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Crystal structure of Zika virus NS2B-NS3 protease in complex with a boronate inhibitor

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Science  07 Jul 2016:
aag2419
DOI: 10.1126/science.aag2419
  • Fig. 1 Crystal structure of the ZIKV NS2B-NS3pro monomer in complex with cn-716.

    (A) Overall structure of the complex. NS3pro (cyan) and NS2B (purple) are shown as ribbons, with secondary-structure elements labeled. NS3pro is made up by two β-barrels with strand order AI-BI-CI-αI-DI-EIa-EIb-FI and AII-BIIa-BIIb-CII-DII-EIIa-EIIb-FII. NS2B includes β-strands β1 - β4. The N- and C- termini of NS2B and NS3pro are indicated by letters in italics and non-italics/ underlined, respectively. The inhibitor cn-716 is shown with carbon atoms in purple and boron in yellow. Residues of the catalytic triad are in dark blue. * denotes residues from NS2B. (B) The inhibitor cn-716 is embedded in the substrate-binding site of ZIKV NS2B-NS3pro (same view as in (A)). The surfaces of NS2B and NS3pro are yellow and purple, respectively. An Fo-Fc difference density contoured at 2.5σ is shown for cn-716. Lys54 from molecule B of the dimer interacts with the inhibitor and is indicated by K54. (C) Chemical structure of cn-716. (D) Schematic drawing and (E), Fo-Fc difference density (2.5σ) for the cyclic diester and its environment in molecule A. (F) Difference density (2.5σ) for the cyclic diester and its environment in molecule B.

  • Fig. 2

    The tight, non-crystallographic 2:2 dimer of quasi-twofold symmetry formed by the ZIKV NS2B-NS3pro:inhibitor complex in the asymmetric unit of the crystal. (A) Front view and back view. The surfaces of NS2B and NS3pro of molecule A are shown in cyan and orange, resp., those of molecule B are dark blue and wheat. Labels of molecule B residues are underlined. Residues of NS2B are marked by *. Residues Leu30 (purple/green) and Leu31 at the tip of the AI-BI loop (Fig. 1A) form a hook making hydrophobic contacts with the opposing monomer. The Cys143 residues forming labile disulfide bonds in the SS-dimer are yellow and pink. (B) A slice through the interior of the dimer, showing the S135 side-chains (blue) covalently bound to the inhibitor molecules. The color code is the same as in (A). Inhibitor molecules are colored purple and red. See fig. S5 for a schematic illustration of the interactions across the dimer interface.

  • Table 1

    Kinetic parameters of variants of ZIKV NS2B-NS3 protease, in comparison to a similar WNV NS2B-NS3pro construct. Data are for the cleavage of the flavivirus protease substrate Bz-Nle-Lys-Lys-Arg-AMC. “Monomer (wt)” [See supplementary materials (p. 4) for details, including definition of “wt” (p. 2)] and “SS-dimer (wt)” indicate enzyme preparations corresponding to the monomer (in the presence of TCEP) and the SS-dimer fraction from gel permeation chromatography. The kinetic parameters for the ZIKV protease with Asp83* replaced by Asn are also included. “WNV NS2B-NS3pro (wt)” is our recombinant preparation of the WNV protease. For comparison, the kcat/Km values for WNV and DENV-2 NS2B-NS3pro and with the substrate Bz-Nle-Lys-Arg-Arg-AMC reported in (6) are given. (dash indicates not reported). All values in this table are obtained at pH 8.5.

    Proteasekcat (s−1)Km (μM)kcat/Km (s−1 M−1)
    ZIKV NS2B-NS3pro
    Monomer (wt)44.6 ± 1.018.3 ± 1.62,440,000 ± 215,000
    SS-dimer (wt)28.5 ± 0.65.9 ± 0.54,850,000 ± 429,000
    Cys80Ser/Cys143Ser28.8 ± 0.55.1 ± 0.55,620,000 ± 546,000
    Asp83*Asn (monomer)38.5 ± 1.435.3 ± 4.21,091,000 ± 136,000
    WNV NS2B-NS3pro
    wt8.7 ± 0.177.4 ± 3.6112,000 ± 5000
    wt (6)37,000 ± 7000
    DENV-2 NS2B-NS3pro
    wt (6)30,000 ± 7000

Supplementary Materials

  • Crystal structure of Zika virus NS2B-NS3 protease in complex with a boronate inhibitor

    Jian Lei, Guido Hansen, Christoph Nitsche, Christian D. Klein, Linlin Zhang, Rolf Hilgenfeld

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S7
    • Table S1
    • Full Reference List