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Mass spectrometry analysis of chemically and collisionally dissociated molecular glue- and PROTAC-mediated protein complexes informs on disassembly pathways.

ABSTRACT: Molecular glues (MG) and proteolysis-targeting chimeras (PROTACs) are used to modulate protein-protein interactions (PPIs), via induced proximity between compounds that have little or no affinity toward each other naturally. They promote either reversible inhibition or selective degradation of a target protein, including ones deemed undruggable by traditional therapeutics. Though native MS (nMS) is capable of analysing multi-protein complexes, the behaviour of these artificially induced compounds in the gas phase is still not fully understood, and the number of publications over the last few years is still rather limited. Here, we studied two MG-induced complexes between mTORFRB and FKBP12, as well as a PROTAC-induced complex between FKBP51 and the von Hippel-Lindau E3 ligase (VHL). Native MS combined with collision-induced dissociation (CID) provided a way of measuring not only the formation of these complexes, but also their dissociation pathways. Both protein complexes seem to eject preferably the centrally-located small (compared to the mass of the proteins) ligand upon CID, rather than dissociating a peripheral subunit, as is often observed for naturally occurring protein complexes. In contrast, chemically-induced dissociation in solution generated complementary data to CID, by disrupting the PPI surface, which resulted in more diverse MS spectra that preserved the stronger interactions in solution.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine) Escherichia Coli Canavalia Brasiliensis

SUBMITTER: Edvaldo Maciiel

LAB HEAD: Prof. Dr. Frederik Lermyte

PROVIDER: PXD059143 | Pride | 2025-02-27

REPOSITORIES: pride

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			Action	DRS
	MSspectradata.zip	Other
	Searchfiles.xlsx	Xlsx

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Project description:Targeted protein degradation (TPD) has emerged as a powerful approach for removing (rather than inhibiting) proteins implicated in diseases. A key step in TPD is the formation of an induced proximity complex where a degrader molecule recruits an E3 ligase to the protein of interest (POI), facilitating the transfer of ubiquitin to the POI and initiating the proteasomal degradation process. Here, we address three critical aspects of the TPD process using atomistic simulations: 1) formation of the ternary complex induced by a degrader molecule, 2) conformational heterogeneity of the ternary complex, and 3) degradation efficiency via the full Cullin Ring Ligase (CRL) macromolecular assembly. We combine experimental biophysical data---in this case hydrogen-deuterium exchange mass spectrometry (HDX-MS, which measures the solvent exposure of protein residues)---with molecular dynamics (MD) simulations aided by enhanced sampling techniques to accurately predict ternary complex structures at atomic resolution. We demonstrate improved efficiency, accuracy, and reliability in the ternary structure predictions of the bromodomain of the cancer target SMARCA2 with the E3 ligase VHL mediated by three different degrader molecules. The simulations accurately reproduce X-ray crystal structures -- including a new structure that we determined in this work (PDB ID: 7S4E) -- with root mean square deviations (RMSD) of 1.1 to 1.6 \r{A}. The simulations also reveal a structural ensemble of low-energy conformations of the ternary complex. Snapshots from these simulations are used as seeds for additional simulations, where we perform 7.1 milliseconds of aggregate simulation time using Folding@home. The detailed free energy surface captures the crystal structure conformation within a low-energy basin and is consistent with solution-phase experimental data (HDX-MS and SAXS). Finally, we graft a structural ensemble of the ternary complexes onto the full CRL and perform enhanced sampling simulations, which suggest that differences in degradation efficiency may be related to the proximity distribution of lysine residues on the POI relative to the E2-loaded ubiquitin. Several of the predicted ubiquitinated lysine residues have been validated through a ubiquitin mapping proteomics experiment. DOI 10.1038/s41467-022-33575-4

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Mass spectrometry analysis of chemically and collisionally dissociated molecular glue- and PROTAC-mediated protein complexes informs on disassembly pathways.

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