Nucleophosmin (NPM1) is a nucleocytoplasmic 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 nuclease-hypersensitive element III region of the c-MYC promoter. These activities are lost by the leukemic variant. Here we analyze the NPM1/G-quadruplex interaction, focusing on residues belonging to both the NPM1 terminal three-helix bundle and a lysine-rich unstructured tail, which has been shown to be necessary for high affinity recognition. We performed extended site-directed mutagenesis and measured 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.

Arcovito, A., Chiarella, S., Della Longa, S., Di Matteo, A., Lo Sterzo, C., Scaglione, G. L., Federici, L., Synergic Role of Nucleophosmin Three-helix Bundle and a Flanking Unstructured Tail in the Interaction with G-quadruplex DNA, <<JOURNAL OF BIOLOGICAL CHEMISTRY>>, 2014; 289 (31): 21230-21241. [doi:10.1074/jbc.M114.565010] [http://hdl.handle.net/10807/59599]

Synergic Role of Nucleophosmin Three-helix Bundle and a Flanking Unstructured Tail in the Interaction with G-quadruplex DNA

Arcovito, Alessandro;Scaglione, Giovanni Luca;
2014

Abstract

Nucleophosmin (NPM1) is a nucleocytoplasmic 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 nuclease-hypersensitive element III region of the c-MYC promoter. These activities are lost by the leukemic variant. Here we analyze the NPM1/G-quadruplex interaction, focusing on residues belonging to both the NPM1 terminal three-helix bundle and a lysine-rich unstructured tail, which has been shown to be necessary for high affinity recognition. We performed extended site-directed mutagenesis and measured 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.
Inglese
Arcovito, A., Chiarella, S., Della Longa, S., Di Matteo, A., Lo Sterzo, C., Scaglione, G. L., Federici, L., Synergic Role of Nucleophosmin Three-helix Bundle and a Flanking Unstructured Tail in the Interaction with G-quadruplex DNA, <<JOURNAL OF BIOLOGICAL CHEMISTRY>>, 2014; 289 (31): 21230-21241. [doi:10.1074/jbc.M114.565010] [http://hdl.handle.net/10807/59599]
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10807/59599
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