## 3 Discussion
However, we did not observe that the presence of the RNA chain had a significant effect on the rate of hydrolysis of the bound compound [12]. In contrast, GppG, which was shown here for the first time as a potent substrate for hNudt16, is an interesting compound for further research on the biological function of this protein because of its high affinity for hNudt16, similar to IDP. Dinucleoside diphosphates are compounds stored in dense granules in human platelets and released into the extracellular space. [PERSON] et al. showed that GppG has a stimulatory effect on vascular smooth muscle cells, representing a new class of mediators with regulatory roles [27]. Dinucleoside polyphosphate are a part of the purinergic signaling system [28] similarly as guanidine-based purines (GTP, GDP, GMP, GUO, GUA). The latter have been shown to modulate important intracellular processes such as growth, differentiation and survival [29]. Given the hydrolytic activity of hNudt16, it may affect the level of compounds that are important components of the purinergic signaling system (e.g., GppG/GMP). Further research in this direction can bring important data about Nudt16 biological role. The ligand-binding modes were elucidated to explain the structural reasons for the large differences in ligand-dependent affinities. Docking of the high-affinity ligands to hNudt16 indicated the existence of two ligand-binding modes. The presence of two binding modes is in agreement with the in silico identification of a potential secondary druggable site for hNudt16 by [PERSON] et al. [37]. Both modes detected in this study are located inside the hNudt16 main binding pocket in the vicinity of the enzyme's active site. Mode 1 is most likely an active conformation adopted during the catalytic event, as the ligands in mode 1 share a common position of phosphates in close proximity to the magnesium ions. They interact with structural elements that are crucial for the catalytic activity of hNudt16, such as Arg50 [17]. We also believe that Arg50 plays a vital role in positioning of the ligands along the magnesium cations, crucial for adopting an active conformation inside the hNudt16 catalytic pocket. The electrostatic attraction between the negatively charged phosphate groups and two Mg cations is a key constituent of the ligand binding pose in docking mode 1. A lack of both Mg\({}^{2+}\) ions may prevent ligand binding in mode 1 and, as a result, inactivate enzyme. Our experiments have shown two distinct binding affinity values for strongly interacting IDP and GppG. In line with experimental data, molecular docking identified two binding modes, with significantly different protein interaction energies. The element common to both modes is the binding of one of the bases in the pocket flanked by His24, Phe57 and Gln170. Because of the considerable overlap between these two modes, the enzyme can only bind one substrate at a time in the active site pocket. In this scenario, the two Kd values could mean that simultaneously a certain population of ligands is bound in mode 1 and another smaller population in mode 2. Another explanation could be a two-step ligand binding. In this case, some ligands would bind directly in mode 1, while others would initially bind in mode 2, in which one of the bases is stably bound in the enzyme pocket while other occupies a variety of positions outside the pocket. Such binding, being less stable, is likely to oscillate between different positions, to eventually flip to the stable and energetically favorable mode 1. In the case of poorly bound and hydrolyzed ligand-AppA, we found that its interactions with hNudt16 in mode 1 are less stable; the nucleobase can easily flip out of the binding pocket, while still interacting with magnesium ions. This observation confirms that adopting a stable nucleobase position inside the pocket flanked by His24, Phe57, and Gln170 is crucial for enzymatic catalysis.