Structure of nucleophosmin DNA-binding domain and analysis of its complex with a G-quadruplex sequence from the c-MYC promoter

Abstract

Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, mainly localized at nucleoli, that plays a key role in several cellular functions, including ribosome maturation and export, centrosome duplication, and response to stress stimuli. More than 50 mutations at the terminal exon of the NPM1 gene have been identified so far in acute myeloid leukemia; the mutated proteins are aberrantly and stably localized in the cytoplasm due to high destabilization of the NPM1 C-terminal domain and the appearance of a new nuclear export signal. We have shown previously that the 70-residue NPM1 C-terminal domain (NPM1-C70) is able to bind with high affinity a specific region at the c-MYC gene promoter characterized by parallel G-quadruplex structure. Here we present the solution structure of the NPM1-C70 domain and NMR analysis of its interaction with a c-MYC-derived G-quadruplex. These data were used to calculate an experimentally restrained molecular docking model for the complex. The NPM1-C70 terminal three-helix bundle binds the G-quadruplex DNA at the interface between helices H1 and H2 through electrostatic interactions with the G-quadruplex phosphate backbone. Furthermore, we show that the 17-residue lysine-rich sequence at the N terminus of the three-helix bundle is disordered and, although necessary, does not participate directly in the contact surface in the complex.

FIGURE 1.

NPM1-C70 quadruplex-binding C-terminal domain encompassing residues 225–294. A, NMR solution structure of NPM1-C70 showing the 20 lowest energy structures. A lysine-rich natively unstructured segment (amino acids 225–242) precedes the terminal three-helix bundle. B, ¹⁵N-¹H R₁ NOE values. C, ¹⁵N-¹H R₂ NOE values. Low R₁ and high R₂ values for segment 225–242 are consistent with the N-terminal tail being unstructured. D, heteronuclear ¹⁵N{¹H} NOEs are positive but smaller than expected for an 8-kDa protein, indicating fast internal motion in the three-helix bundle. Red lines indicate average values for the 225–242 and the 243–294 segments, respectively.

FIGURE 2.

Analysis of Pu27 and Pu24I G-quadruplex conformations. A, superimposition of the one-dimensional ¹H NMR spectra of Pu27 in the free state (blue) and bound (red) to NPM1-C70. Both spectra were acquired at 700 MHz and at 290 K. B, superimposition of the one-dimensional ¹H NMR spectra of Pu24I in the free state (blue) and bound (red) to NPM1-C70. Both spectra were acquired at 700 MHz and 290 K. C, details of the superimposition between ω₁-¹³C-filtered, ω₂-¹³C-filtered NOESY experiments in a two-dimensional plane (¹H-¹H plane) of Pu24I·NPM1-C70 complex (black) and a classical two-dimensional NOESY of Pu24I in its free state (red). Both spectra were acquired at 700 MHz and 290 K.

FIGURE 3.

Interaction of NPM1-C70 with the Pu24I G-quadruplex. A, ¹⁵N HSQC spectra of the protein before (black) and after addition of stoichiometric amounts of unlabeled Pu24I (red). Representative chemical shift variations are labeled, indicating relevant residues. B, chemical shift variations cluster in the three-helix bundle, whereas they are not found at the N-terminal 225–242 segment. The horizontal black line indicates the average chemical shift variation plus one standard deviation upon Pu24I addition. C, residues experiencing chemical shift variations higher than the average plus one standard deviation are highlighted on the structure of the protein. Residues belonging to helices H1 and H2 are solvent-exposed. A few hydrophobic residues belonging to helix H3 are also affected, indicating coupling between the helices upon Pu24I binding. Heteronuclear ¹⁵N-¹H R₁ (D) and heteronuclear ¹⁵N-¹H R₂ values (E) for NPM1-C70 in complex with Pu24I indicate that the N-terminal region flanking the three-helix bundle remains unstructured after Pu24I binding. F, the increase of heteronuclear ¹⁵N{¹H} NOE values in the three-helix bundle upon Pu24I binding (see Fig. 1D for comparison) suggests increased rigidity.

FIGURE 4.

Intermolecular NOEs and HADDOCK calculations. A, an example of NOE cross-peak assignment from ω₁-¹³C-edited, ω₂-¹³C-filtered NOESY experiments in a two-dimensional plane (¹H-¹H plane) of the NPM1-C70·Pu24I complex acquired on a ¹³C,¹⁵N-labeled NPM1-C70·unlabeled Pu24I sample. B, the 20 lowest energy complex structures obtained by NMR data-restrained molecular docking calculations using the HADDOCK protocol. NPM1-C70 is represented in blue, and Pu24I is represented in green.

FIGURE 5.

Structural model of the NPM1-C70·Pu24I complex. A, ribbon representation of the lowest energy model showing helices H1 and H2 of NPM1-C70 approaching the G-quadruplex “laterally” and interacting with a specific segment of the backbone (in orange). B, NPM1-C70 is represented with its electrostatic surface (blue for positive and red for negative), whereas Pu24I is shown in ribbon representation. The Pu24I structure is shown in transparency to highlight the small positively charged groove in between helices H1 and H2 that accommodates a stretch of Pu24I nucleotides (G11–G16; colored in orange). The long unstructured tail is also positively charged and may play a role in long range electrostatic interactions with the approaching oligonucleotide.