Capturing the organisation of the Mycoplasma pneumoniae proteome with in-cell crosslinking mass spectrometry

• Applicant: Professor Dr. Juri Rappsilber
• Subject Area: Biochemistry ; Structural Biology
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 426290502

Cells have complex internal organization which cannot be fully captured via ex vivo experiments. For example, phase transitions and “membrane-less organelles” have been shown by fluorescence microscopy and cryo-electron tomography. A recent paper involving us identified “protein communities” as a potential level of cellular organisation above that of “core” protein complexes that is often lost during purification (Kastritis, O’Reilly et al. 2017) and is ideally studied in situ. Very little is known about this higher-order organisation of the proteome. Currently our most comprehensive understanding for how the proteome is organised has come from large-scale proteomics studies that identified protein complexes by affinity-purification/mass spectrometry or co-fractionation. Importantly these techniques do not produce topological information for the complexes discovered and so complexes have needed to be purified for further structural analysis, potentially causing associated factors to be lost. Additionally, insoluble or membrane associated complexes are usually missed. These are issues that in-cell crosslinking mass spectrometry (CLMS) can address. In this proposal, we will analyze the topology of the proteome of the simple model pathogenic organism Mycoplasma pneumoniae and discover how far biological processes are organised independent of organelles. In particular, we will investigate the expressosome as a previously identified ‘higher-order’ complex in M. pneumoniae for which little structural information is known. Immunoprecipitation experiments have shown that there is direct coupling of transcription and translation in M. pneumoniae. However no in situ structural investigation has been conducted so any associated factors necessary for this interaction are unknown.In-cell CLMS combined with integrative structural modelling techniques will build an organisational model of the M. pneumoniae proteome in situ. The M. pneumoniae proteome is particularly useful here as a model organism for two reasons: firstly, it is simple enough to allow for comprehensive analysis by CLMS; secondly, many previous system-wide studies have been performed and so are a valuable resource for validation. Our preliminary work has already shown that we can detect a large number of previously unknown interactions and can further structurally validate selected interactions through the powerful combination with in-cell cryo-electron tomography in collaboration with the group of Julia Mahamid at EMBL.The systems-level structural insights gathered in this study, while interesting in general for cell biologists, will also indicate potential targets for novel therapies against this pathogen.

• DFG Programme: Research Grants