Dataset Information

Dynamics and Structural Features of eEF1A1 and eEF1A2 Paralogs

ABSTRACT: The aim of the project was to compare the structural dynamics of two tissue-specific variants of human translation elongation factors. eEF1A1 and eEF1A2.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Oryctolagus Cuniculus (rabbit)

TISSUE(S): Liver, Muscle

SUBMITTER: Michał Kistowski

LAB HEAD: Michal Dadlez

PROVIDER: PXD056814 | Pride | 2025-05-07

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 13

Publications

HRaDeX: R Package and Web Server for Computing High-Resolution Deuterium Uptake Rates for HDX-MS Data.

Puchała Weronika W Kistowski Michał M Zhukova Liliya L Burdukiewicz Michał M Dadlez Michał M

Journal of proteome research 20250319 4

Hydrogen-deuterium exchange monitored by mass spectrometry (HDX-MS) is a well-established and powerful technique used to study protein dynamics and stability by capturing local and global unfolding events in protein structures. However, in this technique, obtaining region-specific information requires proteolytic digestion that breaks the protein into peptide fragments, causing the HDX data to reflect averages over these fragments rather than individual amino acids. We propose a new computationa ...[more]

PMID: 40105417

Similar Datasets

Project description:Acrodysostosis represents a group of rare genetic disorders characterized by defective skeletal development and is often accompanied by intellectual disabilities. Mutations in the 3’5’cyclic AMP (cAMP)-dependent Protein Kinase (PKA) type I regulatory subunit isoform α (RIα) and phosphodiesterase (PDE) PDE4D have both been implicated in impaired PKA regulation in Acrodysostosis. How mutations on PDEs and RIα interfere with regulation of cAMP-PKA signaling is not understood. cAMP-PKA signaling can be described in two phases. In the activation phase, cAMP binding to RIα dissociates the free C-subunit. PDEs hydrolyze cAMP bound to RIα, priming the cAMP-free RIα for reassociation with the C-subunit, thereby completing one PKA activation cycle. Signal termination is thus critical for resetting PKA to its basal state and promoting adaptation to hormonal hyperstimulation. This proceeds through formation of a transient signal termination RIα:PDE complex that facilitates cAMP channeling from the cAMP-binding domain of RIα to the catalytic site of PDE. Signal termination of cAMP-PKA proceeds in three steps: Step 1) Channeling: Translocation of cAMP from the CNB of RIα to the PDE catalytic site for hydrolysis Step 2) Processivity: Binding of free cAMP from the cytosol at both CNBs of RIα and Step 3) Product (5’AMP) release from the PDE hydrolysis site through competitive displacement by a new molecule of cAMP that trigger subsequent activation cycles of PKA. We have identified the molecular basis for two acrodysostosis mutants, (PDE8 T690P) and RIα (T207A) that both allosterically impair cAMP-PKA signal termination. A combination of amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and Fluorescence Polarization (FP) reveal that PDE8 T690P and RIα T207A both blocked processive hydrolysis of cAMP by interfering with competitive displacement of product 5’AMP release from the nucleotide channel at the end of each round of cAMP hydrolysis. While T690P blocked product 5’AMP release from the PDE, T207A greatly slowed release of substrate from RIα. These results highlight the role of processivity in cAMP hydrolysis by RIα:PDE termination complexes for adaptation to cAMP from GPCR hyperstimulation. Impairment of the signal termination process provides an alternate molecular basis for acrodysostosis.

Project description:Enzymes play a crucial role in biological systems and biotechnological processes as vital and sustainable catalysts that facilitate and regulate biochemical reactions. Inhibition of product formation of an enzyme by its own substrate was originally dismissed as an artifact of biochemical analysis, but is now a proven fact and occurs in about 20% of known enzymes1. Substrate inhibition is attributed to the formation of an unproductive enzyme-substrate complex with no structural evidence of unproductivity provided to date1–6. Here we show in detail the molecular mechanism of substrate inhibition of the tobacco glucosyltransferase NbUGT72AY1, which transfers glucose from its nucleotide donor substrate to phenol acceptors as a plant protection measure. The peculiarity that the effector β-carotene strongly attenuates the substrate inhibition of NbUGT72AY1, although it unexpectedly acts as a competitive acceptor-substrate inhibitor, allowed us to uncover the conformational changes that occur during substrate binding in the active and substrate-inhibited complexes of this protein. X-ray crystallography revealed structurally different ternary enzyme-substrate complexes that do not conform to the classical compulsory ordered or random mechanism of an enzyme-catalyzed sequential bi-substrate reaction. We suggest an alternative pathway in which the two substrates bind randomly to the enzyme, but the reaction is only catalyzed if a specific order of substrate binding is observed (asymmetric cooperativity). The new paradigm explains substrate inhibition in monomeric multi-substrate enzymes and reactivation of activity by competitive inhibitors. This alternative concept opens up new avenues of research in metabolic regulation as well as a wealth of novel industrial applications. The results enable novel approaches, e.g. to minimize the effects of food components on drug metabolism and to study plant defense mechanisms from a new perspective and offer alternative approaches for the design of more efficient production processes.

			Action	DRS
	A1.csv	Csv
	A1_GDP.raw.zip	Raw
	A1_GTP.raw.zip	Raw
	A1_seq.raw.zip	Raw
	A2.csv	Csv