FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices
Eitan Lerner, Anders Barth, Jelle Hendrix, Benjamin Ambrose, Victoria Birkedal, Scott C Blanchard, Richard Börner, Hoi Sung Chung, Thorben Cordes, Timothy D Craggs, Ashok A Deniz, Jiajia Diao, Jingyi Fei, Ruben L Gonzalez, Irina V Gopich, Taekjip Ha, Christian A Hanke, Gilad Haran, Nikos S Hatzakis, Sungchul Hohng, Seok-Cheol Hong, Thorsten Hugel, Antonino Ingargiola, Chirlmin Joo, Achillefs N Kapanidis, Harold D Kim, Ted Laurence, Nam Ki Lee, Tae-Hee Lee, Edward A Lemke, Emmanuel Margeat, Jens Michaelis, Xavier Michalet, Sua Myong, Daniel Nettels, Thomas-Otavio Peulen, Evelyn Ploetz, Yair Razvag, Nicole C Robb, Benjamin Schuler, Hamid Soleimaninejad, Chun Tang, Reza Vafabakhsh, Don C Lamb, Claus AM Seidel, Shimon Weiss
Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current ‘state of the art’ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of ‘soft recommendations’ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage ‘open science’ practices.
Succinylation of H3K122 destabilizes nucleosomes and enhances transcription
Lara Zorro Shahidian, Mariska Haas, Stephanie Le Gras, Sandra Nitsch, André Mourão, Arie Geerlof, Raphael Margueron, Jens Michaelis, Sylvain Daujat, Robert Schneider
Histone post‐translational modifications (PTMs) are key players in chromatin regulation. The identification of novel histone acylations raises important questions regarding their role in transcription. In this study, we characterize the role of an acylation on the lateral surface of the histone octamer, H3K122 succinylation (H3K122succ), in chromatin function and transcription. Using chromatin succinylated at H3K122 in in vitro transcription assays, we show that the presence of H3K122succ is sufficient to stimulate transcription. In line with this, we found in our ChIP assays H3K122succ enriched on promoters of active genes and H3K122succ enrichment scaling with gene expression levels. Furthermore, we show that the co‐activators p300/CBP can succinylate H3K122 and identify sirtuin 5 (SIRT5) as a new desuccinylase. By applying single molecule FRET assays, we demonstrate a direct effect of H3K122succ on nucleosome stability, indicating an important role for histone succinylation in modulating chromatin dynamics. Together, these data provide the first insights into the mechanisms underlying transcriptional regulation by H3K122succ.
The enzyme subunit SubA of Shiga toxin-producing E. coli strains demonstrates comparable intracellular transport and cytotoxic activity as the holotoxin SubAB in HeLa and HCT116 cells in vitro
Katharina Sessler, Panagiotis Papatheodorou, Fanny Wondany, Maike Krause, Sabrina Noettger, Denise Bernhard, Jens Michaelis, Herbert Schmidt & Holger Barth
The subtilase cytotoxin (SubAB) is secreted by certain Shiga toxin-producing Escherichia coli (STEC) strains and is composed of the enzymatically active subunit SubA and the pentameric binding/transport subunit SubB. We previously demonstrated that SubA (10 µg/ml), in the absence of SubB, binds and intoxicates the human cervix cancer-derived epithelial cell line HeLa. However, the cellular and molecular mechanisms underlying the cytotoxic activity of SubA in the absence of SubB remained unclear. In the present study, the cytotoxic effects mediated by SubA alone were investigated in more detail in HeLa cells and the human colon cancer cell line HCT116. We found that in the absence of SubB, SubA (10 µg/ml) is internalized into the endoplasmic reticulum (ER), where it cleaves the chaperone GRP78, an already known substrate for SubA after its canonical uptake into cells via SubB. The autonomous cellular uptake of SubA and subsequent cleavage of GRP78 in cells is prevented by treatment of cells with 10 µM brefeldin A, which inhibits the transport of protein toxins into the ER. In addition, by analyzing the SubA mutant SubAΔC344, we identified the C-terminal SEEL motif as an ER-targeting signal. Conclusively, our results strongly suggest that SubA alone shares the same intracellular transport route and cytotoxic activity as the SubAB holotoxin.