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

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.

Figures

Figure 1. Structure of human ProRS and of bound ligands

a, Chemical structures of halofuginone (HF) and the ATP analog (ATPa). b, Two ProRS monomers form an asymmetric unit that is a homodimer. HF and ATPa are shown as spherical models at the active site of both subunits. Here and throughout, ATPa and HF are colored as in Fig.1a. c, HF is buried at the bottom of the pocket and covered by co-bound ATPa. A simulated annealing omit map was calculated with Fourier coefficients 2Fo-Fc, and contoured at 1.5σ.

Figure 2. Mechanistic basis for ATP-dependent inhibition of ProRS by halofuginone

a, 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.

Figure 3. HF interacts with both the site for amino acid activation and the site for docking the 3′-end of tRNA

a, Thermal melting 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).

Figure 4. FF blocks ProRS

Febrifugine (FF) docked to the active site of P. falcipaurm ProRS (UniProtKB code Q8I5R7). ProRS and FF are colored as cyan and yellow, respectively. The hydrogen bonds are indicated with dashes. The P. falciparum ProRS structure was generated by the protein structure homology-modeling sever SWISS-MODEL. FF was docked into the P. falciparum ProRS active site based on the structure of ProRS:halofuginone:ATPa complex, while ATP was docked based on the structure of the T. thermophilus ProRS:tRNA:ATP complex (PDB code 1H4Q).