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ATP-directed capture of bioactive herbal-based medicine on human tRNA synthetase.

Zhou H, Sun L, Yang XL, Schimmel P - Nature (2012)

Bottom Line: Thus, HF is a new type of ATP-dependent inhibitor that simultaneously occupies two different substrate binding sites on ProRS.Moreover, our structure indicates a possible similar mechanism of action for febrifugine in malaria treatment.Finally, the elucidation here of a two-site modular targeting activity of HF raises the possibility that substrate-directed capture of similar inhibitors might be a general mechanism that could be applied to other synthetases.

View Article: PubMed Central - PubMed

Affiliation: The Skaggs Institute for Chemical Biology, Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

ABSTRACT
Febrifugine is the active component of the Chinese herb Chang Shan (Dichroa febrifuga Lour.), which has been used for treating malaria-induced fever for about 2,000 years. Halofuginone (HF), the halogenated derivative of febrifugine, has been tested in clinical trials for potential therapeutic applications in cancer and fibrotic disease. Recently, HF was reported to inhibit T(H)17 cell differentiation by activating the amino acid response pathway, through inhibiting human prolyl-transfer RNA synthetase (ProRS) to cause intracellular accumulation of uncharged tRNA. Curiously, inhibition requires the presence of unhydrolysed ATP. Here we report an unusual 2.0 Å structure showing that ATP directly locks onto and orients two parts of HF onto human ProRS, so that one part of HF mimics bound proline and the other mimics the 3' end of bound tRNA. Thus, HF is a new type of ATP-dependent inhibitor that simultaneously occupies two different substrate binding sites on ProRS. Moreover, our structure indicates a possible similar mechanism of action for febrifugine in malaria treatment. Finally, the elucidation here of a two-site modular targeting activity of HF raises the possibility that substrate-directed capture of similar inhibitors might be a general mechanism that could be applied to other synthetases.

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HF interacts with both the site for amino acid activation and the site for docking the 3′-end of tRNAa, Thermal melting19 of ProRS in the presence of different ligands. HF binds in the absence of ATPa, and binds more strongly in the presence of ATPa. b, HF and the comparator Pro-SA block formation of Pro-AMP, in the proline-dependent ATP-PPi exchange reaction. The inset shows that HF had no effect on the alanine-dependent ATP-PPi exchange reaction with AlaRS. c, HF mobilizes the release of Pro-AMP from ProRS. The ProRS:3H-Pro-AMP complex was prepared on ice and then isolated on a column and for 10 min was left untreated, or exposed to HF, or exposed to Pro-SA, respectively. The complex was then re-run on the column and the amount of bound 3H-Pro-AMP was determined. The inset shows that the isolated 3H-Pro was activated (as 3H-Pro-AMP) and could be transferred to tRNA. Error bars are s.e.m. (n=2).
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Figure 3: HF interacts with both the site for amino acid activation and the site for docking the 3′-end of tRNAa, Thermal melting19 of ProRS in the presence of different ligands. HF binds in the absence of ATPa, and binds more strongly in the presence of ATPa. b, HF and the comparator Pro-SA block formation of Pro-AMP, in the proline-dependent ATP-PPi exchange reaction. The inset shows that HF had no effect on the alanine-dependent ATP-PPi exchange reaction with AlaRS. c, HF mobilizes the release of Pro-AMP from ProRS. The ProRS:3H-Pro-AMP complex was prepared on ice and then isolated on a column and for 10 min was left untreated, or exposed to HF, or exposed to Pro-SA, respectively. The complex was then re-run on the column and the amount of bound 3H-Pro-AMP was determined. The inset shows that the isolated 3H-Pro was activated (as 3H-Pro-AMP) and could be transferred to tRNA. Error bars are s.e.m. (n=2).

Mentions: We first determined that the thermal melting of ProRS was barely affected by either proline or ATPa. In contrast, in the presence of HF, the melting curve was shifted to higher temperature by about 10 oC by HF alone, and by about 18 oC with HF plus ATPa (Fig 3a). These data show that HF binds even in the absence of ATP, and also confirm that HF binds much tighter in the presence of ATP. Not surprisingly, the thermal shift obtained with Pro-SA was the largest.


ATP-directed capture of bioactive herbal-based medicine on human tRNA synthetase.

Zhou H, Sun L, Yang XL, Schimmel P - Nature (2012)

HF interacts with both the site for amino acid activation and the site for docking the 3′-end of tRNAa, Thermal melting19 of ProRS in the presence of different ligands. HF binds in the absence of ATPa, and binds more strongly in the presence of ATPa. b, HF and the comparator Pro-SA block formation of Pro-AMP, in the proline-dependent ATP-PPi exchange reaction. The inset shows that HF had no effect on the alanine-dependent ATP-PPi exchange reaction with AlaRS. c, HF mobilizes the release of Pro-AMP from ProRS. The ProRS:3H-Pro-AMP complex was prepared on ice and then isolated on a column and for 10 min was left untreated, or exposed to HF, or exposed to Pro-SA, respectively. The complex was then re-run on the column and the amount of bound 3H-Pro-AMP was determined. The inset shows that the isolated 3H-Pro was activated (as 3H-Pro-AMP) and could be transferred to tRNA. Error bars are s.e.m. (n=2).
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Related In: Results  -  Collection

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Figure 3: HF interacts with both the site for amino acid activation and the site for docking the 3′-end of tRNAa, Thermal melting19 of ProRS in the presence of different ligands. HF binds in the absence of ATPa, and binds more strongly in the presence of ATPa. b, HF and the comparator Pro-SA block formation of Pro-AMP, in the proline-dependent ATP-PPi exchange reaction. The inset shows that HF had no effect on the alanine-dependent ATP-PPi exchange reaction with AlaRS. c, HF mobilizes the release of Pro-AMP from ProRS. The ProRS:3H-Pro-AMP complex was prepared on ice and then isolated on a column and for 10 min was left untreated, or exposed to HF, or exposed to Pro-SA, respectively. The complex was then re-run on the column and the amount of bound 3H-Pro-AMP was determined. The inset shows that the isolated 3H-Pro was activated (as 3H-Pro-AMP) and could be transferred to tRNA. Error bars are s.e.m. (n=2).
Mentions: We first determined that the thermal melting of ProRS was barely affected by either proline or ATPa. In contrast, in the presence of HF, the melting curve was shifted to higher temperature by about 10 oC by HF alone, and by about 18 oC with HF plus ATPa (Fig 3a). These data show that HF binds even in the absence of ATP, and also confirm that HF binds much tighter in the presence of ATP. Not surprisingly, the thermal shift obtained with Pro-SA was the largest.

Bottom Line: Thus, HF is a new type of ATP-dependent inhibitor that simultaneously occupies two different substrate binding sites on ProRS.Moreover, our structure indicates a possible similar mechanism of action for febrifugine in malaria treatment.Finally, the elucidation here of a two-site modular targeting activity of HF raises the possibility that substrate-directed capture of similar inhibitors might be a general mechanism that could be applied to other synthetases.

View Article: PubMed Central - PubMed

Affiliation: The Skaggs Institute for Chemical Biology, Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

ABSTRACT
Febrifugine is the active component of the Chinese herb Chang Shan (Dichroa febrifuga Lour.), which has been used for treating malaria-induced fever for about 2,000 years. Halofuginone (HF), the halogenated derivative of febrifugine, has been tested in clinical trials for potential therapeutic applications in cancer and fibrotic disease. Recently, HF was reported to inhibit T(H)17 cell differentiation by activating the amino acid response pathway, through inhibiting human prolyl-transfer RNA synthetase (ProRS) to cause intracellular accumulation of uncharged tRNA. Curiously, inhibition requires the presence of unhydrolysed ATP. Here we report an unusual 2.0 Å structure showing that ATP directly locks onto and orients two parts of HF onto human ProRS, so that one part of HF mimics bound proline and the other mimics the 3' end of bound tRNA. Thus, HF is a new type of ATP-dependent inhibitor that simultaneously occupies two different substrate binding sites on ProRS. Moreover, our structure indicates a possible similar mechanism of action for febrifugine in malaria treatment. Finally, the elucidation here of a two-site modular targeting activity of HF raises the possibility that substrate-directed capture of similar inhibitors might be a general mechanism that could be applied to other synthetases.

Show MeSH
Related in: MedlinePlus