Limits...
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.

Show MeSH

Related in: MedlinePlus

Mechanistic basis for ATP-dependent inhibition of ProRS by halofuginonea, HF forms extensive hydrophobic contacts and hydrogen-bonding interactions with ProRS and with ATPa. b, Two-dimensional presentation of HF binding. The HF, ATPa and hydrogen-bonded residues are colored as previously, and other residues within 4 Å of HF are colored in gray. c, A stick model showing the binding of HF’s piperidine ring to human ProRS. d, The proline binding pocket of T. thermophilus ProRS in complex with proline (PDB 1H4T, protein is colored as yellow and proline as gray-white). e, Structure superposition of catalytic domain of human ProRS with T. thermophilus ProRS reveals the piperidine ring of HF directly occupies the proline binding pocket. f, The stick model showing the binding of the halogenated 4-quinazolinone group of HF to human ProRS. g, Structure of E. coli ThrRS:tRNA complex, with the pocket for the adenosine group for A76 (PDB 1QF6). h, Overlay of the catalytic domain of human ProRS onto E. coli ThrRS (brown) reveals that HF uses the binding pocket for A76 of the CCA76-3′ end for binding the quinazolinone moiety.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3569068&req=5

Figure 2: Mechanistic basis for ATP-dependent inhibition of ProRS by halofuginonea, HF forms extensive hydrophobic contacts and hydrogen-bonding interactions with ProRS and with ATPa. b, Two-dimensional presentation of HF binding. The HF, ATPa and hydrogen-bonded residues are colored as previously, and other residues within 4 Å of HF are colored in gray. c, A stick model showing the binding of HF’s piperidine ring to human ProRS. d, The proline binding pocket of T. thermophilus ProRS in complex with proline (PDB 1H4T, protein is colored as yellow and proline as gray-white). e, Structure superposition of catalytic domain of human ProRS with T. thermophilus ProRS reveals the piperidine ring of HF directly occupies the proline binding pocket. f, The stick model showing the binding of the halogenated 4-quinazolinone group of HF to human ProRS. g, Structure of E. coli ThrRS:tRNA complex, with the pocket for the adenosine group for A76 (PDB 1QF6). h, Overlay of the catalytic domain of human ProRS onto E. coli ThrRS (brown) reveals that HF uses the binding pocket for A76 of the CCA76-3′ end for binding the quinazolinone moiety.

Mentions: ATPa was located at the canonical ATP binding pocket of class II aaRSs12. Three hydrogen bonds contributed by Thr1164 and Thr1276 and hydrophobic stacking of Phe1167 stabilized the adenosine group (Supplementary Fig. 2). The ribose moiety was fixed by three hydrogen bonds with Gln1237, Thr1240 and Arg1278. The hydrogen bonds from Arg1152 and Arg1163 contributed to stabilizing the α-, β- and γ-phosphate groups of ATPa (Supplementary Fig. 2), which form a cap over the HF binding pocket (Fig. 1c). These extensive hydrophilic and hydrophobic interactions fixed the ATP in a “U”-like bent conformation with the α-phosphate group exposed at the bottom of the U. Superposition of the catalytic domains of human ProRS with that of the previously determined structure of T. thermophilus ProRS showed the conformation and location of ATP was the same (Supplementary Fig. 3), with the α-phosphate group poised for proline activation. In the human ProRS:HF:ATPa ternary complex, the α-phosphate group is proximal to the hydroxypiperidine ring of HF and forms two hydrogen bonds with its hydroxyl group (Fig. 2a, b). It also forms an additional hydrogen bond with the keto group in the bridge between the piperidine ring and the quinazolinone moiety of HF (Fig. 2a, b). These direct hydrogen-bond interactions enable ATP to lock onto and orient HF.


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

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

Mechanistic basis for ATP-dependent inhibition of ProRS by halofuginonea, HF forms extensive hydrophobic contacts and hydrogen-bonding interactions with ProRS and with ATPa. b, Two-dimensional presentation of HF binding. The HF, ATPa and hydrogen-bonded residues are colored as previously, and other residues within 4 Å of HF are colored in gray. c, A stick model showing the binding of HF’s piperidine ring to human ProRS. d, The proline binding pocket of T. thermophilus ProRS in complex with proline (PDB 1H4T, protein is colored as yellow and proline as gray-white). e, Structure superposition of catalytic domain of human ProRS with T. thermophilus ProRS reveals the piperidine ring of HF directly occupies the proline binding pocket. f, The stick model showing the binding of the halogenated 4-quinazolinone group of HF to human ProRS. g, Structure of E. coli ThrRS:tRNA complex, with the pocket for the adenosine group for A76 (PDB 1QF6). h, Overlay of the catalytic domain of human ProRS onto E. coli ThrRS (brown) reveals that HF uses the binding pocket for A76 of the CCA76-3′ end for binding the quinazolinone moiety.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569068&req=5

Figure 2: Mechanistic basis for ATP-dependent inhibition of ProRS by halofuginonea, HF forms extensive hydrophobic contacts and hydrogen-bonding interactions with ProRS and with ATPa. b, Two-dimensional presentation of HF binding. The HF, ATPa and hydrogen-bonded residues are colored as previously, and other residues within 4 Å of HF are colored in gray. c, A stick model showing the binding of HF’s piperidine ring to human ProRS. d, The proline binding pocket of T. thermophilus ProRS in complex with proline (PDB 1H4T, protein is colored as yellow and proline as gray-white). e, Structure superposition of catalytic domain of human ProRS with T. thermophilus ProRS reveals the piperidine ring of HF directly occupies the proline binding pocket. f, The stick model showing the binding of the halogenated 4-quinazolinone group of HF to human ProRS. g, Structure of E. coli ThrRS:tRNA complex, with the pocket for the adenosine group for A76 (PDB 1QF6). h, Overlay of the catalytic domain of human ProRS onto E. coli ThrRS (brown) reveals that HF uses the binding pocket for A76 of the CCA76-3′ end for binding the quinazolinone moiety.
Mentions: ATPa was located at the canonical ATP binding pocket of class II aaRSs12. Three hydrogen bonds contributed by Thr1164 and Thr1276 and hydrophobic stacking of Phe1167 stabilized the adenosine group (Supplementary Fig. 2). The ribose moiety was fixed by three hydrogen bonds with Gln1237, Thr1240 and Arg1278. The hydrogen bonds from Arg1152 and Arg1163 contributed to stabilizing the α-, β- and γ-phosphate groups of ATPa (Supplementary Fig. 2), which form a cap over the HF binding pocket (Fig. 1c). These extensive hydrophilic and hydrophobic interactions fixed the ATP in a “U”-like bent conformation with the α-phosphate group exposed at the bottom of the U. Superposition of the catalytic domains of human ProRS with that of the previously determined structure of T. thermophilus ProRS showed the conformation and location of ATP was the same (Supplementary Fig. 3), with the α-phosphate group poised for proline activation. In the human ProRS:HF:ATPa ternary complex, the α-phosphate group is proximal to the hydroxypiperidine ring of HF and forms two hydrogen bonds with its hydroxyl group (Fig. 2a, b). It also forms an additional hydrogen bond with the keto group in the bridge between the piperidine ring and the quinazolinone moiety of HF (Fig. 2a, b). These direct hydrogen-bond interactions enable ATP to lock onto and orient HF.

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