Search

The b-HLH-LZ domain of c-Myc is a key target for the development of cancer therapies by blunting its binding to DNA with cell penetrant b-HLH-LZs and/or by stabilizing it into a state that cannot recognize Max to activate and amplify transcription of oncogenic genes. Although recent milestones have been reached with DNA binding blunting of c-Myc with the cell penetrant b-HLH-LZ Omomyc, the targeting of its b-HLH-LZ with small molecules, peptides, or proteins is lagging. As reviewed recently, the main problem relies in the intrinsically disordered nature of the b-HLH-LZ of c-Myc. This greatly complicates the classical approach of targeting a docking site with inhibitors. The solution state methods such as NMR are progressing towards the characterization of the ensembles of structures or states the b-HLH-LZ can adopt. However, the delicate balance that dictates the population of these dynamically interchanging states relies on its primary structure and the weak polar, electrostatic and hydrophobic interactions allowed. In this context, it is of the utmost importance to study the b-HLH-LZ of c-Myc in its WT background and avoid the use of tags such as His-tags. These tags could disrupt the balance of forces which could alter the conformational and physical transitions and states it can undergo and adopt. Here, we describe a robust protocol to express the WT b-HLH-LZ in E. coli and purify it, without the need of tags, to obtain the required quantities for solution state biophysical characterization such as NMR.

Miz-1 (ZBTB17) is a poly-Zinc Finger BTB/POZ transcription factor with 12 consecutive C2H2 Zinc Fingers (ZFs). Miz-1 binds transcriptional start sites (TSS), through a consensus sequence, to regulate the expression of genes involved in the cell cycle, autophagy, trafficking as well as T- and B-cell development. Although it is not known how exactly Miz-1 recognises this consensus, evidence and predictions suggest that ZFs 7 to 12 are able to bind it contiguously. As observed in the other members of the ZBTB family, striking divergences in highly conserved positions in the primary structures of the ZFs and linkers of Miz-1 are present. In fact, compared to the conserved TGEKP/Y found in the rest of ZFs 7-12, the linker between ZFs 10 and 11 has the following sequence: DNIRP/H586. We show by solution-state NMR and 15N-relaxation studies that this linker generates two coupled conformational exchange phenomena on the “biological” μs-ms timescale. On the one hand, we demonstrate that the His residue (H586) weakens the βα tertiary structure in ZF11 and promotes the population of an opened state in which the inter-ZF contacts, necessary for canonical DNA binding, is lacking. The existence of this opened state is supported by the local unfolding of the first helical turn of the recognition helix of ZF11. On the other hand, although this local unfolding is quenched by the replacement of the His by the conserved Tyr residue (Y), we found that another conformational exchange is present in the DNIRP linker. Most interestingly, we observe that the DNIRP linker adopts a helical structure that also impedes interZF contacts necessary for canonical and contiguous binding of the consensus. This implies that the recognition of the consensus by ZFs 7-12 is accomplished in a segmented fashion. Supported by bioinformatics predictions and DNA binding assays, our structural and dynamical data are congruent with a model whereby ZFs 7-9 and 11-12 can independently recognize both ends and strands of the consensus. Finally, considering the dynamic nature of DNA and the conformational exchange in ZFs 10-11, we further demonstrate that the simultaneous binding of ZF 7-9 or ZFs 11-12 to their respective subsites is physically possible in a coiled conformation of the consensus.