Abstract
We here investigate the interactions between the DnaB helicase and the C-terminal domain of the corresponding DnaG primase of Helicobacter pylori using solid-state NMR. The difficult crystallization of this 387 kDa complex, where the two proteins interact in a six to three ratio, is circumvented by simple co-sedimentation of the two proteins directly into the MAS-NMR rotor. While the amount of information that can be extracted from such a large protein is still limited, we can assign a number of amino-acid residues experiencing significant chemical-shift perturbations upon helicase-primase complex formation. The location of these residues is used as a guide to model the interaction interface between the two proteins in the complex. Chemical-shift perturbations also reveal changes at the interaction interfaces of the hexameric HpDnaB assembly on HpDnaG binding. A structural model of the complex that explains the experimental findings is obtained.
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Acknowledgments
This work was supported by the French Agence Nationale de la Recherche (ANR-11-BSV8-021-01, ANR-12-BS08- 0013-01, ANR-14-CE09-0024B), the ETH Zurich, the Swiss National Science Foundation (Grants 200020_159707 and 200020_146757). AB and LT are supported by the CIBLE program 2011 from the Région Rhône-Alpes. We thank Professor Gourinath for providing us the coordinates of the HpDnaG-CTD model. We are grateful for support from TGIR-RMN-THC FR3050.
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Carole Gardiennet and Thomas Wiegand have contributed equally to this work.
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Gardiennet, C., Wiegand, T., Bazin, A. et al. Solid-state NMR chemical-shift perturbations indicate domain reorientation of the DnaG primase in the primosome of Helicobacter pylori . J Biomol NMR 64, 189–195 (2016). https://doi.org/10.1007/s10858-016-0018-0
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DOI: https://doi.org/10.1007/s10858-016-0018-0