Abstract
Human guanylate kinase (hGMPK) is the only known enzyme responsible for cellular GDP production, making it essential for cellular viability and proliferation. Moreover, hGMPK has been assigned a critical role in metabolic activation of antiviral and antineoplastic nucleoside-analog prodrugs. Given that hGMPK is indispensable for producing the nucleotide building blocks of DNA, RNA, and cGMP and that cancer cells possess elevated GTP levels, it is surprising that a detailed structural and functional characterization of hGMPK is lacking. Here, we present the first high-resolution structure of hGMPK in the apo form, determined with NMR spectroscopy. The structure revealed that hGMPK consists of three distinct regions designated as the LID, GMP-binding (GMP-BD), and CORE domains and is in an open configuration that is nucleotide binding-competent. We also demonstrate that nonsynonymous single-nucleotide variants (nsSNVs) of the hGMPK CORE domain distant from the nucleotide-binding site of this domain modulate enzymatic activity without significantly affecting hGMPK's structure. Finally, we show that knocking down the hGMPK gene in lung adenocarcinoma cell lines decreases cellular viability, proliferation, and clonogenic potential while not altering the proliferation of immortalized, noncancerous human peripheral airway cells. Taken together, our results provide an important step toward establishing hGMPK as a potential biomolecular target, from both an orthosteric (ligand-binding sites) and allosteric (location of CORE domain-located nsSNVs) standpoint.
Highlights
We focused on a selection of nsSNVs of hGMPK, as reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) database [50], nsSNVs not directly involved in nucleotide binding within the CORE domain
To assess the effect of each nsSNV on hGMPK’s catalytic properties, we evaluated their thermostability with differential scanning fluorimetry (DSF)
We offer support for the existence of allosteric networking linking nonactive site CORE domain residues to the nucleotide-binding domains via sites of nsSNV mutations
Summary
We solved the solution structure of hGMPK using our previously published chemical shift assignment and our measured 15N-resolved 1H,1H NOESY (60-ms mixing time), 13Caliphaticresolved 1H,1H NOESY (80 ms mixing time), and 13Caromaticresolved 1H,1H NOESY (80-ms mixing time) spectra [35] (Fig. 2; Protein Data Bank (PDB) accession code 6NUI; see “Experimental procedures” for a detailed description of the structure determination). As a supplement to these data, we measured backbone 15N heteronuclear NOEs (15N-hetNOEs) and a set of residual dipolar couplings (RDCs) (Fig. 3 and Table S1). The 15N-hetNOE data enabled us to justify removal of dihedral angle and RDC constraints for flexible residues with 15N-hetNOE values lower than 0.70 from the structure calculation. Totals of 2430 NOE, 281 backbone dihedral, 89 NHN-RDCs, and 58 hydrogen bond constraints were used as input for the structure calculation and refinement performed with CYANA [36, 37] and AMBER [39]. The z-scores of the hGMPK structure were analyzed by
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