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Atomic-resolution mapping of transcription factor-DNA interactions by femtosecond laser crosslinking and mass spectrometry

Alexander Reim,R. Ackermann,Jofre Font-Mateu,Robert Kammel,M. Beato,S. Nolte,M. Mann,C. Russmann,M. Wierer

Published 2020 in Nature Communications

ABSTRACT

Transcription factors (TFs) regulate target genes by specific interactions with DNA sequences. Detecting and understanding these interactions at the molecular level is of fundamental importance in biological and clinical contexts. Crosslinking mass spectrometry is a powerful tool to assist the structure prediction of protein complexes but has been limited to the study of protein-protein and protein-RNA interactions. Here, we present a femtosecond laser-induced crosslinking mass spectrometry (fliX-MS) workflow, which allows the mapping of protein-DNA contacts at single nucleotide and up to single amino acid resolution. Applied to recombinant histone octamers, NF1, and TBP in complex with DNA, our method is highly specific for the mapping of DNA binding domains. Identified crosslinks are in close agreement with previous biochemical data on DNA binding and mostly fit known complex structures. Applying fliX-MS to cells identifies several bona fide crosslinks on DNA binding domains, paving the way for future large scale ex vivo experiments. Cross-linking mass spectrometry (MS) is an important tool in structural biology, but its application to protein-DNA complexes has been hampered by low cross-linking efficiency. Here, the authors develop a femtosecond UV-laser induced cross-linking MS workflow to map protein-DNA interactions in vitro and in cells.

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