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Structural Assessment of the Full-Length Tumor Suppressor Protein p53 by Mass Spectrometry-Guided Computational Modeling

ABSTRACT: The tetrameric tumor suppressor p53 represents a great challenge for 3D structural analysis due to its high degree of intrinsic disorder (ca. 40%). We developed and applied an integrative structural biology approach combining complementary techniques of structural mass spectrometry (MS), namely cross-linking mass spectrometry (XL-MS), protein footprinting, and hydrogen/deuterium exchange mass spectrometry (HDX-MS), with advanced protein structure prediction approaches to gain insights into the disordered C-terminal region of p53. Additionally, we evaluate possible differences in p53 regarding solvent accessibility and topology upon DNA binding. Our quantitative XL-MS and lysine labeling data show no major conformational differences in p53 between DNA-bound and DNA-free states. Integration of experimental data generate p53 models for p53’s intrinsically disordered regions (IDRs) that reflect substantial compaction of the molecule. Our models provide the most detailed description of the relationship between p53’s folded regions and IDRs that is available to date. The synergies between complementary structural MS techniques and computational modeling as pursued in our integrative approach is envisioned to serve as general strategy for studying intrinsically disordered proteins (IDPs) and IDRs.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Escherichia Coli

SUBMITTER: Alessio Di Ianni

LAB HEAD: Andrea Sinz

PROVIDER: PXD037030 | Pride | 2023-06-26

REPOSITORIES: Pride

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Different Oligomeric States of the Tumor Suppressor p53 Show Identical Binding Behavior Towards the S100β Homodimer

Project description:The tumor suppressor protein p53 is a transcription factor that is referred to as the “guardian of the genome” and plays an important role in cancer development. P53 is active as a tetramer; the S100β homodimer binds to the intrinsically disordered C-terminus of p53, affecting its transcriptional activity. The p53/S100β complex is regarded as highly promising therapeutic target in cancer. It has been suggested that S100β exerts its oncogenic effects by altering the p53 oligomeric state. Our aim was to study the structures and oligomerization behavior of different p53/S100β complexes by electrospray ionization mass spectrometry (ESI-MS), cross-linking mass spectrometry (XL-MS), and surface plasmon resonance (SPR). For this, wild-type p53 and single amino acid variants, representing different oligomeric states of p53 (tetrameric wild-type, dimeric L344A variant, and monomeric L344P variant) were individually investigated regarding their binding behavior towards S100β. The stoichiometry of the different p53/S100β complexes were determined by ESI-MS showing that tetrameric, dimeric, and monomeric p53 variants all bind to an S100β dimer. In addition, XL-MS revealed the topologies of the p53/S100β complexes to be independent of p53’s oligomeric state. With SPR, the thermodynamic parameters were determined for S100β binding to tetrameric, dimeric or monomeric p53 variants. Our data prove that the S100β homodimer binds to different oligomeric states of p53 with identical stoichiometries and similar binding affinities. This emphasizes the need for alternative explanations to describe the molecular mechanisms underlying p53/S100β interaction.

2022-10-14 | PXD029914 | Pride

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2020-02-28 | PXD017299 | Pride

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Reconstitution of mammalian Cleavage Factor II

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2020-05-26 | PXD010177 | Pride

Integrative structure models of the α-Synuclein dimer conformations

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HspB8 controls molecular aging of FUS condensates by domain-specific protein interactions Part 2

Project description:Biomolecular condensates formed by phase separation offer a confined space where protein-protein interactions (PPIs) facilitate distinct biochemical reactions. As their aberrant behavior is associated with disease, it is crucial to understand the molecular organization of PPIs within condensates. Using quantitative time-resolved crosslinking mass spectrometry (XL-MS) we directly and specifically monitor PPIs and protein dynamics inside condensates formed by the protein fused in sarcoma (FUS) and identify its folded RNA recognition motif (RRM) as a key player of aberrant molecular aging. We find that the chaperone HspB8, but not a disease-associated mutant, prevents FUS aging. In XL-MS and partitioning experiments we find that the disordered region of HspB8 directs its α-crystallin domain (αCD) into FUS droplets where HspB8 prevents aging via condensate-specific αCD–RRM interactions. We propose that chaperones like HspB8 prevent aberrant phase transitions by stabilizing aggregation prone folded domains inside condensates in times of cellular stress.

2021-10-13 | PXD021115 | Pride

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