Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of Brugia malayi and Leishmania major

Abstract

Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery programs, we determined its crystal structure bound to cofactor NAD+. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening format. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHS-encoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibitor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections.

Fig 1. Structure-based sequence alignment of BmDHS, HsDHS, LmDHSp, LmDHSc, TbDHSp, and TbDHSc.

Residues indicated by a light-colored background participate in binding to NAD⁺ (yellow) spermidine/GC7 (blue), or both ligands (green). Background coloring for LmDHSp/DHSc follow that of the T. brucei proteins. Residues indicated by a black background or framed in a box are conserved in all DHS proteins analyzed here. The catalytic lysine is marked with a red star. Gaps are indicated by black dots. For clarity, large gaps in LmDHSc were omitted and their lengths are indicated in parenthesis. The secondary structure (α-helices and β-sheets), and the numbering shown in the top line are for BmDHS. Protein sequences and structures used in the alignment were: BmDHS (UniProt ID A0A0J9XTC4; PDB ID 6W3Z), HsDHS (UniProt ID P49366, PDB ID 1DHS), LmDHSp/DHSc (UniProt IDs Q4QD19 / Q4Q3H5), and TbDHSp/DHSc (UniProt IDs Q4GZD1 / Q38BX0, PDB ID 6DFT). Structural alignment by PROMALS3D [61].

Fig 2. Overall structure of NAD⁺-bound BmDHS.

(A) Cartoon representation of the BmDHS tetramer. Individual protomers within the tetramer are depicted in different colors (A1—blue, A2—pink, B1—yellow, and B2—green). NAD⁺ molecules are shown in a sphere representation. (B) Superposition of a BmDHS protomer (A1 in blue; same orientation as in panel A) onto its human counterpart (magenta).

Fig 3. Interprotomer contacts within a BmDHS homotetramer.

(A, B) Molecular surfaces for protomers A2, B1, and B2 are shown in the same colors as in Fig 2A. Protomer A1 is depicted as a blue cartoon. The position of the N-terminal α1 is indicated. (C) Interactions between the N-terminal α1 from protomer A1 and nearby (4 Å cut-off) residues from protomers B1 and B2. Hydrogen bonds are depicted as dashed black lines.

Fig 4. Details of the BmDHS active site.

(A) Protomer A1 is shown as a white surface, with residues within a 4 Å radius of the NAD⁺ cofactor (spheres) shown as blue sticks and highlighted by pale blue surface. Residues in protomer A2 within a 4 Å radius of the NAD⁺ cofactor bound to protomer A1 are shown as pink sticks. The NAD⁺ cofactor bound to protomer A2 is also shown as pink sticks. Protomers B1 (yellow) and B2 (green) are shown as cartoon. (B, C) Close view showing catalytically-important residues within BmDHS active site. Polder OMIT map [65] for NAD⁺. The grey mesh represents the (mF_obs-DF_model) OMIT difference density contoured at 3.0 σ. Spermidine (yellow stick) was docked following the superposition of the crystal structure of BmDHS onto the crystal structure of spermidine-bound HsDHS (PDB ID 6XXK) [44] using Pymol (Schrödinger, Inc).

Fig 5. Partial reaction mechanism, establishment of a NAD⁺/NADH fluorescence enzymatic assay for the BmDHS partial reaction, and IC₅₀ determination for GC7.

(A) Proposed reaction mechanism for a single turnover catalyzed by BmDHS in the presence of NAD⁺ and spermidine. Maintenance of the covalent adduct between BmDHS Lys326 and the butylimine group from spermidine keeps the enzyme in an inactive form. In the absence of eIF5A, DHS is not regenerated. (B) Reaction progress curve demonstrating the generation of NADH by BmDHS. Fluorescence emission intensity after excitation at 355 ± 15 nm. The final component of the reaction was added to the plate at time 0 and the fluorescence intensity was measured during the indicated time. The reaction (50 μL) was composed of 100 nM BmDHS, 132 μM NAD⁺ and 0.5 μM spermidine. (C) Titration of NAD⁺. Variable concentrations of NAD⁺ (0.195 to 400 μM) and fixed concentrations of BmDHS (100 nM) and spermidine (0.56 μM) were used to carry out the experiment. (D) Titration of spermidine. Variable concentrations of spermidine (0.0005 to 1 μM) and fixed concentrations of BmDHS (100 nM) and NAD⁺ (132 μM) were used to carry out the experiment. (E) Determination of the IC₅₀ for GC7. Variable concentrations of GC7 were used (0.00019 to 10 μM) and fixed concentrations of BmDHS (75 nM), spermidine (0.56 μM) and NAD⁺ (132 μM) were used to carry out the experiment. The estimated value for IC₅₀ is shown in the figure. The individual points represent the mean ± standard error of experimental duplicates.

Fig 6. Functional replacement (complementation) of yeast deoxyhypusine synthase by orthologue enzymes from eukaryotic pathogens.

(A) Ten-fold serial dilutions of the indicated strains (after haploid selection- see Material and Methods) were plated on selective media and grown at 30°C for 3 days. (B) Complementation of the yeast deoxyhypusine synthase deleted strain only by the two isoforms of LmDHS. The indicated strains were plated on selective media during haploid selection and grown at 30°C for 3 days. (C) Hypusination of eIF5A in the yeast strains complemented with the DHS from different organisms.

Fig 7. Yeast-based assay can detect DHS inhibitors.

Bars show the relative growth score of dys1Δ S. cerevisiae strains complemented by the indicated DHS enzymes and the relative growth score of the wild type and isogenic strain VZL1462 (ScDHS—Table 2) in the presence of 1 mM GC7. A single star (*) indicate statistically significant differences (Student’s t-test p < 0.05) between the growth score of individual strains in the presence or absence of GC7.