Crystal Structure of African Swine Fever Virus dUTPase Reveals a Potential Drug Target

Figures

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  FIG 1. Biochemical characterization of ASFV E165R. (A) Analytical gel filtration of E165R protein. The 280-nm absorbance curve from Superdex 200 10/300 GL column and the SDS-PAGE migration profile of the pooled sample are shown. (B) Ultracentrifugation sedimentation profile of E165R. The calculated molecular weight of the indicated protein species is shown. (C) Substrate specificity assay of E165R showing its specificity for the hydrolysis of dUTP. (D) Enzymatic activity assay of E165R. The Km values were calculated using the integrated Michaelis-Menten method (42).

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  FIG 2. Crystal structure of apo-E165R. (A) Side and top views of the trimeric apo-E165R structure. The three protomers are colored in orange, green, and sky blue, respectively. (B) Structure of a protomer of apo-E165R. α, α-helix; β, β-strand; η, 310 helix. The 14 β-strands form 4 β-sheets, which are indicated by salmon, teal, orange, and pale cyan. (C) Phylogenetic tree of dUTPases from different species. Phylogenetic assay shows that the protein sequences of dUTPases from different species cluster into three groups corresponding to the monomeric, dimeric, and trimeric dUTPase families. ASFV E165R (labeled with a red star) belongs to the trimeric dUTPase group.

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  FIG 3. Crystal structure of E165R in complex with dUMP. (A) The overall view of the trimeric E165R-dUMP complex. The three protomers are colored according to the colors shown in Fig. 2A. dUMP substrates are shown as sticks in cyan. (B) Detailed view of one dUMP binding pocket. The binding pocket is formed by two protomers of E165R homotrimer, which are colored in blue and green. (C) The position relations of the motifs (I, II, III, and IV) that surround the substrate binding pockets of E165R.

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  FIG 4. Comparison of the structures of active center motifs of different trimeric dUTPases. (A) The four enzyme active center motifs (I, II, III, and IV) of E165R. The atom names of the dUMP are indicated and side chains of the residues that form atom contacts with the dUMP are shown as sticks. (B). The RMSD values between motifs I, II, III, and IV from ASFV E165R and from other trimeric dUTPases. (C) Superimposition of the active center motifs (I, II, III, and IV) from ASFV E165R (blue) with those of M. tuberculosis dUTPase (yellow). The side chains of the residues that form hydrogen bonds with dUMP in ASFV E165R (please refer to Fig. S1B in the supplemental material) and those of their corresponding aligned residues in M. tuberculosis are shown as sticks. (D) Superimposition of the active center motifs (I, II, III, and IV) from ASFV E165R (blue) with those of P. falciparum dUTPase (pink). The side chains of the residues that form hydrogen bonds with dUMP in ASFV E165R (please refer to Fig. 2) and those of their corresponding aligned residues in P. falciparum are shown as sticks. ECOL, Escherichia coli; MABS, Mycobacterium abscessus; MTUB, Mycobacterium tuberculosis; ATHA, Arabidopsis…

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  FIG 5. Comparison of the E165R structure with those of other trimeric dUTPases in complex with substrates or inhibitors. The substrate binding pockets in E165R in complex with dUMP (A), M. tuberculosis dUTPase in complex with dUTP (PDB ID 1SM8) (B), and P. falciparum dUTPase in complex with dUPNPP (PDB ID 1VYQ) (C) are shown. The pocket-forming residues in panels A, B, and C are colored orange, yellow, and pink, respectively. The solvent accessible areas and volumes of the pockets calculated by CASTp (27) are indicated. The electrostatic potential distribution mapped to the solvent-accessible surface of E165R (D), M. tuberculosis dUTPase (E), and P. falciparum dUTPase (F) are shown. The color gradient bar for the electrostatic surface potential in panels D, E, and F is shown from red (acidic) to blue (basic) at the bottom (k, the Boltzmann constant; T, temperature; e, the charge of an electron).