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. 2016 Nov 15:7:13410.
doi: 10.1038/ncomms13410.

Structure of the NS2B-NS3 protease from Zika virus after self-cleavage

Wint Wint Phoo  1   2   3 , Yan Li  4 , Zhenzhen Zhang  1   3 , Michelle Yueqi Lee  4 , Ying Ru Loh  4 , Yaw Bia Tan  1   3 , Elizabeth Yihui Ng  4 , Julien Lescar  2   3 , CongBao Kang  4 , Dahai Luo  1   2   3
Affiliations

Structure of the NS2B-NS3 protease from Zika virus after self-cleavage

Wint Wint Phoo et al. Nat Commun. .

Abstract

The recent outbreak of Zika virus (ZIKV) infections in the Americas represents a serious threat to the global public health. The viral protease that processes viral polyproteins during infection appears as an attractive drug target. Here we report a crystal structure at 1.84 Å resolution of ZIKV non-structural protein NS2B-NS3 protease with the last four amino acids of the NS2B cofactor bound at the NS3 active site. This structure represents a post-proteolysis state of the enzyme during viral polyprotein processing and provides insights into peptide substrate recognition by the protease. Nuclear magnetic resonance (NMR) studies and protease activity assays unravel the protein dynamics upon binding the protease inhibitor BPTI in solution and confirm this finding. The structural and functional insights of the ZIKV protease presented here should advance our current understanding of flavivirus replication and accelerate structure-based antiviral drug discovery against ZIKV.

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Figures

Figure 1
Figure 1. Crystal structure of eZiPro in complex with the C terminal TGKR tetra-peptide of NS2B.
(a) Full length NS2B and NS3 proteins followed by construct designs for eZiPro, gZiPro, and bZiPro. ZIKV NS3 protease (S1-E177) is covalently linked to NS2B cofactor residues (T45-E96) via K126-R130 of NS2B in eZiPro construct and by G4SG4 linker in gZiPro construct. pETDUET vector with two promoter sites were used for bZiPro resulting in unlinked bZiPro construct. (b) Overall structure of eZiPro showing the TGKR NS2B peptide bound in substrate binding site. NS2B is coloured in magenta and NS3 in yellow. N-terminal residues, C-terminal residues, and residues of the TGKR peptide are shown. (c) Close-up views of the interactions between viral peptide and residues from protease. Hydrogen bonds are shown as dashes. (d) Surface charge density view of the complex. Substrate binding pockets are labelled. (e) A simulated annealing omit map of the TGKR peptide is contoured at 3σ in green mesh. (f) 2mFo-DFc electron density map contoured at 1σ in blue.
Figure 2
Figure 2. Molecular dynamics of the protease constructs in solution.
(a) Dynamics analysis of eZiPro. The T1, T2 and hetNOE values were plotted against residue number. Only assigned residues without signal overlapping were analysed. Error bars represent standard deviations from three values during curve fitting. The measurement was conducted on a magnet with proton frequency of 700 MHz at 310 K. (b) Overlaid 1H-15N-HSQC spectra of eZiPro in black and bZiPro in red. (c) eZiPro, (d) gZiPro and (e) bZiPro before (in black) and after (in red) BPTI binding. Overlaid 1H-15N-HSQC spectra of 0.5 mM protease in the absence and presence of 1 mM BPTI were collected. Only bZiPro exhibited clear interaction with BPTI.
Figure 3
Figure 3. Enzymatic activities and BPTI bindings of protease constructs.
(a) Protease activity of gZiPro, eZiPro and bZiPro were measured using the Bz—nKRR-AMC substrate. Assays were carried out as duplicates at 37 °C at 5 nM enzyme concentration with varying substrate concentrations ranging from 0 to 300 μM. Michaelis–Menten kinetics was plotted using non-linear regression function. Catalytic rates, binding affinity of substrate and catalytic efficiency are mentioned in the table inset the graph. Standard deviations for each data point are represented by error bars. The assays are carried out in duplicates or triplicates. (b) BPTI inhibition against Bz-nKRR-AMC substrate for gZiPro, eZiPro and bZiPro. The IC50s for eZiPro, gZiPro and bZiPro are 350, 76 and 12 nM respectively. The assays are carried out in duplicates or triplicates. Standard deviations for each data point are represented by error bars.
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