GMP kinase

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Introduction

Metabolism is altered in cancer cells, leading to tumor proliferation. A consequence of this metabolic alteration is increased levels of the nucleotides GTP and dGTP due to the upregulation of nucleotide biosynthesis. GTP and dGTP are precursors of RNA and DNA, respectively, and GTP is an important energy source for protein biosynthesis and a key signaling molecule via GTP-binding proteins. An indispensable enzyme in the metabolism of GTP and dGTP is human guanylate kinase (hGMPK, gene name GUK1, ATP: GMP phosphotransferase, EC 2.7.4.8). This enzyme exists at the junction of the salvage and de novo purine nucleotide biosynthesis pathways. hGMPK catalyzes the reversible phosphorylation of GMP to GDP by using ATP as a phosphoryl group donor. Thus, hGMPK is required for all GDP/GTP production, and chemotherapeutic strategies focused on depleting GDP/GTP from cancer cells have targeted two de novo purine nucleotide biosynthesis pathway enzymes, upstream of hGMPK, IMP dehydrogenase (IMPDH) and guanosine monophosphate synthase (GMPS).

The depletion of GDP/GTP levels caused by inhibition of IMPDH can be complemented by the addition of guanosine or guanine to cells, as the salvage pathway still functions through the action of hGMPK and nucleoside diphosphate kinases. So to completely inhibit GDP/GTP production, enzymes located downstream of the intersection of the salvage and de novo pathways must be effectively targeted. Therefore, inhibition of hGMPK may be a potential therapeutic target. Currently, hGMPK is used to activate a variety of antiviral and antineoplastic nucleoside prodrugs, among which 6-thioguanine and its closely related analogue 6-mercaptopurine and 9-β-d-arabinofuranosylguanine are the most effective. Recently, a small-angle X-ray scattering (SAXS) study on hGMPK presented the first reported structural data for hGMPK, demonstrating that ligand binding results in hGMPK compression by approximately 2 Å. Subsequently, solution NMR spectroscopy solved the first high-resolution structure of hGMPK, which is also the first structure with atomic-level resolution of a nucleotide-free mammalian GMPK.

Figure 1. Summary of hGMPK's role in the cell (Khan, N.; et al. 2019)

Solution structure of hGMPK

The electrostatic charge distribution of hGMPK suggests that nucleotide binding is mediated by electrostatic interactions.  The highly positive patch located between the LID domain and the P-loop of the CORE domain includes residues that have been shown to coordinate ATP in the X-ray structure of mGMPK. Residues that interact with phosphate groups and the guanine bases, respectively, are present in positive (GMP-BD) and negative patches (GMP-BD and CORE). In fact, the orientation of the side chains involved in coordinating GMP and ATP are very similar between humans and mice, demonstrating the importance of these electrostatic patches for hGMPK function.

Figure 2. Electrostatic charge distribution of the hGMPK surface (Khan, N.; et al. 2019)

Next, we compared the domain orientation of the hGMPK NMR structure with the yGMPK and mGMPK X-ray structures, using multiple sequence alignment to select the same residues from each species. Notably, hGMPK and mGMPK share 88% sequence identity, equating to a total of 24 of 197 different residues; however, the orientation of the different side chains remains relatively similar. With the exception of the N- and C-termini, the structural differences associated with the substitution of these residues are mainly located around the hinge regions, namely G30S, V86E and Q89R. An interesting exception is L25F, located in the α1 center, where the leucine side chain points away from the interior of hGMPK, whereas the phenylalanine side chain in mGMPK points inward. As is the case for many other enzymes, large-scale domain motions are required for hGMPK to function. Catalytic efficiency was significantly reduced, either by mechanical stress or by mutation of Ser-35 or Ser-37 to proline.

Figure 3. location of the CORE domain, nsSNVs, and active site residues depicted in the lowest-energy conformer of apo-hGMPK (Khan, N.; et al. 2019)