Claudia Bich1, Cédric Bovet1, Natacha Rochel2, Carole Peluso-Iltis2, Andreas Panagiotidis3, Alexis Nazabal4, Dino Moras2, and Renato Zenobi1
- Department of Chemistry and Applied Biosciences, ETH Zürich, Zürich, Switzerland
- Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS, Illkirch, France
- Institute of Molecular Systems Biology (IMSB), ETH Zürich, Zürich, Switzerland
- CovalX AG, Schlieren, Switzerland
Nuclear receptors, such as the retinoic acid receptor (RAR) or the 9-cis retinoic acid receptor (RXR), interact not only with their ligands but also with other types of receptors and with DNA. Here, two complementary mass spectrometry (MS) methods were used to study the interactions between retinoic receptors (RXR/RAR) and DNA: non-denaturing nano-electrospray (nanoESI MS), and high-mass matrix-assisted laser desorption ionization (MALDI MS) combined with chemical cross-linking. The RAR·RXR heterodimer was studied in the presence of a specific DNA sequence (DR5), and a specific RAR·RXR·DNA complex was detected with both MS techniques. RAR by itself showed no significant homodimerization. A complex between RAR and the double stranded DR5 was detected with nanoESI. After cross-linking, high-mass MALDI mass spectra showed that the RAR binds the single stranded DR5, and the RAR dimer binds both single and double stranded DR5. Moreover, the MALDI mass spectrum shows a larger RAR dimer signal in the presence of DNA. These results suggest that a gene-regulatory site on DNA can induce quaternary structural changes in a transcription factor such as RAR.
CovalX Technology Used (Click each option to learn more)
RAR, RAR·RXR, AND DR5 stock solutions were desalted and buffer exchanged against 50 mM NH4OAc (pH 7.4) using columns. The DR5 was incubated with the RAR at room temperature for 15 minutes. The RAR stock solution was diluted to 5.5 μM and the RAR·RXR stock solution was diluted to 5 μM. DR5, non-specific A9, and C9, a 5 μM solution was incubated with the proteins for 15 minutes before the cross-linker was added. The cross-linking reactions were performed using the CovalX K100 Stabilization Kit at approximately 50-fold molar excess in a total volume of 10 μL. The sample/cross-linker mixture was incubated at room temperature for 120 minutes in tubes covered with aluminum foil. 1 μL of the sample was then mixed with 1 μL of the matrix (sinapic acid (10 mg/mnL) in acetonitrile:water (1:1, v/v) with 0.1% TFA before being spotted on a MALDI sample plate. A mass spectrometer that had been modified with a CovalX HM1 detection system was used to analyze the samples. The data was then interpreted using the CovalX Complex Tracker software.