Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Cross-linking mass spectrometry reveals structural insights of the glutamine synthetase from Leishmania braziliensis.

ABSTRACT:

Background

Leishmaniasis is a neglected tropical disease caused by the parasite Leishmania braziliensis, commonly found in Brazil and associated with cutaneous and visceral forms of this disease. Like other organisms, L. braziliensis has an enzyme called glutamine synthetase (LbGS) that acts on the synthesis of glutamine from glutamate. This enzyme plays an essential role in the metabolism of these parasites and can be a potential therapeutic target for treating this disease.

Objectives

Investigate LbGS structure and generate structural models of the protein.

Methods

We use the method of crosslinking mass spectrometry (XLMS) and generate structural models in silico using I-TASSER.

Findings

42 XLs peptides were identified, of which 37 are explained in a monomeric model with the other five indicating LbGS dimerization and pentamers interaction region. The comparison of 3D models generated in the presence and absence of XLMS restrictions probed the benefits of modeling with XLMS highlighting the inappropriate folding due to the absence of spatial restrictions.

Main conclusions

In conclusion, we disclose the conservation of the active site and interface regions, but also unique features of LbGS showing the potential of XLMS to probe structural information and explore new drugs.

SUBMITTER: de Lima JY

PROVIDER: S-EPMC8752055 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

In Situ Structural Restraints from Cross-Linking Mass Spectrometry in Human Mitochondria.

Project description:The field of structural biology is increasingly focusing on studying proteins in situ, i.e., in their greater biological context. Cross-linking mass spectrometry (CLMS) is contributing to this effort, typically through the use of mass spectrometry (MS)-cleavable cross-linkers. Here, we apply the popular noncleavable cross-linker disuccinimidyl suberate (DSS) to human mitochondria and identify 5518 distance restraints between protein residues. Each distance restraint on proteins or their interactions provides structural information within mitochondria. Comparing these restraints to protein data bank (PDB)-deposited structures and comparative models reveals novel protein conformations. Our data suggest, among others, substrates and protein flexibility of mitochondrial heat shock proteins. Through this study, we bring forward two central points for the progression of CLMS towards large-scale in situ structural biology: First, clustered conflicts of cross-link data reveal in situ protein conformation states in contrast to error-rich individual conflicts. Second, noncleavable cross-linkers are compatible with proteome-wide studies.

| S-EPMC7010328 | biostudies-literature

Systems structural biology measurements by in vivo cross-linking with mass spectrometry.

Project description:This protocol describes a workflow for utilizing large-scale cross-linking with mass spectrometry (XL-MS) to make systems-level structural biology measurements in complex biological samples, including cells, isolated organelles, and tissue samples. XL-MS is a structural biology technique that provides information on the molecular structure of proteins and protein complexes using chemical probes that report the proximity of probe-reactive amino acids within proteins, typically lysine residues. Information gained through XL-MS studies is often complementary to more traditional methods, such as X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy. The use of MS-cleavable cross-linkers, including protein interaction reporter (PIR) technologies, enables XL-MS studies on protein structures and interactions in extremely complex biological samples, including intact living cells. PIR cross-linkers are designed to contain chemical bonds at specific locations within the cross-linker molecule that can be selectively cleaved by collision-induced dissociation or UV light. When broken, these bonds release the intact peptides that were cross-linked, as well as a reporter ion. Conservation of mass dictates that the sum of the two released peptide masses and the reporter mass equals the measured precursor mass. This relationship is used to identify cross-linked peptide pairs. Release of the individual peptides permits accurate measurement of their masses and independent amino acid sequence determination by tandem MS, allowing the use of standard proteomics search engines such as Comet for peptide sequence assignment, greatly simplifying data analysis of cross-linked peptide pairs. Search results are processed with XLinkProphet for validation and can be uploaded into XlinkDB for interaction network and structural analysis.

| S-EPMC6777709 | biostudies-literature

Structural insights into leishmanolysins encoded on chromosome 10 of Leishmania (Viannia) braziliensis.

Project description:BackgroundLeishmanolysins have been described as important parasite virulence factors because of their roles in the infection of promastigotes and resistance to host's defenses. Leishmania (Viannia) braziliensis contains several leishmanolysin genes in its genome, especially in chromosome 10. However, the functional impact of such diversity is not understood, but may be attributed partially to the lack of structural data for proteins from this parasite.ObjectivesThis works aims to compare leishmanolysin sequences from L. (V.) braziliensis and to understand how the diversity impacts in their structural and dynamic features.MethodsLeishmanolysin sequences were retrieved from GeneDB. Subsequently, 3D models were built using comparative modeling methods and their dynamical behavior was studied using molecular dynamic simulations.FindingsWe identified three subgroups of leishmanolysins according to sequence variations. These differences directly affect the electrostatic properties of leishmanolysins and the geometry of their active sites. We identified two levels of structural heterogeneity that might be related to the ability of promastigotes to interact with a broad range of substrates.Main conclusionAltogether, the structural plasticity of leishmanolysins may constitute an important evolutionary adaptation rarely explored when considering the virulence of L. (V.) braziliensis parasites.

| S-EPMC5572447 | biostudies-literature

Cross-linking/mass spectrometry at the crossroads.

Project description:Cross-linking/mass spectrometry (XL-MS) has come a long way. Originally, XL-MS was used to study relatively small, purified proteins. Meanwhile, it is employed to investigate protein-protein interactions on a proteome-wide level, giving snapshots of cellular processes. Currently, XL-MS is at the intersection of a multitude of workflows and the impact this technique has in addressing specific biological questions is steadily growing. This article is intended to give a bird's-eye view of the current status of XL-MS, the benefits of using MS-cleavable cross-linkers, and the challenges posed in the future development of this powerful technology. We also illustrate how XL-MS can deliver valuable structural insights into protein complexes when used in combination with other structural techniques, such as electron microscopy. Graphical abstract.

| S-EPMC7442761 | biostudies-literature

Complementary Benzophenone Cross-Linking/Mass Spectrometry Photochemistry.

Project description:Use of a heterobifunctional photoactivatable cross-linker, sulfo-SDA (diazirine), has yielded high-density data that facilitated structure modeling of individual proteins. We expand the photoactivatable chemistry toolbox here with a second reagent, sulfo-SBP (benzophenone). This further increases the density of photo-cross-linking to a factor of 20× over conventional cross-linking. Importantly, the two different photoactivatable groups display orthogonal directionality, enabling access to different protein regions, unreachable with a single cross-linker.

| S-EPMC5441754 | biostudies-literature

Structural analysis of 70S ribosomes by cross-linking/mass spectrometry reveals conformational plasticity.

Project description:The ribosome is not only a highly complex molecular machine that translates the genetic information into proteins, but also an exceptional specimen for testing and optimizing cross-linking/mass spectrometry (XL-MS) workflows. Due to its high abundance, ribosomal proteins are frequently identified in proteome-wide XL-MS studies of cells or cell extracts. Here, we performed in-depth cross-linking of the E. coli ribosome using the amine-reactive cross-linker disuccinimidyl diacetic urea (DSAU). We analyzed 143 E. coli ribosomal structures, mapping a total of 10,771 intramolecular distances for 126 cross-link-pairs and 3,405 intermolecular distances for 97 protein pairs. Remarkably, 44% of intermolecular cross-links covered regions that have not been resolved in any high-resolution E. coli ribosome structure and point to a plasticity of cross-linked regions. We systematically characterized all cross-links and discovered flexible regions, conformational changes, and stoichiometric variations in bound ribosomal proteins, and ultimately remodeled 2,057 residues (15,794 atoms) in total. Our working model explains more than 95% of all cross-links, resulting in an optimized E. coli ribosome structure based on the cross-linking data obtained. Our study might serve as benchmark for conducting biochemical experiments on newly modeled protein regions, guided by XL-MS. Data are available via ProteomeXchange with identifier PXD018935.

| S-EPMC7387497 | biostudies-literature

pH Dependence of Succinimide-Ester-Based Protein Cross-Linking for Structural Mass Spectrometry Applications.

Project description:Within the research field of cross-linking mass spectrometry (XL-MS), the most commonly used cross-linking reagents are succinimide-ester-based (e.g., disuccinimidyl suberate (DSS)). These reagents primarily cross-link lysine side chains. So far, they have predominantly been used to investigate protein structures at neutral to slightly basic pH (7.0-8.5) to ensure the reactivity of the primary amine of the lysine side chain. However, disease-related molecular processes are not limited to such pH ranges; e.g., some important biological pathways are active in acidic intracellular compartments. The applicability of lysine-reactive cross-linking reagents to low-pH conditions remains unclear. Here, we cross-linked a mixture of eight model proteins at eight different pH conditions (pH 4.0-7.5) to investigate the pH dependency of DSS. DSS was able to cross-link proteins even at pH 4.0, but a clear decrease in the cross-linking efficiency was observed when the pH was lowered. Nevertheless, at pH 5.0, approximately half of the number of cross-links observed at pH 7.5 could still be identified. These findings highlight the ability of succinimide-based cross-linking reagents to be useful in probing the structure of proteins in a slightly acidic environment.

| S-EPMC9838815 | biostudies-literature

Selective cross-linking of coinciding protein assemblies by in-gel cross-linking mass-spectrometry

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, demonstrated by experiments on GroEL and purified bovine heart mitochondria, is the substantial reduction of undesired over-length cross-links 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 provide new insights into the initial stages of the terminal complement pathway.

| S-SCDT-EMBOJ-2020-106174 | biostudies-other

Quantitative cross-linking/mass spectrometry using isotope-labelled cross-linkers.

Project description:Dynamic proteins and multi-protein complexes govern most biological processes. Cross-linking/mass spectrometry (CLMS) is increasingly successful in providing residue-resolution data on static proteinaceous structures. Here we investigate the technical feasibility of recording dynamic processes using isotope-labelling for quantitation. We cross-linked human serum albumin (HSA) with the readily available cross-linker BS3-d0/4 in different heavy/light ratios. We found two limitations. First, isotope labelling reduced the number of identified cross-links. This is in line with similar findings when identifying proteins. Second, standard quantitative proteomics software was not suitable for work with cross-linking. To ameliorate this we wrote a basic open source application, XiQ. Using XiQ we could establish that quantitative CLMS was technically feasible.Cross-linking/mass spectrometry (CLMS) has become a powerful tool for providing residue-resolution data on static proteinaceous structures. Adding quantitation to CLMS will extend its ability of recording dynamic processes. Here we introduce a cross-linking specific quantitation strategy by using isotope labelled cross-linkers. Using a model system, we demonstrate the principle and feasibility of quantifying cross-linking data and discuss challenges one may encounter while doing so. We then provide a basic open source application, XiQ, to carry out automated quantitation of CLMS data. Our work lays the foundations of studying the molecular details of biological processes at greater ease than this could be done so far.

| S-EPMC3714596 | biostudies-literature

Selective cross-linking of coinciding protein assemblies by in-gel cross-linking mass spectrometry.

| S-EPMC7883291 | biostudies-literature