Engrailed homeodomain uses an electrostatic spring-loaded mechanism to change conformation upon binding to DNA
Résumé
DNA-binding transcription factors (TFs) recognize and bind specific DNA sequences found at gene control elements to recruit and regulate the transcription machinery. The TF’s recognition of the target site on an ocean of genomic DNA requires an efficient mechanism for DNA scanning. Traditionally, this problem has been formulated in terms of the facilitated diffusive search for one target site on non-specific DNA. However, our group recently found that the engrailed homeodomain (EnHD) binds promiscuously to any variation of its target site with a ladder of affinities. The implication is that the DNA scanning mechanism of homeodomains should be capable of adapting to such a spectrum of binding affinities. In parallel, homeodomains have sequences and structural features that suggest they are intrinsically disordered, including NMR structural studies of the protein alone at moderate salt concentrations. In X-ray structures in complex with DNA or alone at very high salt, a three-helix bundle structure forms with helix 3 inserted in the major groove. Our working hypothesis is that EnHD changes conformation gradually in response to the sequence-dependent interaction energetics with DNA, and that such interplay enables an adaptive DNA scanning mechanism for the efficient navigation of the complex binding signals of eukaryotic gene control elements. We are investigating the coupling between folding and DNA binding of EnHD at atomic resolution using NMR. We have established that, in solution and moderate to physiological ionic strength EnHD does experience a conformational change upon binding to DNA. This structural rearrangement is very apparent by NMR, with ∼60% of the backbone 15N-1H cross-peaks experiencing large chemical shift changes. These changes are largely recapitulated by high ionic strength in the absence of DNA, which indicates that they reflect a conformational change coupled to binding.