Research ArticleSTRUCTURAL BIOLOGY

Crystal structure of 2C helicase from enterovirus 71

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Science Advances  28 Apr 2017:
Vol. 3, no. 4, e1602573
DOI: 10.1126/sciadv.1602573
  • Fig. 1 Overall structure of EV71 2C 116–329.

    (A) Ribbon model of EV71 2C 116–329. The ATPase domain is colored in blue, the zinc finger domain is colored in yellow, and the C-terminal long helix is colored in red. Zinc is shown as a gray sphere. (B) Overview of key features of EV71 2C identified in this study. (C) Topology diagram of EV71 2C. Conserved ATPase motifs Walker A and Walker B and motif C are highlighted in black and labeled with A, B, and C in (A) to (C). (D) Zinc-binding site of EV71 2C. Residues involved in zinc coordination are shown with stick model. PCS predicted from PV 2C sequence are indicated. (E) Structure-based protein sequence alignment of the cysteine-rich domain in enterovirus 2C. Residues with red background are invariant; residues with yellow background are conserved.

  • Fig. 2 Self-oligomerization of EV71 2C.

    (A) Top: 2C in the crystal polymerizes through a specific interaction indicated with arrows. Bottom: Magnified view of the intermolecular interaction. (B) Magnified view of the interaction between two 2C molecules. The C terminus of EV71 2C (orange ribbon model) forms the pocket-binding motif, which binds to the hydrophobic pocket (shown as molecular surface) formed between zinc finger and ATPase domains. Residues 318 to 329 are shown with stick model. Residues lining the pocket (F328, L327, I324, and T323) are highlighted in blue. The molecular surface of EV71 2C harboring the pocket is colored according to the hydrophobicity scale of residues, from white (hydrophobic) to green (hydrophilic). (C) Semitransparent surface of the hydrophobic pocket [color scheme as in (B)]. Residues forming the pocket are shown with stick model. (D) Structure-based sequence alignment of C-terminal amphipathic helix of enteroviral 2C. (E) SAXS profiles of 2C 116–329, 2C mutants without PBD, and mutant F328A. (F to H) Distance distribution function p(r) of the SAXS measurements shown in (E). (I) Molecular weights of EV71 2C 116–329 and various mutants as measured by size exclusion chromatography.

  • Fig. 3 Structural basis for ATP recognition and hydrolysis.

    (A) Adjacent EV71 2C linked by C terminus α6 adopt different orientations in crystal. A hinge region allows significant rotations. EV71 2C molecules on the right side are superimposed. We denote the 2C-2C interactions containing orange, blue, and red molecules as conformation 1, conformation 2, and conformation 3, respectively. (B) ATP-γ-S (cyan) occupies the active site only when adjacent EV71 2C adopt proper orientation [conformation 1 in (A)]. One EV71 2C molecule (green) provides Walker A (pink) and Walker B (blue) motifs to recognize ATP-γ-S. Motif C (red) is located above the Walker B motif. Second EV71 2C molecule (orange) provides R finger (magenta) required for hydrolysis. Adenosine base docks into a hydrophobic cavity at the junction of the two molecules. Residues involved in ATP recognition are shown with stick model. (C) Diagram illustrating recognition of ATP-γ-S by EV71 2C. Hydrogen bonds are shown with dashed lines. (D) ATPase activity of EV71 2C and single mutants (including arginine finger).

  • Fig. 4 Mutagenesis studies of ATPase activity of 2C and EV71 production.

    (A) ATPase activity of WT and mutated EV71 2C with deletions or mutations indicated. (B) Detection of EV71 in Vero cells by immunofluorescence analysis. The cells were infected with the supernatant from RD cell culture transfected with RNA transcripts from an EV71 infectious clone containing indicated mutations or deletions. The infectious clone with mutation K135A (Walker A motif) and D176N (Walker B motif) serve as the negative controls. (C) Histogram representation of results from (B). Production of EV71 mutants is expressed as percentage of the number of positive dots in comparison to WT EV71. The results are the mean value averaged from eight randomly selected visual fields. (D) A model illustrating the processing of polyprotein and formation of functional oligomer of 2C through C terminus–mediated oligomerization.

  • Fig. 5 The hexameric ring model of EV71 2C 116–329.

    (A) Top view of the modeled EV71 2C 116–329 hexamer, colored by chains. ATP-γ-S molecules are shown with stick model in red. C-terminal helices (α6), zinc atoms, and predicted C-terminal RNA binding motif are labeled. Inset: Magnified view of the interaction between PBD and the pocket. Slight rotation around the hinge allows binding of the C terminus of EV71 2C to the pocket on the adjacent subunit. Blue ribbon indicates the structure before rotation, and gray ribbon indicates the structure after rotation. (B) Side view of the modeled EV71 2C 116–329 hexamer with the dimension of the hexameric ring indicated. (C) Superposition of EV71 2C 116–329 hexameric model (red ribbon model) onto cryo-EM structure of human p97 (PDB ID: 5FTK), which is colored by domains (green, N-domain; blue, D1-domain; gray, D2-domain). (D) Electrostatic surface of the cytoplasmic side of EV71 2C 113-329 hexamer model (left) and membrane proximal side (right; negative charge, red; positive charge, blue). The central channel has a funnel-like shape. The diameters of the narrowest and widest openings are indicated. (E) Structure comparison 2C-2C conformation 1 observed in the crystal structure with the 2C-2C interaction in hexameric model of EV71 2C 116–329. The 2C monomers providing Walker motifs to the active site are superimposed. The 2C-2C interaction in the hexameric model is shown with gray and red molecules; the 2C-2C interaction in conformation 1 is shown with gray and green molecules. ATP-γ-S and the R finger residues are shown with stick model.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/4/e1602573/DC1

    fig. S1. Structure-based multiple sequence alignment of 2C proteins from different enteroviruses.

    fig. S2. Structure of three-liganded zinc finger of EV71 2C.

    fig. S3. SAXS experiments of various EV71 2C with mutations or deletions at pocket-binding motifs.

    fig. S4. Circular dichroism spectroscopy of EV71 2C mutants and truncations.

    fig. S5. Structure of ATP-binding site of EV71 2C.

    fig. S6. Model of the C terminus of EV71 2C bound to 3C proteinase.

    fig. S7. Structural comparison of 2C-2C interactions.

    table S1. Data collection and refinement statistics.

    table S2. Parameters in SAXS experiments.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Structure-based multiple sequence alignment of 2C proteins from different enteroviruses.
    • fig. S2. Structure of three-liganded zinc finger of EV71 2C.
    • fig. S3. SAXS experiments of various EV71 2C with mutations or deletions at pocket-binding motifs.
    • fig. S4. Circular dichroism spectroscopy of EV71 2C mutants and truncations.
    • fig. S5. Structure of ATP-binding site of EV71 2C.
    • fig. S6. Model of the C terminus of EV71 2C bound to 3C proteinase.
    • fig. S7. Structural comparison of 2C-2C interactions.
    • table S1. Data collection and refinement statistics.
    • table S2. Parameters in SAXS experiments.

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