Monitoring of the retinoic acid receptor–retinoid X receptor dimerization upon DNA binding by native mass spectrometry

Identifying protein–DNA interactions is essential to understand the regulatory networks of cells and their influence on gene expression. In this study, we use native electrospray mass spectrometry (ESI-MS) to investigate how the heterodimerization of retinoic acid receptor–retinoid X receptor (RAR-RXR) is mediated by DNA sequence. In presence of various RAR response elements (RAREs), three oligomeric states of RAR–RXR DNA binding domains (DBDs) bound to RAREs (monomer, homo- or heterodimers) were detected and individually monitored to follow subunit assembly and disassembly upon RAREs' abundancy or sequence. In particular, a cooperative heterodimerization was shown with RARb2 DR5 (5 base pair spaced direct repeat) while a high heterogeneity reflecting random complex formation could be observed with the DR0 response elements, in agreement with native gel electrophoresis data or molecular modeling. Such MS information will help to identify the composition of species formed in solution and to define which DR sequence is specific for RAR–RXR heterodimerization. [Display omitted] •The composition of RAR–RXR complexes varies with direct repeat sequences.•The heterodimerization of RAR–RXR bound to DR0 elements is non-cooperative.•Protein/DNA molar ratios determine the relative abundancy of RAR–RXR–DNA complexes.•The dimerization of RAR–RXR upon DNA binding was monitored by native ESI-MS.