• BMB Reports
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• v.40 no.5
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• pp.649-655
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• 2007

The nsp14 protein is an exoribonuclease that is encoded by severe acute respiratory syndrome coronavirus (SARS-CoV). We have cloned and expressed the nsp14 protein in Escherichia coli, and characterized the nature and the role(s) of the metal ions in the reaction chemistry. The purified recombinant nsp14 protein digested a 5'-labeled RNA molecule, but failed to digest the RNA substrate that is modified with fluorescein group at the 3'-hydroxyl group, suggesting a 3'-to-5' exoribonuclease activity. The exoribonuclease activity requires $Mg^{2+}$ as a cofactor. Isothermal titration calorimetry (ITC) analysis indicated a two-metal binding mode for divalent cations by nsp14. Endogenous tryptophan fluorescence and circular dichroism (CD) spectra measurements showed that there was a structural change of nsp14 when binding with metal ions. We propose that the conformational change induced by metal ions may be a prerequisite for catalytic activity by correctly positioning the side chains of the residues located in the active site of the enzyme.

• Journal of Ginseng Research
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• v.44 no.3
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• pp.461-474
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• 2020

Background: Ginseng effectively reduces fatigue in both animal models and clinical trials. However, the mechanism of action is not completely understood, and its molecular targets remain largely unknown. Methods: By screening for proteins that interact with the primary components of ginseng (ginsenosides) in an affinity chromatography assay, we have identified muscle-type creatine kinase (CK-MM) as a potential target in skeletal muscle tissues. Results: Biolayer interferometry analysis showed that ginsenoside metabolites, instead of parent ginsenosides, had direct interaction with recombinant human CK-MM. Subsequently, 20(S)-protopanaxadiol (PPD), which is a ginsenoside metabolite and displayed the strongest interaction with CK-MM in the study, was selected as a representative to confirm direct binding and its biological importance. Biolayer interferometry kinetics analysis and isothermal titration calorimetry assay demonstrated that PPD specifically bound to human CK-MM. Moreover, the mutation of key amino acids predicted by molecular docking decreased the affinity between PPD and CK-MM. The direct binding activated CK-MM activity in vitro and in vivo, which increased the levels of tissue phosphocreatine and strengthened the function of the creatine kinase/phosphocreatine system in skeletal muscle, thus buffering cellular ATP, delaying exercise-induced lactate accumulation, and improving exercise performance in mice. Conclusion: Our results suggest a cellular target and an initiating molecular event by which ginseng reduces fatigue. All these findings indicate PPD as a small molecular activator of CK-MM, which can help in further developing better CK-MM activators based on the dammarane-type triterpenoid structure.

• Journal of Ginseng Research
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• v.45 no.4
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• pp.465-472
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• 2021

Background: Ginseng can help regulate brain excitability, promote learning and memory, and resist cerebral ischemia in the central nervous system. Ginsenosides are the major effective compounds of Ginseng, but their protein targets in the brain have not been determined. Methods: We screened proteins that interact with the main components of ginseng (ginsenosides) by affinity chromatography and identified the 14-3-3 ζ protein as a potential target of ginsenosides in brain tissues. Results: Biolayer interferometry (BLI) analysis showed that 20(S)-protopanaxadiol (PPD), a ginseng saponin metabolite, exhibited the highest direct interaction to the 14-3-3 ζ protein. Subsequently, BLI kinetics analysis and isothermal titration calorimetry (ITC) assay showed that PPD specifically bound to the 14-3-3 ζ protein. The cocrystal structure of the 14-3-3 ζ protein-PPD complex showed that the main interactions occurred between the residues R56, R127, and Y128 of the 14-3-3 ζ protein and a portion of PPD. Moreover, mutating any of the above residues resulted in a significant decrease of affinity between PPD and the 14-3-3 ζ protein. Conclusion: Our results indicate the 14-3-3 ζ protein is the target of PPD, a ginsenoside metabolite. Crystallographic and mutagenesis studies suggest a direct interaction between PPD and the 14-3-3 ζ protein. This finding can help in the development of small-molecular compounds that bind to the 14-3-3 ζ protein on the basis of the structure of dammarane-type triterpenoid.