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J Biol Chem. 2017 Feb 24;292(8):3323-3340. doi: 10.1074/jbc.M116.748699. Epub 2016 Dec 29.

Structural Insights into c-Myc-interacting Zinc Finger Protein-1 (Miz-1) Delineate Domains Required for DNA Scanning and Sequence-specific Binding.

Author information

1
From the Département de Biochimie, Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke J1H 5N4, Canada, the Regroupement Stratégique sur la Fonction, la Structure, et l'Ingénierie des Protéines (PROTEO), Université Laval, Québec G1V 0A6, Canada, and the Groupe de Recherche Axé sur la Structure des Protéines (GRASP), McGill University, Montréal, Québec H3G 0B1, Canada.
2
From the Département de Biochimie, Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke J1H 5N4, Canada, the Regroupement Stratégique sur la Fonction, la Structure, et l'Ingénierie des Protéines (PROTEO), Université Laval, Québec G1V 0A6, Canada, and the Groupe de Recherche Axé sur la Structure des Protéines (GRASP), McGill University, Montréal, Québec H3G 0B1, Canada pierre.lavigne@usherbrooke.ca.

Abstract

c-Myc-interacting zinc finger protein-1 (Miz-1) is a poly-Cys2His2 zinc finger (ZF) transcriptional regulator of many cell cycle genes. A Miz-1 DNA sequence consensus has recently been identified and has also unveiled Miz-1 functions in other cellular processes, underscoring its importance in the cell. Miz-1 contains 13 ZFs, but it is unknown why Miz-1 has so many ZFs and whether they recognize and bind DNA sequences in a typical fashion. Here, we used NMR to deduce the role of Miz-1 ZFs 1-4 in detecting the Miz-1 consensus sequence and preventing nonspecific DNA binding. In the construct containing the first 4 ZFs, we observed that ZFs 3 and 4 form an unusual compact and stable structure that restricts their motions. Disruption of this compact structure by an electrostatically mismatched A86K mutation profoundly affected the DNA binding properties of the WT construct. On the one hand, Miz1-4WT was found to bind the Miz-1 DNA consensus sequence weakly and through ZFs 1-3 only. On the other hand, the four ZFs in the structurally destabilized Miz1-4A86K mutant bound to the DNA consensus with a 30-fold increase in affinity (100 nm). The formation of such a thermodynamically stable but nonspecific complex is expected to slow down the rate of DNA scanning by Miz-1 during the search for its consensus sequence. Interestingly, we found that the motif stabilizing the compact structure between ZFs 3 and 4 is conserved and enriched in other long poly-ZF proteins. As discussed in detail, our findings support a general role of compact inter-ZF structures in minimizing the formation of off-target DNA complexes.

KEYWORDS:

fluorescence anisotropy; nuclear magnetic resonance (NMR); protein dynamic; protein-DNA interaction; protein-protein interaction; structural biology; transcription factor; zinc finger

PMID:
28035002
PMCID:
PMC5336166
DOI:
10.1074/jbc.M116.748699
[Indexed for MEDLINE]
Free PMC Article

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