Comprehensive profiling of the catalytic conformations of human Guanylate kinase

Abstract

Human guanylate kinase (GMPK) as the sole enzyme for GDP biosynthesis plays pivotal roles in antiviral prodrug activation and tumorigenesis. Despite its biological significance, the catalytic mechanism remains poorly understood. Here, we resolve crystal structures of GMPK in free and GMP-bound form, revealing the interdomain motions of GMPBD and LID relative to the CORE domain. Biochemical assays demonstrate potassium's dual functionality in substrate recognition and phosphoryl transfer catalysis. Structural analyses uncover intradomain conformational motion within the LID domain and essential interactions for ADP/ATP binding. Notably, the cooperative ATPγS binding potentiated by prior GMP binding are structurally elucidated. Three key complexes, pre-reaction state (GMP/ATPγS), transition state (AlF₄^- mimic), and post-reaction state (GDP/ADP), collectively delineate the reversible catalytic pathway. This comprehensive structural characterization of GMPK's dynamic landscape establishes a foundation for developing conformation-specific inhibitors through structure-guided drug design.

Fig. 1. GMP induces the closure of the GMP binding domain.

a Histogram of GMP-induced chemical shift perturbations on amino acid residues with a molar ratio of GMP to GMPK of 5:1. b Cartoon representation of the apo form of GMPK: aquamarine for the CORE domain; pink for the GMPBD domain; slate for the LID domain and wheat for the Hinge region. c Cartoon representation of the GMPK-GMP complex with the mFo-DFc omit map density (black mesh) for GMP shown at a contour level of 3σ. d Electrostatic surface representation of the GMPK-GMP complex. e, f Detailed views of the interaction between the GMP and GMPK. Dashed lines indicate interatomic distances. g, h Binding affinities between GMPK mutants and the GMP, measured by ITC. The displayed values represent the fitted K_d and their corresponding errors. i Kinetic parameters (K_M, k_cat and k_cat/K_M) for GMP were determined for both the wild-type and mutant GMPK. N.D. indicates no detectable activity.

Fig. 2. Potassium ion binding site.

a, b ITC analysis of GMPK binding to GMP in buffers containing 150 mM NaCl (blue curve) and 150 mM KCl (magenta curve), respectively. The displayed values represent the K_d and errors from triplicate experiments. c Kinetic parameters (K_M, K_i, k_cat and k_cat/K_M) for GMP obtained under various potassium and sodium ion conditions. d Cartoon representation of the GMPK-GMP-K⁺ complex, with a detailed view of the potassium ion binding site. Potassium ion is depicted as purple spheres, water molecules as red spheres, and the residues S37, D101, and GMP are shown in stick representation. The mFo-DFc omit map density (black mesh) for the Potassium ion and water molecules is shown at a contour level of 10σ. Dashed lines indicate interatomic distances. e Structural superposition of GMPK-GMP (white) and GMPK-GMP-K⁺ (aquamarine). Water molecules near the potassium binding site are shown as spheres. D101 and GMP are shown as sticks.

Fig. 3. Structural basis for ATP recognition.

a Histogram of ATPγS-induced chemical shift perturbations in GMPK at a molar ratio of 2:1. b Mapping of ATPγS-induced CSPs larger than mean plus 2σ (red) onto the apo state of the GMPK. c Cartoon representation of the GMPK-GMP-ATPγS complex structure with the mFo-DFc omit map density (black mesh) for GMP and ATPγS contoured at 3σ. d Electrostatic surface potential of the GMPK-GMP-ATPγS complex. e Structural superposition of GMPK-GMP-ATPγS and GMPK-ADP. pink: GMPK-ADP, aquamarine: GMPK-GMP-ATPγS. f Detailed view of the adenine moiety and α/β -phosphate moiety interactions with GMPK with dashed lines indicating interatomic distances. g Detailed view of the γ-phosphate moiety interactions with GMPK. h Binding affinity between GMPK mutants with ATPγS as determined by ITC with fitted values and errors presented. i Kinetic parameters (K_M, k_cat and k_cat/K_M) for ATP were determined for both the wild-type and mutant GMPK. N.D. indicates no detectable activity.

Fig. 4. GMP facilitates the binding of ATP to GMPK.

a Raw ITC binding isotherms of ATPγS binding to GMPK (blue curve) and GMPK-GMP mixtures (magenta curve). b Fitted ITC binding isotherms, with K_d values and associated errors presented from triplicate experiments. c¹ H-¹⁵N HSQC spectra of ATPγS titration against GMPK and GMPK-GMP mixtures, with colors representing molar ratios of ATPγS to GMPK: red (0), cyan (0.5), pink (1), and purple (2). d Raw ITC binding isotherms of ADP binding to GMPK (gray curve) and GMPK-GMP mixtures (pink curve). e Structural superposition of apo, ADP-bound and GMP-ATPγS-bound GMPK. f Structural superposition of apo, GMP-bound and GMP-ATPγS-bound GMPK.

Fig. 5. Structural basis of reversible phosphoryl transfer reactions.

a Structural superposition of the GMPK-GDP-ADP and GMPK-GMP-ATPγS complexes. b Interaction of the GDP β-phosphate with GMPK. c ITC analysis of GMPK mutants binding to GDP. d Reverse reaction recorded at different time points by ¹H NMR. e Cartoon representation of the GMPK-GMP-ADP-AlF₄^--Mg²⁺-K⁺ complex with the mFo-DFc omit map density (black mesh) for GMP, ADP, AlF₄^-, Mg²⁺ and K⁺ contoured at 3σ. f AlF₄^- and magnesium ion binding site. Potassium ion is depicted as purple sphere, water molecule as red sphere, magnesium ion as green sphere and AlF₄^- is shown in stick representation. g Structural superposition of the transition state analogue and GMPK-GMP-ATPγS-K⁺ complexes. h Proposed catalysis model of GMPK.