Zinc Fingers 10 and 11 of Miz-1 undergo conformational exchange to achieve specific DNA binding.

Miz-1 (ZBTB17) is a poly-zinc finger BTB/POZ transcription factor with 12 consecutive C2H2 zinc fingers (ZFs) that binds transcriptional start sites (TSSs) to regulate the expression of genes involved in cell development and proliferation. As of now, it is not known which of the 12 consecutive ZFs are responsible for the recognition of the 24 base pair consensus sequence found at these TSSs. Evidence suggests ZFs 7-12 plays this role. We provide validation for this and describe the structural and dynamical characterization of unprecedented conformational exchange in the linker between ZFs 10 and 11. This conformational exchange uncouples ZFs 7-10 from 11 and 12 and promotes a scanning-recognition mechanism through which the two segments cooperate to bind two sub-sites at both ends of the consensus. We further show that this can result in the coiling of TSSs as part of Miz-1's mechanism of transcriptional transactivation.

Methods of Expression, Purification, and Preparation of the c-Myc b-HLH-LZ for Its Biophysical Characterization.

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.