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Structural Analysis of 70S Ribosomes by Cross-Linking/Mass Spectrometry Reveals Conformational Plasticity

ABSTRACT: We analyzed 143 E. coli ribosomal structures, mapping a total of 10,771 intramolecular distances for 126 cross-link-pairs and 3,405 intermolecular distances for 97 protein pairs. Our study might serve as benchmark for conducting biochemical experiments on newly modeled protein regions, guided by XL-MS.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Escherichia Coli

SUBMITTER: Daniele Ubbiali

LAB HEAD: Andrea Sinz

PROVIDER: PXD018935 | Pride | 2020-08-13

REPOSITORIES: Pride

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			Action	DRS
	Ribosome_1A.mzML	Mzml
	Ribosome_1A.mzid	Mzid
	Ribosome_1A.raw	Raw
	Ribosome_1B.mzML	Mzml
	Ribosome_1B.mzid	Mzid

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Structural analysis of 70S ribosomes by cross-linking/mass spectrometry reveals conformational plasticity.

Tüting Christian C Iacobucci Claudio C Ihling Christian H CH Kastritis Panagiotis L PL Sinz Andrea A

Scientific reports 20200728 1

The ribosome is not only a highly complex molecular machine that translates the genetic information into proteins, but also an exceptional specimen for testing and optimizing cross-linking/mass spectrometry (XL-MS) workflows. Due to its high abundance, ribosomal proteins are frequently identified in proteome-wide XL-MS studies of cells or cell extracts. Here, we performed in-depth cross-linking of the E. coli ribosome using the amine-reactive cross-linker disuccinimidyl diacetic urea (DSAU). We ...[more]

PMID: 32724211

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2022-09-12 | PXD032739 | Pride

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Epitope and Paratope Mapping of TNFα/Infliximab complex by cross-linking mass spectrometry and integrative modeling reveals high level of interaction specificity

Project description:Mass spectrometry (MS) has become one of the core technologies to study protein structures, protein-ligand and protein-protein interactions. Chemical cross-linking combined with mass spectrometry (XL-MS) is quickly spreading over different biochemistry laboratories for the continuous increase of its biological applications and standardized protocols that make it attractive for academia and industry research scientists. In recent years, XL-MS has developed as a powerful technique in structural biology, from studying structures of purified proteins in vitro to deciphering intricate protein-protein interaction (PPI) networks in cells, organelles and tissues on a system-wide scale. However, the number of XL-MS studies for the inves-tigation of epitope/paratope mapping of antigen-antibody complexes is still limited up to now. In this manuscript, we devel-oped a simple, fast and automated workflow to define the interaction interface between the chimeric antibody Infliximab (IFX) and its own target, the Tumor Necrosis Factor alpha (TNFα). This workflow integrates XL-MS data to identify areas in proximity to the binding regions and epitope/paratope probability calculation to pinpoint the antigen-antibody binding sites. The data are combined to generate refined 3D models of the antigen-antibody complex which closely resemble the X-ray IFX/TNFα structure and capture their correct interaction surface. The present workflow can be applied to investigate any antigen-antibody complex, even when no previous structural knowledge is available. This strategy is a fascinating oppor-tunity to thoroughly characterize antigen-antibody interactions and to allow the understanding of their binding mechanisms in order to properly design better antibody therapeutics in the future

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