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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): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

DISEASE(S): Parkinson's Disease

SUBMITTER: 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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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 parameter settings enhance proteome-wide formation of crosslinks on low-abundant proteins

Project description:Studies using crosslinking coupled to mass spectrometry on the proteome-wide level have spurred great interest as they facilitate structural probing of protein interactions in living cells or even organisms. Here we show, by using both an in-vitro mimic of a crowded cellular environment and eukaryotic cell lysates, that current proteome-wide crosslinking protocols have a bias for high abundant proteins. We demonstrate that this bias can be explained by kinetics that govern the formation of a crosslink between two polypeptides. We further show that optimized parameter settings, in particularly an excess of crosslinker, leadto a significant overall increase in the detection of lower abundant proteins within cellular lysates on a proteome-wide scale. Our study therefore explains the cause of a major limitation in current proteome-wide crosslinking studies and demonstrates a way forward how to address a larger part of the proteome by crosslinking

2020-02-17 | PXD014759 | Pride

Utilising crosslinking MS to explore protein structure and interactions

Project description:In this study, we performed proteome-wide crosslinking mass spectrometry (XLMS) on human HEK293 intact organelles and examined the combined utility of these crosslinks with AlphaFold.

2023-04-24 | PXD035844 | Pride

Structure and molecular assembly of a MuvB transcription factor complex with the nucleosome

Project description:The core cell cycle machinery genes are transcriptionally regulated by the MuvB family of protein complexes in a cell cycle specific manner. During cell cycle exit in quiescence or senescence, the DREAM complex, which is the repressive form of MuvB, directs transcriptional repression of cell cycle genes; conversely during cell proliferation, the complex of MuvB with the transcription factors (TFs) B-MYB and FOXM1 activate mitotic genes during the G2 phase of the cell cycle. The mechanisms of transcriptional regulation of these complexes are still poorly characterised. Here we combine biochemical analysis and in vitro reconstitution, with structural analysis by cryo-electron microscopy (cryo-EM) and cross-linking mass spectrometry (XL-MS), to functionally examine these complexes. Our data suggests that MuvB is a chromatin regulator whereby a core region binds the nucleosome and remodels it, thereby exposing nucleosomal DNA. This remodelling activity is supported by B-MYB which directly binds the remodelled DNA. Given the remodelling activity on the nucleosome, we propose that the MuvB complex with B-MYB (MMB) function as a pioneer transcription factor complex. Our data rationalises prior biochemical and cellular studies and provides a molecular framework of interactions on a protein complex, which is key for cell cycle regulation.

2022-08-17 | PXD031421 | Pride

Identifying and characterising Thrap3, Bclaf1 and Erh direct interactions using cross-linking mass spectrometry

Project description:Cross-linking mass spectrometry (XL-MS) is a powerful technology capable of yielding structural insights across the complex cellular protein interaction network. We performed XL-MS using MS-cleavable cross-linker disuccinimidyl sulfoxide (DSSO) on endogenous affinity-purified BAF complex. We identified numerous crosslinks between three BAF co-purifying proteins, namely Thrap3, Bclaf1 and Erh. Our data suggests that Bclaf1, Erh and Thrap3 interact closely and might represent a stable complex. The XL data allowed us to map interaction surfaces on Thrap3 and B, which overlap with the non-disordered portions of both proteins.

2021-09-24 | PXD027611 | Pride

Cryo-EM structures of the human SIN3B histone deacetylase complex unveils assembly, enzymatic activation and histone lysine selection mechanisms

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2023-03-31 | PXD040006 | Pride

Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro (XL-MS)

Project description:SARS-CoV-2, a human coronavirus, is the causative agent of the COVID-19 pandemic, entailing enormous disruption worldwide. Its ~30 kb RNA genome is translated into two large polyproteins subsequently cleaved by viral papain-like protease and main protease (Mpro/nsp5). Polyprotein processing is essential yet incompletely understood. We studied Mpro-mediated processing of the nsp7-10/11 polyprotein, whose mature products are cofactors of the viral replicase, identifying the order of cleavages: 1) nsp9-10, 2) nsp8-9/nsp10-11, and 3) nsp7-8. Integrative modeling based on mass spectrometry (including hydrogen-deuterium exchange and cross-linking) and X-ray scattering yielded three-dimensional models of the nsp7-10/11 polyprotein, and the data suggest that the nsp7-10/11 structure in complex with Mpro strongly resembles the unbound polyprotein. Our array of techniques supports the notion that interplay between polyprotein conformation and junction accessibility determine the preference and order of cleavages. Finally, we leveraged assays of limited proteolysis by Mpro of nsp7-11 using a series of inhibitors/binders to characterize Mpro inhibition using a polyprotein substrate.

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