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Solution structure and functional investigation of human guanylate kinase reveals allosteric networking and a crucial role for the enzyme in cancer.

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.

SUBMITTER: Khan N

PROVIDER: S-EPMC6682748 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Solution structure and functional investigation of human guanylate kinase reveals allosteric networking and a crucial role for the enzyme in cancer.

Khan Nazimuddin N Shah Parag P PP Ban David D Trigo-Mouriño Pablo P Carneiro Marta G MG DeLeeuw Lynn L Dean William L WL Trent John O JO Beverly Levi J LJ Konrad Manfred M Lee Donghan D Sabo T Michael TM

The Journal of biological chemistry 20190614 31

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 functio ...[more]

PMID: 31201273

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Project description:Since the introduction of the induced-fit theory by D. E. Koshland Jr., it has been established that conformational motion invariably accompanies the execution of protein function. The catalytic activity of kinases, specifically, is associated with large conformational changes ( approximately 1 nm amplitude). In the case of guanylate kinase, upon substrate binding, the LID and nucleotide-monophosphate-binding domains are brought together and toward the CORE with large concerted movements about the alpha3 (helix 3) axis. However, whether the change in conformation mostly affects the catalytic rate or mostly increases binding affinities for one or the other substrate is unclear. We investigate this question using a nanotechnology approach based on mechanical stress. Using an "allosteric spring probe", we bias conformational states in favor of the "open" (substrate-free) conformation of the enzyme; the result is that the binding constant for the substrate guanosine monophosphate (GMP) is reduced by up to a factor of 10, whereas the binding constant for adenosine triphosphate (ATP) and the catalytic rate are essentially unaffected. The results show that the GMP-induced conformational change, which promotes catalysis, does not promote ATP binding, consistent with previous mutagenesis studies. Furthermore, they show that this conformational change is of the induced-fit type with respect to GMP binding (but not ATP binding). We elaborate on this point by proposing a quantitative criterion for the classification of conformational changes with respect to the induced-fit theory. More generally, these results show that the allosteric spring probe can be used to affect enzymatic activity in a continuously controlled manner, and also to affect specific steps of the reaction mechanism while leaving others unaffected. It is presumed that this will enable informative comparisons with the results of future molecular dynamics or statistical mechanics computations.

Dataset Information

Solution structure and functional investigation of human guanylate kinase reveals allosteric networking and a crucial role for the enzyme in cancer.

Publications

Solution structure and functional investigation of human guanylate kinase reveals allosteric networking and a crucial role for the enzyme in cancer.

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