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Structural and dynamical characterization of the Miz-1 zinc fingers 5–8 by solution-state NMR

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Abstract

Myc-interacting zinc finger protein-1 (Miz-1) is a BTB/POZ transcription factor that activates the transcription of cytostatic genes, such as p15INK4B or p21CIP1. The C-terminus of Miz-1 contains 13 consensus C2H2 zinc finger domains (ZF). ZFs 1–4 have been shown to interact with SMAD3/4, while the remaining ZFs are expected to bind the promoters of target genes. We have noted unusual features in ZF 5 and the linker between ZFs 5 and 6. Indeed, a glutamate is found instead of the conserved basic residue two positions before the second zinc-coordinating histidine on the ZF 5 helix, and the linker sequence is DTDKE in place of the classical TGEKP sequence. In a canonical ββα fold, such unusual primary structure elements should cause severe electrostatic repulsions. In this context, we have characterized the structure and the dynamics of a Miz-1 construct comprising ZFs 5–8 (Miz 5–8) by solution-state NMR. Whilst ZFs 5, 7 and 8 were shown to adopt the classical ββα fold for C2H2 ZFs, the number of long-range NOEs was insufficient to define a classical fold for ZF 6. We show by using 15N-relaxation dispersion experiments that this lack of NOEs is due to the presence of extensive motions on the μs–ms timescale. Since this negatively charged region would have to be located near the phosphodiester backbone in a DNA complex, we propose that in addition to promoting conformational searches, it could serve as a hinge region to keep ZFs 1–4 away from DNA.

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Abbreviations

CD:

Circular dichroism

Miz 5-8:

Miz-1 zinc fingers 5–8

NMR:

Nuclear magnetic resonance spectroscopy

Water-TFA:

0.1 % trifluoroacetic acid in water

ZF:

Zinc finger

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Acknowledgments

This work was supported by the Natural Science and Engineering Research Council (NSERC) of Canada (grant to P.L. and studentships to M.B. and D.B.) and by the Regroupement stratégique sur la fonction, la structure et l’ingénierie des protéines (PROTEO). D.B. also acknowledges PROTEO for the award of graduate studentship. The authors thank Dr. Frank Hänel (Hans-Knöll-Institut für Naturstoff-Forschung e.V, Germany) for kindly providing us with the Miz-1 cDNA, Dr. Yves L. Dory (Université de Sherbrooke, Canada) for giving access and advice to use his HPLC system, Dr. Jean-François Naud for the initial cloning and Dr. Martin Montagne for his helpful comments on the manuscript. Finally, we thank Prof. Jim Omichinski (U. de Montréal) for his help with the refolding protocol of Zinc Fingers.

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Authors and Affiliations

  1. Département de Biochimie, Faculté de Médecine et des Sciences de la Santé, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, 3001, 12e avenue Nord, Sherbrooke, QC, J1H 5N4, Canada

    David Bernard, Mikaël Bédard, Josée Bilodeau & Pierre Lavigne

  2. Regroupement Stratégique sur la Fonction, la Structure et l’Ingénierie des Protéines (PROTEO), Québec, Canada

    David Bernard, Mikaël Bédard, Josée Bilodeau & Pierre Lavigne

  3. Groupe de Recherche Axé sur la Structure des Protéines (GRASP), Montréal, Canada

    David Bernard, Mikaël Bédard, Josée Bilodeau & Pierre Lavigne

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  1. David Bernard
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  2. Mikaël Bédard
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  4. Pierre Lavigne
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Correspondence to Pierre Lavigne.

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Bernard, D., Bédard, M., Bilodeau, J. et al. Structural and dynamical characterization of the Miz-1 zinc fingers 5–8 by solution-state NMR. J Biomol NMR 57, 103–116 (2013). https://doi.org/10.1007/s10858-013-9770-6

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