Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Oct 10;22(20):10929.
doi: 10.3390/ijms222010929.

Insight into the Binding and Hydrolytic Preferences of hNudt16 Based on Nucleotide Diphosphate Substrates

Magdalena Chrabąszczewska  1 , Maria Winiewska-Szajewska  2 , Natalia Ostrowska  3 , Elżbieta Bojarska  3 , Janusz Stępiński  3 , Łukasz Mancewicz  1 , Maciej Łukaszewicz  1 , Joanna Trylska  3 , Michał Taube  4 , Maciej Kozak  4   5 , Edward Darżynkiewicz  1   3 , Renata Grzela  1   3
Affiliations

Insight into the Binding and Hydrolytic Preferences of hNudt16 Based on Nucleotide Diphosphate Substrates

Magdalena Chrabąszczewska et al. Int J Mol Sci. .

Abstract

Nudt16 is a member of the NUDIX family of hydrolases that show specificity towards substrates consisting of a nucleoside diphosphate linked to another moiety X. Several substrates for hNudt16 and various possible biological functions have been reported. However, some of these reports contradict each other and studies comparing the substrate specificity of the hNudt16 protein are limited. Therefore, we quantitatively compared the affinity of hNudt16 towards a set of previously published substrates, as well as identified novel potential substrates. Here, we show that hNudt16 has the highest affinity towards IDP and GppG, with Kd below 100 nM. Other tested ligands exhibited a weaker affinity of several orders of magnitude. Among the investigated compounds, only IDP, GppG, m7GppG, AppA, dpCoA, and NADH were hydrolyzed by hNudt16 with a strong substrate preference for inosine or guanosine containing compounds. A new identified substrate for hNudt16, GppG, which binds the enzyme with an affinity comparable to that of IDP, suggests another potential regulatory role of this protein. Molecular docking of hNudt16-ligand binding inside the hNudt16 pocket revealed two binding modes for representative substrates. Nucleobase stabilization by Π stacking interactions with His24 has been associated with strong binding of hNudt16 substrates.

Keywords: DSF; GppG; IDP; MST; SAXS; dinucleoside diphosphates; hNudt16; nudix family.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
HPLC chromatograms for the hydrolysis of GppG catalyzed by hNudt16. (A) GppG in buffer (without hNudt16), (B) after 2 min reaction with hNudt16, (C) after 8 min reaction with hNudt16. Substrate concentration was 20 µM, enzyme concentration 0.002 µM. Chromatogram (A) was obtained after five-minute substrate incubation at 95 °C to show that heat inactivation of the enzyme in the assay buffer does not lead nonenzymatic degradation of GppG.
Figure 2
Figure 2
(A) CD spectra for wild-type hNudt16 and studied mutants. (B) Contribution of secondary structures in the polypeptide chain obtained from CD measurements (calculated in BeStSel) in comparison to the corresponding crystal structure.
Figure 3
Figure 3
Representative MST pseudo-titration data for binding of GppG to hNudt16E76Q. Squares represent experimental points, solid lines represent results of fitting for two independent binding sites model, and gray area bounded by a dashed line represents 95% confidence bands for this model. See Figure S4 for other ligands data.
Figure 4
Figure 4
Comparison of the free energy associated with the dissociation of the complex (ΔGdiss) calculated for stronger binding site (with Kd1 obtained from MST) with ΔTm obtained from dye-free and DSF with dye measurements for tested hNudt16E76Q-ligand pairs.
Figure 5
Figure 5
SAXS data analysis for hNudt16: (A) Comparison of experimental SAXS data for hNudt16 in solution with a fit for all crystal structures of hNudt16 dimer available in the PDB database. Theoretical scattering curves for PDB structures and discrepancy χ value (compared to SAXS data) were obtained in CRYSOL. PDB: 2XSQ had the lowest discrepancy with experimental SAXS data; (B) pair distance distribution function (p(R)) determined based on SAXS scattering curve for the hNudt16 (calculated in GNOM software). The calculated structural parameters were Rg = 24.16 Å and Dmax = 79.22 Å; (C) superposition of low-resolution 3D model of hNudt16 in solution (semitransparent shape) with the crystal structure of dimeric hNudt16 PDB: 2XSQ in projections along the two axes of the molecule. Monomer-like subunits of 2XSQ dimer are marked with different colors (light and dark blue). Figures obtained in PyMOL.
Figure 6
Figure 6
Top: IDP and GppG bound to hNudt16 in docking (A) mode 1 and (B) mode 2. Bottom: Schematic illustration of IDP interactions in (C) mode 1 and (D) mode 2.
Figure 7
Figure 7
(A,B) Conformations of GppG and AppA bound to hNudt16 in mode 1. GppG remains in its active conformation, whereas adenine positions are less stabilized and one of the AppA adenines either stays inside (A) or flips out (B) of the pocket (marked with three residues, His24, Phe57 and Gln170 in ball and stick representation in gray). (C,D) Schematic illustration of GppG and AppA interactions in active conformations of mode 1 (ligand conformations as in (A)). (E) Comparison of the ligand positions in active conformations of mode 1: a very good hNudt16 substrate, GppG, vs. the conformation of a poor substrate, ADPr (ADPr position is from the PDB:5W6X structure). (F) Schematic illustration of ADPr interactions with hNudt16.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bessman M.J., Frick D., O’Handley S.F. The MutT proteins or “nudix” hydrolases, a family of versatile, widely distributed, “housecleaning” enzymes. J. Biol. Chem. 1996;271:25059–25062. doi: 10.1074/jbc.271.41.25059. - DOI - PubMed
    1. Mildvan A., Xia Z., Azurmendi H., Saraswat V., Legler P., Massiah M., Gabelli S., Bianchet M., Kang L.-W., Amzel L.M. Structures and mechanisms of Nudix hydrolases. Arch. Biochem. Biophys. 2005;433:129–143. doi: 10.1016/j.abb.2004.08.017. - DOI - PubMed
    1. Xu W., Dunn C.A., Jones C.R., D’Souza G., Bessman M.J. The 26 Nudix hydrolases of Bacillus cereus, a close relative of Bacillus anthracis. J. Biol. Chem. 2004;279:24861–24865. doi: 10.1074/jbc.M403272200. - DOI - PubMed
    1. Zheng Q.-C., Li Z.-S., Sun M., Zhang Y., Sun C.-C. Homology modeling and substrate binding study of Nudix hydrolase Ndx1 from Thermos thermophilus HB. Biochem. Biophys. Res. Commun. 2005;333:881–887. doi: 10.1016/j.bbrc.2005.05.169. - DOI - PubMed
    1. Peculis B.A., Reynolds K., Cleland M. Metal determines efficiency and substrate specificity of the nuclear NUDIX decapping proteins X29 and H29K (Nudt16) J. Biol. Chem. 2007;282:24792–24805. doi: 10.1074/jbc.M704179200. - DOI - PubMed