Synergic Role of Nucleophosmin Three-Helix Bundle and of a Flanking Unstructured Tail in the Interaction with G-Quadruplex DNA [Molecular Biophysics]

June 21st, 2014 by Arcovito, A., Chiarella, S., Della Longa, S., Di Matteo, A., Lo Sterzo, C., Scaglione, G. L., Federici, L.

Nucleophosmin (NPM1) is a nucleo-cytoplasmic shuttling protein, mainly localized at nucleoli, that plays a number of functions in ribosome biogenesis and export, cell cycle control and response to stress stimuli. NPM1 is the most frequently mutated gene in Acute Myeloid Leukemia; mutations map to the C-terminal domain of the protein and cause its denaturation and aberrant cytoplasmic translocation. NPM1 C-terminal domain binds G-quadruplex regions at ribosomal DNA and at gene promoters, including the well characterized sequence from the NHEIII region of the c-MYC promoter. These activities are lost by the leukemic variant. Here we analyze the NPM1-G-quadruplex interaction, focussing both on residues belonging to the NPM1 terminal three-helix bundle and to a lysine-rich unstructured tail, which is shown to be necessary for high affinity recognition. We perform extended site-directed mutagenesis and measure binding rate constants through surface plasmon resonance analysis. These data, supported by molecular dynamics simulations, suggest that the unstructured tail plays a double role in the reaction mechanism. On the one hand, it facilitates the formation of an encounter complex through long range electrostatic interactions; on the other hand, it directly contacts the G-quadruplex scaffold, through multiple and transient electrostatic interactions, significantly enlarging the contact surface.
  • Posted in Journal of Biological Chemistry, Publications
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