## 2 Results
### hNudt16 Stability in the Presence of Different Ligands and Its Substrate Affinity Studies
#### 2.3.4 Molecular Docking Analysis  
To elucidate the structural reasons for the large differences observed in ligand affinities to hNudt16 dependent on the ligand nucleobase, we performed molecular docking of the chosen ligands to the hNudt16 crystal structure. The following three ligands were docked: IDP and GppG, which were found to strongly interact with hNudt16E76Q, and AppA, which showed weaker interactions with hNudt16E76Q (Tables 2 and 3). Docking revealed two distinguishable ligand binding modes (Figure 6) inside the hNudt16 binding pocket, differing in the location of the phosphate groups and one of the nucleobases. All predicted ligand poses can be classified as one of these modes. In both modes, one of the bases was located inside the pocket flanked by His24, Phe57, and Gln170. The same pocket was  
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 \(\chi\) value (compared to SAXS data) were obtained in CRYSOL. PDB: 2 XSQ 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 \(R_{\mathrm{g}}=24.16\) Å and \(D_{\mathrm{max}}=79.22\) Å; (**C**) superposition of low-resolution 3D model of hNudt16 in solution (semitransparent shape) with the crystal structure of dimeric hNudt16 PDB: 2 XSQ in projections along the two axes of the molecule. Monomer-like subunits of 2 XSQ dimer are marked with different colors (light and dark blue). Figures obtained in PyMOL. occupied by the nucleobase of IMP, crystallized with hNudt16 (Figure S5). His24 interacts with the base via \(\pi\) stacking, while Phe57 and Gln170 participate in a hydrogen-bond network stabilizing the interacting ligand. The estimated free energies of binding in mode 1 of approximately \(-16\) kcal/mol are two times more favorable than in mode 2 with \(\Delta\)Gs of \(-6\) to \(-8\) kcal/mol (Table S1). In mode 1, which was found to be energetically favored in the case of all docked ligands, two Mg\({}^{2+}\) ions were closely aligned with the phosphate groups, showing strong charge-charge interactions (Figures 6 and 7). Arg50, which appeared indispensable in the adoption of mode 1 conformations, in the most energetically favorable poses, bonded with the phosphate groups and ribose ring via hydrogen bonds (Figure 7). Therefore, we believe that Arg50 plays a vital role in positioning the ligands along the magnesium cations. In mode 1, the ligands interacted with structural elements that are crucial for the catalytic activity of hNudt16, such as Mg\({}^{2+}\) and Arg50. In binding mode 1, this was likely the ligand conformation adopted during the cleavage event. 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. In mode 2, the phosphate groups and one of the bases moved away from the magnesium cations towards the exit of the hNudt16 active site. However, in mode 2, the ligand conformation and its interaction network varied and were not as conserved as those in mode 1. In dinucleotide ligands, one of the nucleobases fit into the hNudt16 pocket (Figure 6), while the other nucleobase adopted a range of conformations, interacting with various amino acids on the hNudt16 surface, including Gly101, Ser102, Ala4, Ser 166, and His99 (Figure S6). 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).