Journal Article

Protein complexes in cells by AI‐assisted structural proteomics

Stülke, Jörg, Rappsilber, Juri, Forbrig, Christian, Bremenkamp, Rica, Elfmann, Christoph, Charles, Kristine, O'Reilly, Francis J, Graziadei, Andrea, Lenz, Swantje, Fischer, Lutz

2023

Overview

Accurately modeling the structures of proteins and their complexes using artificial intelligence is revolutionizing molecular biology. Experimental data enable a candidate‐based approach to systematically model novel protein assemblies. Here, we use a combination of in‐cell crosslinking mass spectrometry and co‐fractionation mass spectrometry (CoFrac‐MS) to identify protein–protein interactions in the model Gram‐positive bacterium Bacillus subtilis . We show that crosslinking interactions prior to cell lysis reveals protein interactions that are often lost upon cell lysis. We predict the structures of these protein interactions and others in the Subti Wiki database with AlphaFold‐Multimer and, after controlling for the false‐positive rate of the predictions, we propose novel structural models of 153 dimeric and 14 trimeric protein assemblies. Crosslinking MS data independently validates the AlphaFold predictions and scoring. We report and validate novel interactors of central cellular machineries that include the ribosome, RNA polymerase, and pyruvate dehydrogenase, assigning function to several uncharacterized proteins. Our approach uncovers protein–protein interactions inside intact cells, provides structural insight into their interaction interfaces, and is applicable to genetically intractable organisms, including pathogenic bacteria. Synopsis An integrative approach using crosslinking mass spectrometry (MS), co‐fractionation MS and Alphafold‐Multimer discovers novel protein complexes and their topologies in the model gram‐positive bacterium Bacillus subtillis. Crosslinking mass spectrometry and co‐fractionation mass spectrometry identify protein interactions from intact cells. AlphaFold‐Multimer confidently predicts the structure of dimeric complexes which can be validated with crosslinks. The binding site of YneR on the E1 subunit of the pyruvate dehydrogenase complex identifies it as an inhibitor of pyruvate dehydrogenase activity, PdhI. The approach can assign structure, function, and interactors of uncharacterized proteins in whole cells without requiring genetic manipulation. Graphical Abstract An integrative approach using crosslinking mass spectrometry (MS), co‐fractionation MS, and AlphaFold‐Multimer discovers novel protein complexes and their topologies in the model Gram‐positive bacterium Bacillus subtillis .

• Publisher: Nature Publishing Group UK,EMBO Press,John Wiley and Sons Inc,Springer Nature
• Subject: AlphaFold‐Multimer / Artificial Intelligence / Assemblies / Bacteria / Crosslinking / crosslinking mass spectrometry / Datasets / DNA-directed RNA polymerase / EMBO23 / EMBO40 / EMBO56 / Fractionation / Gram-positive bacteria / Interfaces / Lysis / Mass spectrometry / Mass Spectrometry - methods / Mass spectroscopy / Molecular Biology / Protein interaction / Proteins / Proteins - chemistry / protein–protein interactions / Proteomics / Proteomics - methods / pyruvate dehydrogenase / Pyruvic acid / RNA polymerase / Scientific imaging / Structural models / uncharacterized proteins