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An UBX domain in UBXD1 organizes Ub binding at the C-terminus of the VCP/p97 AAA-ATPase

ABSTRACT: Identification of crosslinked peptides between UBXD1, Ubiquitin and p97 using different cross-linking approaches (chemical cross-linking with DSSO, photo crosslinking with incorporated BPA or photo-Met)

INSTRUMENT(S): Orbitrap Fusion Lumos, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Farnusch Kaschani

LAB HEAD: Farnusch Kaschani

PROVIDER: PXD039606 | Pride | 2023-05-25

REPOSITORIES: Pride

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An UBX domain in UBXD1 organizes Ub binding at the C-terminus of the VCP/p97 AAA-ATPase

Project description:Identification of crosslinked peptides between UBXD1 and HR23b using different cross-linking approaches (chemical cross-linking with DSSO and photo crosslinking with incorporated BPA).

2023-05-25 | PXD040984 | Pride

Structure of Anabaena flos-aquae gas vesicles revealed by cryo-ET

Project description:Gas Vesicles (GVs) are gas-filled protein nanostructures employed by several species of bacteria and archaea as flotation devices to enable access to optimal light and nutrients. The unique physical properties of GVs have led to their use as genetically-encodable contrast agents for ultrasound and MRI. Currently, however, the structure and assembly mechanism of GVs remain unknown. Here we employ cryo-electron tomography to reveal how the GV shell is formed by a helical filament of highly-conserved GvpA subunits. This filament changes polarity at the center of the GV cylinder, a site that may act as an elongation center. High-resolution subtomogram averaging reveals a corrugated pattern of the shell arising from polymerization of GvpA into a β-sheet. The accessory protein GvpC forms a helical cage around the GvpA shell, providing reinforcement. Together, our results help explain the remarkable mechanical properties of GVs and their ability to adopt different diameters and shapes.

2023-03-30 | PXD038631 | Pride

Optimized TMT-based quantitative cross-linking mass spectrometry strategy for large-scale interactomic studies

Project description:Cross-linking mass spectrometry is a powerful method for the investigation of protein-protein interactions from highly complex samples. XL-MS combined with tandem mass tag labeling holds the promise of large-scale PPI quantification. However, a robust and efficient TMT-based XL-MS quantification method has not yet been established due to the lack of a benchmarking dataset and thorough evaluation of various MS parameters. To tackle these limitations, we generate a two-interactome dataset by spiking-in TMT-labeled cross-linked E. coli lysate into TMT-labeled cross-linked HEK293T lysate using a defined mixing scheme. Using this benchmarking dataset, we assess the efficacy of cross-link identification and accuracy of cross-link quantification using different MS acquisition strategies. For identification, we compare various MS2- and MS3-based XL-MS methods, and optimize stepped HCD energies for TMT-labeled cross-links. We observed a need for notably higher fragmentation energies compared to unlabeled cross-links. For quantification, we assess the quantification accuracy and dispersion of MS2-, MS3- and synchronous precursor selection-MS3-based methods. We show that a stepped HCD-MS2 method with stepped collision energies 36-42-48 provides a vast number of quantifiable cross-links with high quantification accuracy. This widely applicable method paves the way for multiplexed quantitative PPI characterization from complex biological systems.

2022-04-07 | PXD031114 | Pride

Structural and functional analyses identify unique features of the Smc5/6 complex among SMC family of complexes

Project description:Cross-linking mass spectrometry data for integrative structural analyses of the Smc5/6 holo-complex from budding yeast. Electron microscopy, crosslinking mass spectrometry, and computational modeling reveal that while the complex shares an overall organization with other SMC complexes, it possesses several strikingly different features.

2021-05-05 | PXD023164 | Pride

Orphan quality control shapes network dynamics and gene expression

Project description:All eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb interactions between network components often result in disease, but how composition and dynamics of complex networks are established remains poorly understood. Here, we identify the E3 ligase UBR5 as a signaling hub that helps degrade unpaired subunits of multiple transcriptional regulators that act within a network centered on the c-MYC oncoprotein. Biochemical and structural analyses show that UBR5 binds motifs that only become available upon complex dissociation. By rapidly turning over orphan transcription factor subunits, UBR5 establishes dynamic interactions between transcriptional regulators that allow cells to effectively execute gene expression, while remaining receptive to environmental signals. We conclude that orphan quality control plays an essential role in establishing dynamic protein networks, which may explain the conserved need for protein degradation during transcription and offers unique opportunities to modulate gene expression in disease. Crosslinking mass spec experiment with UBR5 HECT domain STREP-SUMO-MCRS1 and DSSO.

2023-05-30 | PXD041519 | Pride

Conformation of the AP-3 complex explains its role in cargo recruitment at the Golgi

Project description:Crosslinking mass spectrometry study of an important adapter protein complex at the Golgi AP-3. We performed crosslinking mass spectrometry study using crosslinker DSSO and analyzed the digested peptides by LC-MS. The crosslinking study provided important information the subunit connectivity of AP-3, which facilitated the structural analysis of AP-3 by cyro-EM.

2022-02-17 | PXD028005 | Pride

Normal respiratory chain capacity and physiology in mice with severely reduced levels of mitochondrial respirasomes; Cross-linking of intact mitochondria

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2023-03-11 | PXD031345 | Pride

Structure of herpes simplex virus pUL7:pUL51, a conserved complex required for efficient herpesvirus assembly

Project description:Herpesviruses are an ancient family of highly-prevalent human and animal pathogens that acquire their membrane envelopes in the cytoplasm of infected cells. While multiple conserved viral proteins are known to be required for efficient herpesvirus production, many of these proteins lack identifiable structural homologues and the molecular details of herpesvirus assembly remain unclear. We have characterized the complex of assembly proteins pUL7 and pUL51 from herpes simplex virus (HSV)-1, an α-herpesvirus, using multi-angle light scattering and small-angle X-ray scattering with chemical crosslinking. HSV-1 pUL7 and pUL51 form a stable 1:2 complex that is capable of higher-order oligomerization in solution. We solved the crystal structure of this complex, revealing a core heterodimer comprising pUL7 bound to residues 41–125 of pUL51. While pUL7 adopts a previously-unseen compact fold, the extended helix-turn-helix conformation of pUL51 resembles the cellular endosomal complex required for transport (ESCRT)- III component CHMP4B, suggesting a direct role for pUL51 in promoting membrane scission during virus assembly. We demonstrate that the interaction between pUL7 and pUL51 homologues is conserved acrosshuman α-, β- and γ-herpesviruses, as is their association with trans-Golgi membranes in cultured cells. However, pUL7 and pUL51 homologues do not form complexes with their respective partners from different virus families, suggesting that the molecular details of the interaction interface have diverged. Our results demonstrate that the pUL7:pUL51 complex is conserved across the herpesviruses and provide a structural framework for understanding its role in herpesvirus assembly.

2020-05-11 | PXD015941 | Pride

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

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2024-04-23 | PXD046634 | Pride

dentification of Cross-linking partners of BpA modified p37 and p97 by photo cross-linking MS

Project description:we wanted to see where and how the protein p37 interacts with its partner proteins p97, PPI,Sds22 and I3. To this end p37 derivatives with genetically encoded crosslinking amino acid (p-benzoyl-L-phenylalanine) were generated. After incubation with the partner proteins, photocross-linking and tryptic digest the resulting cross-linked peptides were analysed by mass spectrometry.

2020-10-19 | PXD021182 | Pride

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