Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structural perturbation at the N-terminal domain of human phosphoglycerate kinase 1 reduces the kinetic stability and unfolding cooperativity of a two-domain enzyme

ABSTRACT: Phosphoglycerate kinase has been a model for the stability, folding cooperativity and catalysis of a two-domain protein. The human isoform 1 (hPGK1) is associated with cancer development and rare genetic diseases that affect several of their features. To investigate how mutations affect hPGK1 folding landscape, we have designed and introduced mutations at a buried site in the N-terminal domain (F25 mutants) that either created cavities (F25L, F25V, F25A), enhanced conformational entropy (F25G) or introduced structural strain (F25W) and evaluated their effects using biophysical experimental and theoretical methods. All F25 mutants folded well, but showed reduced unfolding cooperativity, kinetic stability and altered activation energetics according to the results from thermal and chemical denaturation analyses. These alterations correlated well with the structural perturbation caused by mutations and the destabilization caused in the interdomain interface. Importantly, experimental and theoretical analyses showed that these effects are significant even when the perturbation is mild and local based on biophysical and HDX-MS analyses. Our approach will be useful to establish the molecular basis of hPGK1 genotype-phenotype correlations due to phosphorylation events and single amino acid substitutions associated with disease in vitro and inside cells

INSTRUMENT(S): solariX

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Dmitry Loginov

LAB HEAD: Petr Man

PROVIDER: PXD035628 | Pride | 2022-11-03

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

Counterintuitive structural and functional effects due to naturally-occurring mutations targeting the active site of the disease-associated NQO1 enzyme

Project description:Our knowledge on the genetic diversity of the human genome is exponentially growing. However, our capacity to establish genotype-phenotype correlations in a large-scale requires a combination of detailed experimental and computational work. This is a remarkable task in human proteins, which are typically multifunctional and structurally complex. In addition, mutations often prevent the determination of mutant high-resolution structures by X-ray crystallography. We have characterized here the effects of five mutations in the vicinity of the active site of the disease-associated NQO1 protein which are found either in cancer cell lines or in massive exome sequencing analysis in human population. Using a combination of H/D exchange, rapid-flow enzyme kinetics, binding energetics and conformational stability, we show that mutations in both sets may cause counterintuitive functional effects that are explained well by their effects on local stability regarding different functional features. Importantly, mutations predicted to be highly deleterious (even those affecting the same protein residue) may cause mild to catastrophic effects on protein function. These functional effects are not well explained by current predictive bioinformatic tools and evolutionary models that account for site conservation and physicochemical changes upon mutation. Our study also reinforces the notion that naturally-occurring mutations not (yet?) identified as disease-associated can be highly deleterious. Our approach, combining protein biophysics and structural biology tools is readily accessible to broadly increase our understanding of genotype-phenotype correlations and to improve predictive computational tools aimed at distinguishing disease-prone against neutral missense variants in the human genome.

2022-11-23 | PXD036417 | Pride

Phenotypic divergence due to site-specific phosphorylation in the antioxidant and cancer-associated NQO1 enzyme

Project description:Aims: to evaluate the effects of site-specific phosphorylation (using phosphomimetic mutations at sites S40, S82 and T128) on multiple functional aspects of the antioxidant and disease-associated human flavoprotein NQO1. Results: in vitro biophysical studies revealed effects of phosphorylation at different sites such as decreased binding affinity for FAD and structural stability of its binding site (S82), conformational stability (S40 and S82) and reduced catalytic efficiency and functional cooperativity (T128). Local stability measurements by HDX in different ligation states provided insight into these effects. Transfection of eukaryotic cells showed that phosphorylation at sites S40 and S82 may reduce steady-levels of NQO1 protein by enhanced proteasome-induced degradation. Innovation: our approach allows to establish relationships between site-specific phosphorylation, functional and structural stability effects in vitro and inside cells paving the way for more detailed analyses of phosphorylation at the flavoproteome scale. Conclusions: we show that site-specific phosphorylation of human NQO1 may cause pleitropic and counterintuitive effects on this multifunctional protein with potential implications for its relationships with human disease.

2022-09-20 | PXD034600 | Pride

Structural analysis of human cis-prenyltransferase

Project description:Isoprenoids are synthesized by the prenyltransferases superfamily, which is subdivided according to the product stereoisomerism and length. In short- and medium-chain isoprenoids, product length correlates with active site volume. However, enzymes synthesizing long-chain products and rubber synthases fail to conform to this paradigm, due to an unexpectedly small active site. Here, we focused on the human cis-prenyltransferase complex (hcis-PT), residing at the endoplasmic reticulum membrane and playing a crucial role in protein glycosylation. Crystallographic investigation of hcis-PT along the reaction cycle revealed an outlet for the elongating product. Hydrogen-deuterium exchange mass spectrometry analysis showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Finally, using a fluorescence substrate analog, we show that product elongation and membrane association are closely correlated. Together, our results support direct membrane insertion of the elongating isoprenoid during catalysis, uncoupling active site volume from product length.

2022-06-09 | PXD031707 | Pride

Structural dynamics of Na+ and Ca 1 2+ interactions with full-size mammalian NCX

Project description:NCX proteins (Na+/Ca2+ exchangers) are allosterically regulated by cytosolic Ca2+ and Na+ to feedback ion signaling in diverse cell types. The recently discovered cryo-EM structure of the cardiac NCX1.1 provided breakthrough information on the mammalian NCX structure, although the structure-dynamic basis of allosteric regulation remains unresolved. Here, we analyzed the apo, Ca2+-, and Na+ -bound species of the brain NCX1.4 variant using hydrogen-deuterium exchange mass spectrometry (HDX-MS) in conjunction with molecular dynamics (MD) simulations. We show that Ca2+ binding to the regulatory CBD domains dramatically rigidifies the intracellular regulatory loop (5L6) and promotes its interaction with the membrane. Either Na+ or Ca2+ stabilizes the intracellular portions of TM3, TM4, TM9, TM10, and their connecting loops (3L4 and 9L10), exposing a putative cation binding site for allosteric regulation. Either Ca2+ or Na+ rigidifies TMH2, encompassing the palmitoylation domain, and the neighboring two-helix TM1/TM6 bundle(governing the alternating access transitions) to control the ion transport rates. Collectively, our results uncovered important details of allosteric regulation in mammalian NCX, while highlighting structure-dynamic features of functional regions not resolved in the cryo-EM structure. The present outcomes provide useful clues toward resolving the structure-dynamic determinants of regulatory diversity in NCX isoform/splice variants.

2024-04-17 | PXD047098 | Pride

Insights into PH1 disease mechanisms from the analyses of the structural stability and dynamics of AGT variants

Project description:Primary hyperoxaluria type I (PH1) is a genetic disease caused by a deficiency in the peroxisomal alanine:glyoxylate aminotransferase (AGT) activity. Mutations in AGT mostly cause protein mistargeting and enhanced aggregation, although the molecular and structural basis of these mechanisms are unknown. In this work, we use hydrogen-deuterium exchange monitored by mass spectrometry (HDX-MS) to provide novel insight into these pathogenic mechanisms. We characterize the wild-type (WT) protein, the LM variant (containing the mutations P11L and I340M, a haplotype more frequent in PH1 patients) and the LM G170R (the most common genotype in PH1, introducing the G170R mutation on the LM background). Our study provides the first experimental analysis of the local stability and dynamics of AGT, showing that stability is heterogeneous in the native state and providing a blueprint for frustrated regions with potentially functional relevance. The LM and LM G170R variants destabilize locally the structure. Enzymatic transamination of the PLP bound to AGT hardly affects stability. Our study thus supports that AGT misfolding is not caused by dramatic effects on the stability and dynamics of the holo-protein.

2024-01-25 | PXD046749 | Pride

Characterization of ISG65-Cd3 complex

Project description:Identification of disulfide bonds of ISG65 protein and hydrogen-deuteium exchange with mass spectrometry for characterization of protein-protein interaction area.

2023-05-10 | PXD033606 | Pride

Chloride intracellular channel (CLIC) proteins function as fusogens

Project description:The Cl- intracellular channel (CLIC) family members uniquely transition between soluble and membrane-associated conformations. Despite decades of extensive functional and structural studies, CLICs’ function as ion channels remains debated, rendering our understanding of their physiological role incomplete. Here, we expose a novel function of CLIC5 as a fusogen. We demonstrate that purified CLIC5 directly interacts with the membrane and induces fusion, as reflected by increased liposomal diameter and lipid and content mixing between liposomes. Moreover, we show that this activity is facilitated by acidic pH, a known trigger for CLICs’ transition to a membrane-associated conformation and that increased exposure of the hydrophobic inter-domain interface is crucial for this process. Finally, mutation of a conserved hydrophobic interfacial residue diminishes the fusogenic activity of CLIC5 in vitro and impairs excretory canal formation in C. elegans in vivo. Together, our results unravel the long-sought physiological role of these enigmatic proteins.

2024-03-10 | PXD045186 | Pride

TTYH family members form tetrameric complexes at the cell membrane

Project description:The conserved Tweety homolog (TTYH) family consists of three paralogs in vertebrates, displaying a ubiquitous expression pattern. Although considered as ion channels for almost two decades, recent structural and functional analyses refuted this role. Intriguingly, while all paralogs shared a dimeric stoichiometry following detergent solubilization, their structures revealed divergence in their relative subunit orientation. Here, we determined the stoichiometry of intact mouse TTYH (mTTYH) complexes in cells. Using cross-linking and single-molecule fluorescence microscopy, we demonstrate that mTTYH1 and mTTYH3 form tetramers at the plasma membrane, stabilized by interactions between their extracellular domains. Using blue-native PAGE, fluorescence-detection size-exclusion chromatography and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we reveal that detergent solubilization results in tetramers destabilization, leading to their dissolution into dimers. Moreover, HDX-MS demonstrates that the extracellular domains are stabilized in the context of the tetrameric mTTYH complex. Together, our results expose the innate tetrameric organization of TTYH complexes at the cell membrane. Future structural analyses of these assemblies in native membranes are required to illuminate their long-sought cellular function.

2022-08-31 | PXD031833 | Pride

SARS-CoV-2 S protein ACE2 interaction reveals novel allosteric targets

Project description:SARS-CoV-2 spike protein binding to receptor ACE2 allosterically enhances furin proteolysisat distal S1/S2 cleavage sites

2021-08-09 | PXD023138 | Pride

A conserved tryptophan in the acylated segment controls β2- integrin-independent cell penetration of RTX toxins

Project description:The post-translationally acylated Repeats in ToXins (RTX) leukotoxins, such as the adenylate cyclase (CyaA) or α-hemolysin (HlyA), bind β2-integrins of leukocytes, but also penetrate cells lacking these receptors. We show that the indole of conserved tryptophans within the acylated segments, e.g. W876 in CyaA and W579 in HlyA, is crucial for β2-integrin-independent membrane penetration of toxins. Substitutions of W876 by aliphatic or aromatic residues did not affect acylation, folding or activities of CyaA W876L/F/Y toxin variants on CR3-expressing cells. In contrast, toxin activity of CyaA W876L/F/Y on cells lacking CR3 was strongly impaired. Similarly, a W579L substitution selectively reduced HlyA W579L cytotoxicity towards cells lacking β2-integrins. Intriguingly, the W876L/F/Y substitutions increased the thermal stability (Tm) of CyaA by 4 to 8 °C. In addition, the hydrogen-deuterium exchange revealed structural changes in the acylated segment of the CyaA W876F variant, compared to intact CyaA. The substitution of W876 with a polar glutamine (W876Q), not increasing the Tm, or combining of the W876F substitution with a cavity-filling V822M substitution, decreasing the Tm of CyaA W876F+V822M to a value closer to that of CyaA, yielded a milder defect of toxin activity on erythrocytes lacking CR3. Furthermore, the activity of CyaA was selectively impaired on erythrocytes when the interaction of the pyrrolidine of P848 with the indole of W876 was ablated. These results suggest that the bulky indole of the W876 of CyaA, or W579 of HlyA, rules the local structural flexibility of the acylated segment. This may facilitate adoption of a membrane-penetrating conformation of the toxins in the absence of β2-integrin-mediated positioning of the toxin towards the cell membrane.

2023-07-11 | PXD041207 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

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