Ligand-bound structures provide atomic snapshots for the catalytic mechanism of D-amino acid deacylase

Abstract

D-tyrosyl-tRNA(Tyr) deacylase (DTD) is an editing enzyme that removes D-amino acids from mischarged tRNAs. We describe an in-depth analysis of the malaria parasite Plasmodium falciparum DTD here. Our data provide structural insights into DTD complexes with adenosine and D-amino acids. Bound adenosine is proximal to the DTD catalysis site, and it represents the authentic terminal adenosine of charged tRNA. DTD-bound D-amino acids cluster at three different subsites within the overall active site pocket. These subsites, called transition, active, and exit subsites allow docking, re-orientation, chiral selection, catalysis, and exit of the free D-amino acid from DTD. Our studies reveal variable modes of D-amino acid recognition by DTDs, suggesting an inherent plasticity that can accommodate all D-amino acids. An in-depth analysis of native, ADP-bound, and D-amino acid-complexed DTD structures provide the first atomic snapshots of ligand recognition and subsequent catalysis by this enzyme family. We have mapped sites for the deacylation reaction and mark possible routes for entry and egress of all substrates and products. We have also performed structure-based inhibitor discovery and tested lead compounds against the malaria parasite P. falciparum using growth inhibition assays. Our studies provide a comprehensive structural basis for the catalytic mechanism of DTD enzymes and have implications for inhibition of this enzyme in P. falciparum as a route to inhibiting the parasite.

FIGURE 1.

Expression and activity of PfDTD. A, schematic representation of deacylation process. B, enzyme activity and selectivity of PfDTD. Rate of l-Tyr-tRNA deacylation was significantly lower than of d-Tyr-tRNA hydrolysis. C, localization of PfDTD in different intra-erythrocytic stages of P. falciparum by immunofluorescence staining, Ring stage (R), trophozoite stage (T), and schizont stage (S) are shown. In each panel are shown: (i) image of cell stained with DAPI (blue), (ii) anti-PfDTD antibodies, (iii) anti-PfNapL antibodies, (iv) merged image, (v) merge with phase contrast.

FIGURE 2.

Structure of PfDTD. A, 2-fold-related dimeric molecules A and B are colored in cornflower blue and purple, respectively. Each monomer contains eight β-strands (B1–B8), two α-helices (A1 and A2), and three long loops (IL, L1, and L2). The low complexity insertion loop IL residues 17–26, loop L1 residues 55–65, and loop L2 residues 90–110 are highlighted in orange, yellow, and brown, respectively. The N and C termini are marked. B, ordered insertion loop (IL) in PfDTD-d-Lys complex structure. Difference Fourier (Fo-Fc) and final 2Fo-Fc maps are contoured at 2.5 and 1.0σ levels, respectively. C, orthogonal views showing electrostatic potential at the molecular surface of the PfDTD dimer. The orientation of the dimer is similar to the one shown in A. The electrostatic surface is displayed as a color gradient in red (electronegative, ≤ −12 kTe⁻¹) and blue (electropositive, ≥ 12 kTe⁻¹). D, view of reduced thiols in PfDTD. The final 2Fo-Fc map is contoured at the 2.5σ level.

FIGURE 3.

Sequence conservation in DTDs. A, structure-based sequence alignment of DTDs. Proteins are: P. falciparum, A. aeolicus, E. coli, H. influenzae, and H. sapiens (also known as the DNA-unwinding element-binding protein, DUEB). Identical/well conserved residues, conserved residues, and semi-conserved residues are marked with asterisks, semicolons, and dots, respectively. The two conserved active site motifs (blue), tRNA recognition residues (green), and 3′-end of tRNA and d-amino acid binding residues (red) are highlighted. B, superimposition of crystal structures of PfDTD (cyan), HsDTD (purple), AaDTD (yellow), EcDTD (pink), and HiDTD (green). C, residue conservation in DTDs is shown as a schematic diagram. Identical, well-conserved, semi-conserved, and weakly conserved residues are rendered in red, pink, gray, and blue, respectively.

FIGURE 4.

Dimer interface. A, native DTD. The 2-fold-related molecule is labeled with an asterisk symbol. B, dimer interface of ADP-bound PfDTD structure. Asn-147 adopts different orientations, and an additional water molecule W1 is observed at the dimeric interface.

FIGURE 5.

Omit electron density map and surface representation of bound ADP in PfDTD-ATP complex I. A, omit map is contoured at the 1.5σ level near the active site residues in the PfDTD-ATP complex I structure. B, surface representation of the PfDTD active site, and bound ADP molecules are shown as a ball-and-stick model. The right side panel represents the 3′-end of tRNA. Active site residues Phe-89, Thr-90, and Met-141 are shown in orange, blue, and red, respectively.

FIGURE 6.

Additional water molecules in the PfDTD active site. Superimposition of active sites of molecules A and B of the PfDTD-ADP complex II structure. In molecule B, the active site is occupied by partially bound adenine ring (yellow), and there are five additional water molecules (orange spheres) between the adenine entry point and the enantio-selectivity checkpoint. Active site residues are colored as described in the legend to Fig. 5.

FIGURE 7.

Omit electron density map near the active site in PfDTD-d-Arg complex and subsite classifications of bound d-amino acids. A, omit map is contoured at the 1.5 σ level around the active site residues. Hydrogen bonds and N-H…π are marked with black and red dashed lines, respectively. B, bound d-amino acids in PfDTD cluster at four different subsites within the active pocket, and we have termed these sites as transition subsites (T1 and T2), active subsite A, and exit subsite E.

FIGURE 8.

Surface representation of bound d-amino acids in various subsites. d-amino acids (ball-and-stick) located in different subsites (T1, T2, A, and E) within the active site pocket of PfDTD. Active site residues are colored as in Fig. 5.

FIGURE 9.

Views of the PfDTD-ADP structure with the substrate analog of Pab-NTD along with PfDTD subsites. A, superimposition of pre-transfer substrate analog (seryl-3′-aminoadenosine, A3S, yellow) complex structure of Pab-NTD onto ADP-bound PfDTD. B, bound adenine ring in PfDTD and in Pab-NTD complexes mimics entry point of tRNA. We termed these subsites as transition sites B1 and B2, respectively. C, classification of various subsites in the active site pocket of DTDs. Active site residues are colored as in Fig. 5.

FIGURE 10.

Surface representation of open and close conformations of Phe-89 at the adenosine-docking site. A, open conformation of active site Phe-89 in native DTDs, in d-amino acid-bound and ADP-bound structures. The stick model of Phe-89 is shown in the right side panel. Color codes of active site residues are as in Fig. 5. B, closed conformation of Phe-89 is observed in HiDTD, molecule D of EcDTD, and molecule C of HsDTD.

FIGURE 11.

Modeled 3′-end of d-Tyr charged tRNA (tyrosyl-3′-aminoadenosine, A3Y) in the active site of PfDTD. A, ball-and-stick representation of A3Y that mimics the 3′-end of d-amino acid (yellow)-charged tRNA (green). B, modeled A3Y at B2 subsite in PfDTD. The position of the carbonyl carbon atom of tyrosyl is near the active Thr-90. Active site residues are colored as in Fig. 5.

FIGURE 12.

Proposed catalytic mechanism. A, hydroxyl group of Thr-90 attacks carbonyl carbon of A3Y in a nucleophilic manner, followed by cleavage of ester bond and formation of acyl enzyme (d-amino acid bound to DTD). B, movement of catalytic water Wa1 close to carbonyl carbon of d-amino acid. C, cleavage of ester bond between d-amino acid and DTD. Thus, free d-amino acid may migrate to subsite E from where it could diffuse.

FIGURE 13.

Parasite inhibition assays. A, growth of cultures of P. falciparum after 48 h in the presence of four different inhibitors at varying concentrations ranging from 0.01 to 100 μm. B, parasites growth at varying concentrations of d-isoleucine in presence or absence of 0.1 μm concentration of compound 2 in the medium. The structure of compound 2 is shown.