Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Ensured auxin sensing by degron flexibility

ABSTRACT: Using a structural proteomics approach via chemical cross-linking combined with mass spectrometry (XL-MS), we have resolved SCFTIR auxin·AUX/IAA complex overall topology, and established the power of IDRs modulating auxin receptor assemblies.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Michael Niemeyer

LAB HEAD: Luz Irina Calderón Villalobos

PROVIDER: PXD015285 | Pride | 2020-05-11

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	12rep1_minIAA_minT1.raw	Raw
	12rep1_minIAA_plusT1.raw	Raw
	12rep1_minlAA_minT1.mzML	Mzml
	12rep1_minlAA_plusT1.mzML	Mzml
	12rep1_plusIAA_minT1.raw	Raw

Items per page:

1 - 5 of 69

Publications

Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin co-receptor assemblies.

Niemeyer Michael M Moreno Castillo Elena E Ihling Christian H CH Iacobucci Claudio C Wilde Verona V Hellmuth Antje A Hoehenwarter Wolfgang W Samodelov Sophia L SL Zurbriggen Matias D MD Kastritis Panagiotis L PL Sinz Andrea A Calderón Villalobos Luz Irina A LIA

Nature communications 20200508 1

Cullin RING-type E3 ubiquitin ligases SCF<sup>TIR1/AFB1-5</sup> 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 ...[more]

PMID: 32385295

Similar Datasets

Epitope and Paratope Mapping of TNFα/Infliximab complex by cross-linking mass spectrometry and integrative modeling reveals high level of interaction specificity

Project description:Mass spectrometry (MS) has become one of the core technologies to study protein structures, protein-ligand and protein-protein interactions. Chemical cross-linking combined with mass spectrometry (XL-MS) is quickly spreading over different biochemistry laboratories for the continuous increase of its biological applications and standardized protocols that make it attractive for academia and industry research scientists. In recent years, XL-MS has developed as a powerful technique in structural biology, from studying structures of purified proteins in vitro to deciphering intricate protein-protein interaction (PPI) networks in cells, organelles and tissues on a system-wide scale. However, the number of XL-MS studies for the inves-tigation of epitope/paratope mapping of antigen-antibody complexes is still limited up to now. In this manuscript, we devel-oped a simple, fast and automated workflow to define the interaction interface between the chimeric antibody Infliximab (IFX) and its own target, the Tumor Necrosis Factor alpha (TNFα). This workflow integrates XL-MS data to identify areas in proximity to the binding regions and epitope/paratope probability calculation to pinpoint the antigen-antibody binding sites. The data are combined to generate refined 3D models of the antigen-antibody complex which closely resemble the X-ray IFX/TNFα structure and capture their correct interaction surface. The present workflow can be applied to investigate any antigen-antibody complex, even when no previous structural knowledge is available. This strategy is a fascinating oppor-tunity to thoroughly characterize antigen-antibody interactions and to allow the understanding of their binding mechanisms in order to properly design better antibody therapeutics in the future

2024-02-27 | PXD043347 | Pride

In vivo and in vitro characterization and structural analysis of human DPCD protein in complex with RUVBL1/RUVBL2 AAA-ATPases

Project description:DPCD is a protein that may play a role in cilia formation and whose absence leads to primary ciliary dyskinesia (PCD), a rare disease caused by impairment of ciliated cells. Except for high-throughput studies that identified DPCD as a possible RUVBL1 (R1) and RUVBL2 (R2) partner, no in-depth cellular, biochemical, and structural investigations involving DPCD have been reported so far. R1 and R2 proteins are ubiquitous highly conserved AAA+ family ATPases that assemble and mature a plethora of macromolecular complexes and are pivotal in numerous cellular processes, especially by guaranteeing a co-chaperoning function within R2TP or R2TP-like machineries. In the present study, we identified DPCD as a new R1R2 partner in vivo. We show that DPCD interacts directly with R1 and R2 in vitro and in cells. We characterized the physico-chemical properties of DPCD in solution and built 3D structural models of DPCD alone and in complex with R1R2 validated by experimental data combining Small-Angle X-ray Scattering and Cross-linking Mass Spectrometry, among others. Interestingly, DPCD disrupts the dodecameric state of R1R2 complex upon binding and this interaction occurs mainly via the DII domains of R1R2.

2023-04-06 | PXD031339 | Pride

RNxQuest: an extension to the xQuest pipeline enabling analysis of protein-RNA cross-linking/mass spectrometry data

Project description:The recently introduced cross-linking of isotope-labelled RNA coupled with mass spectrometry (CLIR-MS) technique enables protein-RNA cross-links to be used as precisely localized distance restraints in de novo structural modelling. The novel data type requires a bespoke data analysis approach. The new RNxQuest Python package supports this approach. Here we demonstrate the performance of the new package using a mixture of novel and published datasets.

2023-09-06 | PXD039754 | Pride

2) Twisting Urea- to Imide-Based Mass Spectrometry-Cleavable Cross-Linkers to Embed Affinity Tags

Project description:Disuccinimidyl dibutyric urea (DSBU) is a mass spectrometry (MS)-cleavable cross-linker that has been applied to multiple applications in structural biology, ranging from isolated protein complexes to comprehensive system-wide interactomics. DSBU facilitates a rapid and reliable identification of cross-links through the dissociation of its urea group in the gas-phase. In this study, we further advance the structural capabilities of DSBU by twisting the urea group into an imide, thus introducing a novel class of cross-linkers. This modification preserves the MS-cleavability of the amide bond, granted by the two acyl groups of the imide function. The central nitrogen atom enables the introduction of affinity purification tags. Here, we introduce disuccinimidyl dibutyric imide (DSBI) as prototype of this class of cross-linkers. It features a phosphonate handle for immobilized metal ion affinity chromatography (IMAC) enrichment. We detail DSBI synthesis and describe its behavior in solution and in the gas-phase while cross-linking isolated proteins and human cell lysates. DSBI and DSBU cross-links are compared at the same enrichment depths to bridge these two cross-linker classes. We validate DSBI cross-links by mapping them in high-resolution structures of large protein assemblies . The cross-links observed yield insights into the morphology of intrinsically disordered proteins (IDPs) and their complexes. The DSBI linker might spearhead a novel class of MS-cleavable and enrichable cross-linkers

2024-09-03 | PXD050960 | Pride

First direct observation of elongated conformational states in alpha-synuclein upon liquid-liquid phase separation

Project description:α-Synuclein (α-syn) is an intrinsically disordered protein (IDP) that undergoes liquid-liquid phase separation (LLPS), fibrillation, and forms insoluble intracellular Lewy’s bodies in neurons, which are the hallmark of Parkinson’s Disease (PD). Neurotoxicity precedes the formation of aggregates and is probably related to LLPS of α-syn in the cell. The molecular mechanisms underlying the early stages of LLPS are still elusive. To obtain structural insights into α-syn upon LLPS, we take advantage of cross-linking/mass spectrometry (XL-MS) and introduced an innovative new approach, termed COMPASS (COMPetitive PAiring StatisticS). COMPASS unravels transient interactions between α-syn molecules in liquid droplets to derive structural information of α-syn upon LLPS. In this work, we show that the conformational ensemble of α-syn shifts towards more elongated conformational states upon LLPS. We obtain insights into the critical initial stages of PD and establish a novel mass spectrometry-based approach that will aid to solve open questions in LLPS structural biology.

2024-03-18 | PXD033205 | Pride

"In-gel cross-linking mass spectrometry (IGX-MS) of proteins and protein complexes"

Project description:Cross-linking mass spectrometry has developed into an important method to study protein structures and interactions. The in-solution cross-linking workflows involve time and sample consuming steps and do not provide sensible solutions for differentiating cross-links obtained from co-occurring protein oligomers, complexes, or conformers. Here we developed a cross-linking workflow combining blue native PAGE with in-gel cross-linking mass spectrometry (IGX-MS). This workflow circumvents steps, such as buffer exchange and cross-linker concentration optimization. Additionally, IGX-MS enables the parallel analysis of co-occurring protein complexes using only small amounts of sample. Another benefit of IGX-MS observed by experiments on GroEL and purified bovine heart mitochondria, is the substantial reduction of artificial over-length cross-links when compared to in-solution cross-linking. We next used IGX-MS to investigate the complement components C5, C6, and their hetero-dimeric C5b6 complex. The obtained cross-links were used to generate a refined structural model of the complement component C6, resembling C6 in its inactivated state. This finding shows that IGX-MS can be used to provide new insights into the initial stages of the terminal complement pathway.

2020-12-14 | PXD020014 | Pride

Structural basis for the assembly of Mis18a/b core modules

Project description:The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18a, Mis18b and Mis18BP1), which recruits the CENP-A specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18a, 2 Mis18b and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18a can associate with centromeres and deposit CENP-A independently of Mis18b, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.

2024-05-15 | PXD047345 | Pride

In vivo protein interaction network analysis reveals porin-localized toxin inactivation in antibiotic resistant bacteria

Project description:The nosocomial pathogen Acinetobacter baumannii is a frequent cause of hospital acquired infections worldwide, and a challenge for treatment due to its evolved resistance to antibiotics, including carbapenems. To gain insight on A. baumannii antibiotic resistance mechanisms, we analyzed the protein interaction network of a multidrug-resistant A. baumannii clinical strain Ab5075. Using in vivo chemical cross-linking and mass spectrometry, we identified 2,068 non-redundant cross-linked peptide pairs containing 245 intra- and 398 inter- molecular interactions. Outer membrane proteins OmpA and YiaD, and carbapenemase Oxa-23 are hubs of the identified interaction network. Eighteen novel interactors of Oxa-23 were identified. Interactions of Oxa-23 with outer membrane porins OmpA and CarO were verified with co-immunoprecipitation analysis. Furthermore, transposon mutagenesis of oxa-23 or interactors of Oxa-23 demonstrated changes in meropenem or imipenem sensitivity in Ab5075. These results provide the first view of a porin-localized toxin inactivation model and increase understanding of bacterial antibiotic resistance mechanisms.

2016-12-22 | PXD004236 | Pride

An experimentally generated peptide database increases the sensitivity in XL-MS of complex samples

Project description:The application of non-cleavable cross-linkers to complex samples in XL-MS workflows is limited by the n² problem, a quadratic expansion of the search space with increasing database size. Here, a peptide-focused approach is proposed, for which cross-linking peptide candidates are identified in a parallel experiment by using a thiol-cleavable cross-linker with equal reactivity. Samples are reduced and alkylated, thereby releasing cross-linked peptides with a variable modification on the initially cross-linked residue. Modified peptides are identified by database search and concatenated to a peptide database, which is finally used for the analysis of the sample cross-linked with a non-cleavable cross-linker. This way, the search space is dramatically reduced leading to a higher sensitivity, because there is less chance for random hits to false positive sequences. The approach was benchmarked on cross-linked purified protein complexes (20 S Proteasome, yeast PolII, and TFIIH), and in vivo cross-linked bacteria (Bacillus subtilis, Bacillus cereus) by comparing it to the conventional approach of searching against the protein sequences.

2020-03-26 | PXD016554 | Pride

Cellular interactome dynamics during heat stress

Project description:Quantitative in vivo cross-linking-mass spectrometry was used to identify significant time-dependent interactome changes that occur prior to large-scale proteome abundance remodeling in cells subjected to heat stress. Interactome changes were identified within minutes of applied heat stress, including changes in chaperone systems as expected due to altered functional demand. Global analysis of all interactome changes revealed the largest significant enrichment in the class of RNA binding proteins, providing new time-dependent conformational insight on complex relationships that exist between transcription, translation and cellular stress response mechanisms upstream of transcriptional reprogramming. Observed interactome dynamics of RNA binding proteins that are also HSP90 clients constitute mechanistic changes that increase HSP90 functional demand, evident from observed HSP90 interactome changes early in heat stress. However, these changes together with observed heat stress-induced ATP level reduction result in observed complex time-dependent HSP90 interactome dynamics, indicating HSP90 conformational bottleneck formation with increased stress duration.

2024-11-25 | PXD058191 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data