title: Structural Insights into c-Myc-interacting Zinc Finger Protein-1 (Miz-1) Delineate Domains Required for DNA Scanning and Sequence-specific Binding.
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, Miz1-4WTwas 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 Miz1-4A86Kmutant 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.
authors.affiliation: Département de Biochimie, Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke J1H 5N4, Canada; Regroupement Stratégique sur la Fonction, la Structure, et l'Ingénierie des Protéines (PROTEO), Université Laval, Québec G1V 0A6, Canada; Groupe de Recherche Axé sur la Structure des Protéines (GRASP), McGill University, Montréal, Québec H3G 0B1, Canada.
authors.forename: Mikaël
authors.initials: M
authors.lastname: Bédard
authors.affiliation: Département de Biochimie, Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke J1H 5N4, Canada; Regroupement Stratégique sur la Fonction, la Structure, et l'Ingénierie des Protéines (PROTEO), Université Laval, Québec G1V 0A6, Canada; Groupe de Recherche Axé sur la Structure des Protéines (GRASP), McGill University, Montréal, Québec H3G 0B1, Canada.
authors.forename: Vincent
authors.initials: V
authors.lastname: Roy
authors.affiliation: Département de Biochimie, Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke J1H 5N4, Canada; Regroupement Stratégique sur la Fonction, la Structure, et l'Ingénierie des Protéines (PROTEO), Université Laval, Québec G1V 0A6, Canada; Groupe de Recherche Axé sur la Structure des Protéines (GRASP), McGill University, Montréal, Québec H3G 0B1, Canada.
authors.forename: Martin
authors.initials: M
authors.lastname: Montagne
authors.affiliation: Département de Biochimie, Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke J1H 5N4, Canada; Regroupement Stratégique sur la Fonction, la Structure, et l'Ingénierie des Protéines (PROTEO), Université Laval, Québec G1V 0A6, Canada; Groupe de Recherche Axé sur la Structure des Protéines (GRASP), McGill University, Montréal, Québec H3G 0B1, Canada. Electronic address: pierre.lavigne@usherbrooke.ca.
authors.forename: Pierre
authors.initials: P
authors.lastname: Lavigne
mesh_terms: D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism*; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D016415:Sequence Alignment; D016335:Zinc Fingers
chemical_list: D051741:Kruppel-Like Transcription Factors; C107961:ZBTB17 protein, human; D004247:DNA
keywords: fluorescence anisotropy; nuclear magnetic resonance (NMR); protein dynamic; protein-DNA interaction; protein-protein interaction; structural biology; transcription factor; zinc finger