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Mapping of nanobody binding sites on human LRRK2 by CX-MS

ABSTRACT: As part of an in-depth characterization of nanobodies directed against the human Leucine-rich repeat kinase 2 (LRRK2), epitopes of the nanobodies bound to LRRK2 were mapped by chemical crosslinking combined with mass spectrometry using the CID-cleavable crosslinker disuccinimidyl sulfoxide (DSSO).

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

DISEASE(S): Parkinson's Disease

SUBMITTER: Christian Johannes Gloeckner

LAB HEAD: Christian Johannes Gloeckner

PROVIDER: PXD030063 | Pride | 2022-02-27

REPOSITORIES: Pride

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			Action	DRS
	GUA3120-S02_13606_fraction1.raw	Raw
	GUA3120-S03_13606_fraction2.raw	Raw
	GUA3120-S06_14133_fraction1.raw	Raw
	GUA3120-S07_14133_fraction2.raw	Raw
	GUA3120-S08_14259_fraction1.raw	Raw

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1 - 5 of 26

Publications

Nanobodies as allosteric modulators of Parkinson's disease-associated LRRK2.

Singh Ranjan K RK Soliman Ahmed A Guaitoli Giambattista G Störmer Eliza E von Zweydorf Felix F Dal Maso Thomas T Oun Asmaa A Van Rillaer Laura L Schmidt Sven H SH Chatterjee Deep D David Joshua A JA Pardon Els E Schwartz Thomas U TU Knapp Stefan S Kennedy Eileen J EJ Steyaert Jan J Herberg Friedrich W FW Kortholt Arjan A Gloeckner Christian Johannes CJ Versées Wim W

Proceedings of the National Academy of Sciences of the United States of America 20220301 9

Mutations in the gene coding for leucine-rich repeat kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson's disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multidomain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common, disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for drug discovery. Currently, drug development has mainly fo ...[more]

PMID: 35217606

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Analysis of conformational changes induced by binding of allosteric nanobodies to a bacterial LRRK2 homologue by CX-MS

Project description:As part of an in-depth characterization of nanobodies directed against the Chlorobium tepidum protein CtRoco, a bacterial LRRK2 homologue, chemical crosslinking combined with mass spectrometry (CX-MS) was performed. Two allosteric nanobodies binding to two different epitopes in the LRR and the Roc domain of CtRoco, respectively were analysed using the CID-cleavable crosslinker disuccinimidyl sulfoxide (DSSO).

2024-04-23 | PXD046634 | Pride

Selennoprotein S interacts with the replication and transcription complex of the SARS-CoV-2 by binding nsp7

Project description:The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) replicates and evades detection by using endoplasmic reticulum (ER) membranes and their associated protein machineries. Among these hijacked human ER proteins is selenoprotein S, which usually takes part in the ER protein degradation pathway, NFkB signaling, and regulation of cytokines secretion. While the role of selenoprotein S in the viral life cycle is not yet known, it has been reported that it interacts with SARS-CoV-2 non-structural protein 7 (nsp7), a viral protein essential for the reproduction of SARS-CoV-2. However, it was unknown if selenoprotein S and nsp7 interact directly and whether the two proteins can still interact when nsp7 is bound to the virus' replication machinery. Using biochemical assays, we show that selenoprotein S indeed binds directly to nsp7. In addition, we find that selenoprotein S can also bind nsp7 when it is in a complex with the coronavirus's minimal replication complex: nsp7, nsp8 and the RNA-dependent RNA polymerase. Using cross-linking experiments, we mapped the interactions of selenoprotein S and nsp7 in the replication complex and show that the hydrophobic segment of selenoprotein S is essential for binding nsp7. This arrangement leaves an extended helix and the intrinsically disordered segment of selenoprotein S exposed and free to potentially recruit additional proteins to the replication complex. Thus, selenoprotein S is shown to directly interact with the viral replication complex, which places this human protein at the heart of viral replication.

2023-03-11 | PXD038629 | Pride

Optimized fragmentation improves the identification of peptides cross-linked using MS-cleavable reagents

Project description:Cross-linking mass spectrometry (XLMS) is becoming increasingly popular, and current advances are widening the applicability of the technique so that it can be utilized by non-specialist laboratories. Specifically, the use of novel mass spectrometry-cleavable (MS-cleavable) reagents dramatically reduces complexity of the data by providing i) characteristic reporter ions and ii) the mass of the individual peptides, rather than that of the cross-linked moiety. However, optimum acquisition strategies to obtain the best quality data for such cross-linkers with higher energy C-trap dissociation (HCD) alone is yet to be achieved. Therefore, we have carefully investigated and optimized MS parameters to facilitate the identification of disuccinimidyl sulfoxide (DSSO)-based cross-links on HCD-equipped mass spectrometers. From the comparison of 9 different fragmentation energies we chose several stepped- HCD fragmentation methods that were evaluated on a variety of cross-linked proteins. The optimal stepped-HCD method was then directly compared with previously described methods using an Orbitrap Fusion™ Lumos™ Tribrid™ instrument using a high-complexity sample. The final results indicate that our stepped-HCD method is able to identify more cross-links than other methods, mitigating the need for multistage MS (MSn) enabled instrumentation and alternative dissociation techniques.

2019-02-01 | PXD011861 | Pride

Development of functional anti-Gn nanobodies specific for SFTSV based on next generation sequencing and proteomics

Project description:Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by novel bunyavirus (SFTSV), with a mortality rate of 6.3% ~ 30%. To date, there is no specific treatment for SFTS. Previously, our studies demonstrate that SFTSV surface glycoprotein (Glycoprotein N, Gn) is a potential target for the development of SFTS vaccine or therapeutic antibodies, and anti-Gn neutralizing antibodies play a protective role in SFTS infection. Compared with traditional antibodies, nanobodies from camelids have various advantages including small molecular weight, high affinity, low immunogenicity and convenient production by gene engineering, etc. In this study, we combined next generation sequencing (NGS) with proteomics technology and bioinformatics analysis to high-throughput screen monoclonal anti-Gn nanobodies from camel immunized with Gn protein. We identified 19 anti-Gn monoclonal nanobody sequences, and selected 6 of them for recombinant protein expression and purification. Among these 6 anti-Gn nanobodies, nanobody 57493 was validated to be highly specific for Gn.

2024-01-26 | PXD032731 | Pride

Cross-linking mass spectrometry uncovers interactions between high-density lipoproteins and the SARS-CoV-2 spike glycoprotein

Project description:Multiple studies to date have shown that high-density lipoprotein (HDL) levels are reduced in patients with coronavirus disease 2019 (COVID-19), and that the extent of this reduction is associated with poor clinical outcomes. While lipoproteins are known to play a key role during the lifecycle of hepatitis C virus, the direct influence they have upon coronavirus (CoV) infections remains poorly understood. In this study we utilise cross-linking mass spectrometry (XL-MS) to determine circulating protein interactors of the severe acute respiratory syndrome (SARS)-CoV-2 spike glycoprotein. Spike was revealed to interact with HDL, through direct interactions with apolipoprotein (Apo)-A1, ApoC3 and ApoD, highlighting multiple modalities of how SARS-CoV-2 and HDL may interact. XL-MS of plasma isolated from COVID-19 patients uncovered HDL protein interaction networks, dominated by acute phase serum amyloid proteins (SAA), whereby serum amyloid A2 (SAA2) was shown to bind to ApoD. ApoD was confirmed to also interact with core apolipoproteins of both LDL and VLDL, suggesting that SARS-CoV-2 may bind multiple lipoprotein species. The interaction between ApoD and spike was further validated in cells using immunoprecipitation-MS, which uncovered a novel interaction between both ApoD and spike with membrane-associated progesterone receptor component 1 (PGRMC1). Mechanistically, XL-MS coupled with data-driven structural modelling determined that ApoD may interact within the receptor-binding domain (RBD) of spike, posing the hypothesis that ApoD may interfere with binding to the angiotensin-converting enzyme 2 (ACE2) receptor. However, utilising multiple cell-based assays we determined ApoD to have no effect upon viral replication or infectivity.

2023-07-20 | PXD041831 | Pride

Cross-linking mass spectrometry uncovers interactions between high-density lipoproteins and the SARS-CoV-2 spike glycoprotein

2023-07-20 | PXD036640 | Pride

Cross-linking mass spectrometry uncovers interactions between high-density lipoproteins and the SARS-CoV-2 spike glycoprotein

2023-07-20 | PXD041438 | Pride

Cross-linking mass spectrometry uncovers interactions between high-density lipoproteins and the SARS-CoV-2 spike glycoprotein

2023-07-20 | PXD042625 | Pride

Monolinks Matter: Rational Modification of a Cross-Linker for Improved Flexible Protein Structure Modeling

Project description:Chemical cross-linking/mass spectrometry (XL-MS) has emerged as a complementary tool for mapping interaction sites within protein networks as well as gaining moderate-resolution native structurale insight with minimal interference. XL-MS technology mostly relies on chemoselective reactions (crosslinking) between protein residues and a linker. Although complementary to crosslinks, monolinks are less commonly recognised as adjuvants for protein structure elucidation. Herein we report the rational design and synthesis of DSSO-carbamate, a novel cross-linker with increased selectivity for monolinks. Monolinks represent useful information for the elucidation of protein conformational changes and recent work has exploited their application in experimental crosslinking MS integrated protein structure prediction algorithms such as XL-MS-AlphaFold2 . its application supported by AlphaFold2 . Herein we report the rational design and synthesis of DSSO-carbamate, a novel cross-linker with increased selectivity for monolinks. Further, we demonstrate DSSO-carbamate’s capacity to facilitate improved protein structure prediction on account of its superior monolink generation.

2025-05-06 | PXD051742 | Pride

Structural proteomics defines a sequential priming mechanism for the progesterone receptor (XL-MS)

Project description:The progesterone receptor (PR) is a steroid-responsive nuclear receptor with two isoforms: PR-A and PR-B. Disruption of PR-A:PR-B signaling is associated with breast cancer through interactions with oncogenic co-regulatory proteins (CoRs). However, molecular details of isoform-specific PR-CoR interactions remain poorly understood. Using structural mass spectrometry, we investigate the sequential binding mechanism of purified full-length PR and intact CoRs, steroid receptor coactivator 3 (SRC3) and p300, as complexes on target DNA. Our findings reveal selective CoR NR-box binding by PR and novel interaction surfaces between PR and CoRs during complex assembly, providing a structural basis for CoR sequential binding on PR. Antagonist-bound PR showed persistent CoR interactions, challenging the classical model of nuclear receptor activation and repression. Collectively, we offer a peptide-level perspective on the organization of the PR transcriptional complex and infer the mechanisms behind the interactions of these proteins, both in active and inactive conformations.

2025-04-07 | PXD056360 | Pride

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