020-16147-2

Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their AUX/IAA targets perceive the phytohormone auxin. The F-box protein TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, by adopting biochemical, structural proteomics and in vivo approaches we unveil how flexibility in AUX/IAAs and regions in TIR1 affect their conformational ensemble allowing surface accessibility of degrons. We resolve TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron’s vicinity, cooperatively position AUX/IAAs on TIR1. We identify essential residues at the TIR1 N- and C-termini, which provide non-native interaction interfaces with IDRs and the folded PB1 domain of AUX/IAAs. We thereby establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for a multiplicity of functional states.
Preprints

Niemeyer, M.; Moreno Castillo, E.; Ihling, C. H.; Iacobucci, C.; Wilde, V.; Hellmuth, A.; Hoehenwarter, W.; Samodelov, S. L.; Zurbriggen, M. D.; Kastritis, P. L.; Sinz, A.; Calderón Villalobos, L. I. A.; Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin receptor assemblies bioRxiv (2019) DOI: 10.1101/787770

Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their ubiquitylation targets, AUX/IAAs, sense auxin concentrations in the nucleus. TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, we resolved TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron′s vicinity, cooperatively position AUX/IAAs on TIR1. The AUX/IAA PB1 interaction domain also assists in non-native contacts, affecting AUX/IAA dynamic interaction states. Our results establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation and might provide conformational flexibility for adopting a multiplicity of functional states. We postulate IDRs in distinct members of the AUX/IAA family to be an adaptive signature for protein interaction and initiation region for proteasome recruitment.
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 Nat. 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.
Publikation

López-Carrasco, A.; Ballesteros, C.; Sentandreu, V.; Delgado, S.; Gago-Zachert, S.; Flores, R.; Sanjuán, R.; Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing PLOS Pathog. 13, e1006547, (2017) DOI: 10.1371/journal.ppat.1006547

Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplast-replicating viroid. However, whether this is a general feature of viroids remains unclear. Here, we have used high-fidelity ultra-deep sequencing to determine the mutation rate in a common host (eggplant) of two viroids, each representative of one family: the chloroplastic eggplant latent viroid (ELVd, Avsunviroidae) and the nuclear potato spindle tuber viroid (PSTVd, Pospiviroidae). This revealed higher mutation frequencies in ELVd than in PSTVd, as well as marked differences in the types of mutations produced. Rates of spontaneous mutation, quantified in vivo using the lethal mutation method, ranged from 1/1000 to 1/800 for ELVd and from 1/7000 to 1/3800 for PSTVd depending on sequencing run. These results suggest that extremely high mutability is a common feature of chloroplastic viroids, whereas the mutation rates of PSTVd and potentially other nuclear viroids appear significantly lower and closer to those of some RNA viruses.
Publikation

López-Carrasco, A.; Gago-Zachert, S.; Mileti, G.; Minoia, S.; Flores, R.; Delgado, S.; The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in their in vivo RNA conformations RNA Biol. 13, 83-97, (2016) DOI: 10.1080/15476286.2015.1119365

Eggplant latent viroid (ELVd), like other members of family Avsunviroidae, replicates in plastids through a symmetric rolling-circle mechanism in which elongation of RNA strands is most likely catalyzed by a nuclear-encoded polymerase (NEP) translocated to plastids. Here we have addressed where NEP initiates transcription of viroid strands. Because this step is presumably directed by sequence/structural motifs, we have previously determined the conformation of the monomeric linear (+) and (−) RNAs of ELVd resulting from hammerhead-mediated self-cleavage. In silico predictions with 3 softwares led to similar bifurcated conformations for both ELVd strands. In vitro examination by non-denaturing PAGE showed that they migrate as prominent single bands, with the ELVd (+) RNA displaying a more compact conformation as revealed by its faster electrophoretic mobility. In vitro SHAPE analysis corroborated the ELVd conformations derived from thermodynamics-based predictions in silico. Moreover, sequence analysis of 94 full-length natural ELVd variants disclosed co-variations, and mutations converting canonical into wobble pairs or vice versa, which confirmed in vivo most of the stems predicted in silico and in vitro, and additionally helped to introduce minor structural refinements. Therefore, results from the 3 experimental approaches were essentially consistent among themselves. Application to RNA preparations from ELVd-infected tissue of RNA ligase-mediated rapid amplification of cDNA ends, combined with pretreatments to modify the 5′ ends of viroid strands, mapped the transcription initiation sites of ELVd (+) and (−) strands in vivo at different sequence/structural motifs, in contrast with the situation previously observed in 2 other members of the family Avsunviroidae.
Bücher und Buchkapitel

Hellmuth, A.; Calderón Villalobos, L. I. A.; Radioligand Binding Assays for Determining Dissociation Constants of Phytohormone Receptors (Lois, L. M. & Matthiesen, R., eds.). Methods Mol. Biol. 1450, 23-34, (2016) ISBN: 978-1-4939-3759-2 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 (Ki) 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.
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

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, 905-909, (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.
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. L.; 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 facilitates ubiquitination of auxin (aux)/indole-3-acetic acid (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 (Saccharomyces cerevisiae) 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 (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

Flores, R.; Grubb, D.; Elleuch, A.; Nohales, M.-?.; Delgado, S.; Gago, S.; Rolling-circle replication of viroids, viroid-like satellite RNAs and hepatitis delta virus: Variations on a theme RNA Biol. 8, 200-206, (2011) DOI: 10.4161/rna.8.2.14238

Viroids and viroid-like satellite RNAs from plants, and the human hepatitis delta virus (HDV) RNA share some properties that include small size, circularity and replication through a rolling-circle mechanism. Replication occurs in different cell compartments (nucleus, chloroplast and membrane-associated cytoplasmatic vesicles) and has three steps: RNA polymerization, cleavage and ligation. The first step generates oligomeric RNAs that result from the reiterative transcription of the circular templates of one or both polarities, and is catalyzed by either the RNA-dependent RNA polymerase of the helper virus on which viroid-like satellite RNAs are functionally dependent, or by host DNA-dependent RNA polymerases that, remarkably, viroids and HDV redirect to transcribe RNA templates. Cleavage is mediated by host enzymes in certain viroids and viroid-like satellite RNAs, while in others and in HDV is mediated by cis-acting ribozymes of three classes. Ligation appears to be catalyzed mainly by host enzymes. Replication most likely also involves many other non-catalytic proteins of host origin and, in HDV, the single virus-encoded protein.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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