Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structure of the DDB1-AMBRA1 E3 ligase receptor complex linked to cell cycle regulation

ABSTRACT: AMBRA1 is a tumor suppressor protein that functions as a substrate receptor of the ubiquitin conjugation system with roles in autophagy and the cell cycle regulatory network. The intrinsic disorder of AMBRA1 has thus far precluded its structural determination. To solve this problem, we analyzed the dynamics of AMBRA1 using hydrogen deuterium exchange mass spectrometry (HDX-MS). The HDX results indicated that AMBRA1 is a highly flexible protein and can be stabilized upon interaction with DDB1, the adaptor of the Cullin4A/B E3 ligase. Here, we present the cryo-EM structure of AMBRA1 in complex with DDB1 at 3.08 Å resolution. The structure shows that parts of the N- and C-terminal structural regions in AMBRA1 fold together into the highly dynamic WD40 domain and reveals how DDB1 engages with AMBRA1 to create a binding scaffold for substrate recruitment. The N-terminal helix-loop-helix motif and WD40 domain of AMBRA1 associate with the double-propeller fold of DDB1. We also demonstrate that DDB1 binding-defective AMBRA1 mutants prevent ubiquitination of the substrate Cyclin D1 in vitro and increase cell cycle progression. Together, these results provide structural insights into the AMBRA1-ubiquitin ligase complex and suggest a mechanism by which AMBRA1 acts as a hub involved in various physiological processes.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Goran Stjepanovic

LAB HEAD: Goran Stjepanovic

PROVIDER: PXD046056 | Pride | 2023-11-16

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	AMBRA1_0s.raw	Raw
	AMBRA1_10s_replicate1.raw	Raw
	AMBRA1_10s_replicate2.raw	Raw
	AMBRA1_10s_replicate3.raw	Raw
	AMBRA1_15min_replicate1.raw	Raw

Items per page:

1 - 5 of 39

Similar Datasets

Study of the TRAPP complexes by HDX-MS

Project description:Use of HDX-MS to study the allosteric and structural differences between the TRAPPII and TRAPPIII complex in the presence and absence of membrane and rab GTPases

2021-09-22 | PXD025928 | Pride

Ligand induced conformational changes in the B1-Adrenergic Receptor Revealed by Hydrogen-Deuterium Exchange Mass Spectrometry

Project description:Project employ Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS), to characterise the structural dynamics of turkey β1-adrenergic receptor (tβ1AR) in complex with nine ligands, including agonists, partial agonists and antagonists.We show that dynamic signatures across the GPCR structure can be grouped by compound modality. Surprisingly, we discovered repeated destabilisation of the intracellular loop 1 (ICL1) upon full agonist binding and stabilisation upon antagonist binding, suggesting that increased dynamics in this region are an essential component for G protein recruitment. Multiple sequence alignments and molecular dynamics simulations indicate that L72 in ICL1 plays an important structural role. Differential HDX-MS experiment of tβ1AR and tβ1AR L72A construct in complex with miniGs, in response to various ligands, suggests involvement of ICL1 in stabilising the GDP bound state by influencing the stability of HG helix of miniGs.

2024-08-30 | PXD051297 | Pride

Pulse-SILAC and interactomics reveal distinct DDB1-CUL4 associated factors (DCAFs), cellular functions, and protein substrates

Project description:Cullin-RING finger ligases (CRLs) represent the largest family of ubiquitin ligases. They are responsible for the ubiquitination of ~20% of cellular proteins degraded through the proteasome, by catalyzing the transfer of E2-loaded ubiquitin to a substrate. Seven Cullins are described in vertebrates. Among them, CUL4 associates with DDB1 to form the CUL4-DDB1 ubiquitin ligase complex, which is involved in protein ubiquitination and in the regulation of many cellular processes. Substrate recognition adaptors named DDB1/CUL4 associated factors (DCAF) mediate the specificity of CUL4-DDB1 and have a short structural motif of approximately forty amino acids terminating in a tryptophan (W)-aspartic acid (D) dipeptide, called the WD40 domain. Using different approaches (bioinformatics/structural analyses), independent studies suggested that at least sixty WD40-containing proteins could act as adaptors for the DDB1/CUL4 complex. To better define this association and classification, the interaction of each DCAFs with DDB1 was determined, and new partners and potential substrates were identified. Using BioID and AP-MS approaches, we demonstrated that seven WD40 proteins can be considered DCAFs with a high confidence level. Identifying protein interactions does not always lead to identifying protein substrates for E3-ubiquitin ligases, so we measured changes in protein stability or degradation by pulse-SILAC to identify changes in protein degradation following the expression of each DCAF. In conclusion, these results provide new insights into the roles of DCAFs in regulating the activity of the DDB1-CUL4 complex, in protein targeting, and characterized the cellular processes involved

2023-10-11 | PXD042903 | Pride

The Conformational Stability of Pro-apoptotic BAX is Dictated by Discrete Residues of the Protein Core

Project description:BAX is a pro-apoptotic member of the BCL-2 family, which regulates the balance between cellular life and death. During homeostasis, BAX predominantly resides in the cytosol as a latent monomer but, in response to stress, transforms into an oligomeric protein that permeabilizes the mitochondria, leading to cell death. Because renegade BAX activation poses a grave risk to the cell, the architecture of BAX must ensure monomeric stability yet simultaneously enable conformational change upon stress signaling. The specific structural features that afford both stability and dynamic flexibility remain ill-defined and represent a critical control point of BAX regulation. Here, we identified a nexus of interactions involving four discrete residues of the BAX core α5 helix that are individually essential to maintaining the structure and latency of monomeric BAX and are collectively required for dimeric assembly. We compared the HDX MS profile of full-length recombinant wild-type BAX in solution with that of the BAX L113A, F114A, Y115A, and F116A single point mutants. In each case, we observed striking, regiospecific consequences of replacing the bulky hydrophobic residue with alanine. We also compared HDX in a construct of α2-α5 for the wt protein as well as mutants. The dual yet distinct roles of these residues reveals the intricacy of BAX conformational regulation and opportunities for therapeutic modulation.

2021-08-26 | PXD024479 | Pride

HDX analysis of PI3K oncogenic mutants

Project description:We used Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to examine the dynamic structural changes caused by oncogenic mutations. We performed HDX-MS comparing the full-length complex against the catalytic core which suggest that addition of pY leads to mixed two state population where the state 1 consists of iSH2 bound p110α and the state 2 consists of p110α where the iSH2 is completely disengaged.Therefore, we propose that the non-kinase domain mutations push the equilibrium towards state 2 leading to enhanced enzyme activity.We also propose that the kinase domain mutations lead to the re-orientation of the c-terminal tail leading to exposure of the activation loop and enhanced membrane binding.

2023-02-22 | PXD031080 | Pride

Identification of protein substrates of the Cullin-RING E3 ubiquitin ligase CRL4 through its substrate recognition factors DCAFs

Project description:Cullin-RING finger Ligases (CRLs) represent the largest family of E3 ubiquitin ligases and are responsible for ubiquitination of ~20% of cellular proteins degraded through the proteasome, catalyzing the transfer of E2-loaded ubiquitin to a substrate. Eight Cullins were described in vertebrates and among them, CUL4 associates with DDB1 acting as an adaptor to form the CUL4-DDB1 ubiquitin ligase complex, involved in protein ubiquitination and regulation of many cellular processes. The specificity of CUL4-DDB1 is mediated by substrate recognition adaptors named DDB1/CUL4 associated factors (DCAFs), which are characterized by the presence of a short structural motif of approximately 40 amino acids terminating in a tryptophan (W)-aspartic acid (D) dipeptide, the WD40 domain. Using different approaches (bioinformatics/structural analyses), independent studies suggested at least 60 different WD40 containing proteins that could act as adaptors for the DDB1/CUL4 complex. In this study, we aimed to validate the DCAFs based on their interaction with DDB1 and to define new partners and potential substrates of each DCAF using affinity purification followed by mass spectrometry. Using BioID and AP-MS approaches, we confirmed seven WDR40 protein that can be considered as DCAF with a high confidence level. Because identification of protein substrates of E3-ubiquitin ligases is not always guaranteed by identifying protein interactions, changes in protein stability and protein degradation was measured by pulse-SILAC to identify the protein substrates targeted for proteasomal degradation by each DCAFs. In conclusion, this work provides new insights into the roles of DCAFs as substrate adaptors for the DDB1-CUL4 complex, identifies the proteins targeting and the cellular processes that are involved.

2023-10-24 | PXD037482 | Pride

Protection of the prodomain α1-helix correlates with latency in the transforming growth factor-β family

Project description:The 33 members of the transforming growth factor beta (TGF-β) family are fundamentally important for organismal development and homeostasis. Family members are synthesized and secreted as pro-complexes of non-covalently associated prodomains and growth factors (GF). Pro-complexes from a subset of family members are latent and require activation steps to release the GF for signaling. Why some members are latent while others are non-latent is incompletely understood, particularly because of large family diversity. Here, we have examined representative family members in negative stain electron microscopy (nsEM) and hydrogen deuterium exchange (HDX) to identify features that differentiate latent from non-latent members. nsEM showed three overall pro-complex conformations that differed in prodomain arm domain orientation relative to the bound growth factor. Two cross-armed members, TGF-β1 and TGF-β2, were each latent. However, among V-armed members, GDF8 was latent whereas ActA was not. All open-armed members, BMP7, BMP9, and BMP10, were non-latent. Family members exhibited remarkably varying HDX patterns, consistent with large prodomain sequence divergence. A strong correlation emerged between latency and protection of the prodomain α1-helix from exchange. Furthermore, latency and protection from exchange correlated structurally with increased α1-helix buried surface area, hydrogen bonds, and cation-pi bonds. Moreover, a specific pattern of conserved basic and hydrophobic residues in the α1-helix and aromatic residues in the interacting fastener were found only in latent members. Thus, this first comparative survey of TGF-β family members reveals not only diversity in conformation and dynamics but also unique features that distinguish latent members.

2022-01-19 | PXD026841 | Pride

An HDX-MS optimised approach to characterise nanobodies as tools for biochemical and structural studies

Project description:Given the far-reaching applications of nanobodies, a stream-lined approach to characterizing nanobodies tailored to its intended application is warranted. Camelids can produce a large number of antibodies that can bind to a host of epitopes spanning the entire protein of interest, making identification of binders appropriate to your study slow and cumbersome. To overcome this challenge, we propose the use of hydrogen-deuterium mass spectrometry (HDX-MS) to rapidly characterize nanobody epitopes. HDX-MS is a technique that measures the rate of exchange of amide hydrogens in the protein backbone and can provide valuable insight into interaction surfaces with binding partners. Using the example of the lipid signalling complex PI3K, we employed HDX-MS to characterize the epitopes of a large cohort of nanobodies simultaneously with reasonable accuracy. Some of these nanobodies proved to be extremely useful, stabilizing a flexible region in structural studies and blocking signalling inputs from activating partners in in-vitro kinase assays. These experiments were in turn, instrumental in obtaining the first high resolution structure of a PI3K complex, besides providing novel tools to study PI3K signalling in-vivo.

2021-09-22 | PXD025207 | Pride

HDX-MS Analysis of Conformation Selection by ATP-competitive Inhibitors and Allosteric Communication in ERK2

Project description:Activation of the extracellular signal regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states, named “L” and “R”, where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.

2024-03-20 | PXD048311 | Pride

Activation loop phosphorylation tunes conformational dynamics underlying Pyk2 tyrosine kinase activation

Project description:Pyk2 is a multidomain non-receptor tyrosine kinase that undergoes a complex, multi-stage activation process. Ca2+-flux induces conformational rearrangements that relieve autoinhibitory FERM domain interactions. The kinase domain phosphorylates a key linker residue to recruit Src kinase. Pyk2 and Src mutually phosphorylate activation loop residues to confer full activation. While the mechanisms of autoinhibition are established, the conformational dynamics associated with autophosphorylation and Src recruitment remain unclear. Here, we employ hydrogen/deuterium exchange mass spectrometry (HDX-MS) to map the conformational changes associated with Src-mediated activation segment phosphorylation in a Pyk2 construct encompassing FERM and kinase domains (residues 20-692). Results reveal increased dynamics at regulatory interfaces spanning FERM and kinase domains. Phosphorylation of the activation segment stabilizes two antiparallel beta strands linking activation and catalytic loops. Increased dynamics of the C-terminal end of the activation loop propagate to the F-helix, explaining how phosphorylation prevents reversion to the autoinhibitory FERM interaction. HDX-guided site-directed mutagenesis and kinase activity profiling establish a mechanism for phosphorylation-induced active site sculpting to confer high activity.

2023-03-08 | PXD034028 | 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