Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

HDX-MS Study of Conformational Changes in ASK1(269-658) upon H43 Binding

ABSTRACT: This project investigates the conformational dynamics of the ASK1(269-658) protein fragment and the structural changes induced by its interaction with the small molecule H43. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS), we compared the deuterium uptake profiles of apo ASK1(269-658) and its complex with H43. Samples were prepared with different exchange times (30, 90, 600, and 1800 seconds) in D₂O buffer, quenched, enzymatically digested with immobilized pepsin, and analyzed by LC-MS on a Thermo Orbitrap QE mass spectrometer. Peptide identification was performed using PEAKS Online, and HDX data analysis was conducted with HDExaminer software. The results provide insights into dynamic regions within ASK1(269-658) and reveal ligand-induced conformational changes.

ORGANISM(S): Homo Sapiens

SUBMITTER: Fang Bai

PROVIDER: PXD069646 | iProX | Fri Oct 17 00:00:00 BST 2025

REPOSITORIES: iProX

ACCESS DATA

Json Xml

Similar Datasets

Substrate-induced conformational dynamics of the dopamine transporter

Project description:The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na+-driven reuptake of neurotransmitter from the extracellular space. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. Here we probe the global Na+- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. We identify Na+- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, we detect ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a molecular mechanism that assists in the transporter function. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter.

2019-11-12 | PXD013841 | Pride

Local and Global Breathing Motions Prime the Access to Buried Binding Site in an Antibiotic-Sequestering Protein

Project description:Proteins frequently undergo large-scale conformational excursions in their native ensemble. Such structural transitions are particularly critical for enabling access to binding sites when they are buried in the protein interior. Here, we map the conformational landscape of AlbAS, a natural isoform of the transcription factor AlbA from the gut microbe Klebsiella oxytoca, which sequesters the antibiotic albicidin in a solvent-inaccessible binding tunnel. Combining equilibrium, time-resolved experiments, structural mass spectrometry and calorimetry with statistical modeling, we show that AlbAS displays large differences in local and global stability and dynamics, with ~600-fold difference in unfolding rates across different parts of the structure. Several residues lining the ligand-binding pocket and the inter-sub-domain residues rapidly exchange protons with the solvent in hydrogen-deuterium exchange mass spectrometry experiments, indicative of anisotropic distributions of local stabilities, with the N-terminal subdomain being less stable. The AlbAS conformational landscape is thus quite rugged encompassing numerous partially structured states in equilibrium, including partial unlocking of the N-terminal sub-domain at a time-constant of 6 milliseconds that exposes the binding sites to aid in albicidin binding.

2025-08-04 | PXD061296 | Pride

Dynamics of an LPS translocon induced by substrate and an antimicrobial peptide

Project description:We investigated the conformational dynamics of the LPS translocon LptDE was using hydrogen-deuterium exchange mass spectrometry. We evaluated the conformational changes of LptDE upon binding of LPS, Re-LPS (an LPS substructure), thanatin, and the membrane lipid POPG. Our data reveal that LPS induces opening of the LptD β-taco domain, coupled with conformational changes on β-strands adjacent to the putative lateral exit gate. Conversely, an antimicrobial peptide, thanatin, stabilises the β-taco, thus blocking these conformational fluctuations and preventing LPS transport. Our results illustrate that LPS insertion into the OM relies on concerted opening movements of both β-barrel and β-taco domains.

2020-11-16 | PXD021743 | Pride

Unravelling the mechanism of neurotensin recognition by neurotensin receptor 1

Project description:The conformational ensembles of G-protein coupled receptors (GPCRs) include inactive and active states. Spectroscopy techniques, including NMR, show that agonists, antagonists and other ligands shift the ensemble toward specific states depending on the pharmacological efficacy of the ligand. How receptors recognize ligands and the kinetic mechanism underlying this population shift is poorly understood. Here, we investigate the kinetic mechanism of neurotensin recognition by neurotensin receptor 1 (NTS1) using 19F-NMR, hydrogen-deuterium exchange mass spectrometry and stopped-flow fluorescence spectroscopy. Our results indicate slow-exchanging conformational heterogeneity on the extracellular surface of ligand-bound NTS1. Numerical analysis of the kinetic data of neurotensin binding to NTS1 shows that ligand recognition follows an induced fit mechanism, in which conformational changes occur after neurotensin binding. This approach is applicable to other GPCRs to provide insight into the kinetic regulation of ligand recognition by GPCRs.

2023-12-09 | PXD045464 | Pride

Targeting of disease-linked mutations in PI3K gamma

Project description:Using hydrogen deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics we have identified mutation specific conformational changes, and provide a molecular framework for how they alter kinase activity.Many of the activating conformational changes mimicked previously described changes that occur upon membrane binding and allosteric inhibitor binding. We surveyed a diverse panel of PI3K inhibitors using HDX-MS and X-ray crystallography, and find that inhibitors that interact with the activation loop lead to allosteric conformational changes that partially mimic the R1021C mutation. Intriguingly, inhibitors that showed similar conformational changes to the activating mutant showed increased potency (~3 fold) for the mutant over wild type PI3K. Overall this work provides unique insight into the regulatory mechanisms that control PI3K kinase activity, and reveals a framework for the potential design of PI3K isoform and mutant selective inhibitors.

2021-03-26 | PXD021132 | Pride

Molecular mechanism of β-arrestin-2 pre-activation by phosphatidylinositol 4,5-bisphosphate

Project description:Arrestins are a protein family that regulate G protein-coupled receptor (GPCR) signaling, with four distinct members in mammals (arrestin 1–4). Phosphorylated residues of G protein-coupled receptors bind to the N-domain of arrestin, resulting in C-terminus release. This induces further allosteric conformational changes, such as polar core disruption, alteration of interdomain loops, and domain rotation, which transform arrestins into the receptor-activated state. It is widely accepted that arrestin activation occurs by conformational changes propagated from the N- to the C-domain. However, recent studies have revealed that binding of phosphatidylinositol 4,5-bisphosphate (PIP2) to the C-domain transforms arrestins into an active state. In this study, we aimed to elucidate the mechanisms underlying PIP2-induced arrestin activation. We compared the conformational changes of β-arrestin-2 upon binding of PIP2 or phosphorylated C-tail peptide of vasopressin receptor type 2 (V2Rpp) using hydrogen/deuterium exchange mass spectrometry (HDX-MS). Introducing point mutations on the potential routes of the allosteric conformational changes and analyzing these mutant constructs with HDX-MS revealed that PIP2-binding at the C-domain affects the back loop, which destabilizes the gate loop and β-strand XX to transform β-arrestin-2 into the pre-active state.

2024-07-15 | PXD049391 | Pride

Investigating the dynamics of the SecA–SecYEG complex dynamic by HDX-MS

Project description:We employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the conformational dynamics required to facilitate based Brownian ratchet mechanism for protein secretion (by the SecA-SecYEG complex).

2019-07-16 | PXD013594 | Pride

Hydrogen-deuterium exchange mass spectrometry for molecular glue characterisation

Project description:Molecular glues are powerful bioactive molecules that stabilize protein-protein interactions. Yet, the precise mechanisms by which many molecular glues exert their adhesive effects are still not well understood. Native mass spectrometry is an established technique used to monitor the stoichiometry and binding equilibria of molecular glue-induced protein-protein interactions. However, knowledge is lacking on how protein interaction dynamics change upon molecular glue-induced stabilization, and what conformational changes occur that enhance the stability of the resulting protein-protein-glue ternary complex. Here, we showcase hydrogen-deuterium exchange mass spectrometry (HDX-MS) as an analytical tool for molecular glue characterization. Using a broadly applicable molecular glue system involving the eukaryotic regulatory protein 14-3-3, its binding partners, and the molecular glue fusicoccin A, we show the power of HDX-MS in revealing not only molecular glue binding sites, but also conformational changes upon glue binding that result in differentially stabilized protein-protein complexes. Overall, we envisage our HDX-MS approach will become highly valuable for the characterization of molecular glues, guiding their optimization towards successful design.

2025-07-30 | PXD065689 | Pride

HDX-MS analysis of PLIN3 membrane binding

Project description:Hydrogen deuterium exchange mass spectrometry of PLIN3 in the presence of three different membrane vesicles to analyze structural changes induced by membrane binding.

2023-04-14 | PXD025717 | 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