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. 2016 Dec 1;44(21):10505-10514.
doi: 10.1093/nar/gkw941. Epub 2016 Oct 19.

Molecular mechanism of divalent-metal-induced activation of NS3 helicase and insights into Zika virus inhibitor design

Xiaocong Cao  1 , Yajuan Li  2 , Xiangyu Jin  1 , Yuelong Li  1 , Feng Guo  3 , Tengchuan Jin  4
Affiliations

Molecular mechanism of divalent-metal-induced activation of NS3 helicase and insights into Zika virus inhibitor design

Xiaocong Cao et al. Nucleic Acids Res. .

Abstract

Zika virus has attracted increasing attention because of its potential for causing human neural disorders, including microcephaly in infants and Guillain-Barré syndrome. Its NS3 helicase domain plays critical roles in NTP-dependent RNA unwinding and translocation during viral replication. Our structural analysis revealed a pre-activation state of NS3 helicase in complex with GTPγS, in which the triphosphate adopts a compact conformation in the absence of any divalent metal ions. In contrast, in the presence of a divalent cation, GTPγS adopts an extended conformation, and the Walker A motif undergoes substantial conformational changes. Both features contribute to more extensive interactions between the GTPγS and the enzyme. Thus, this study provides structural evidence on the allosteric modulation of MgNTP2- on the NS3 helicase activity. Furthermore, the compact conformation of inhibitory NTP identified in this study provides precise information for the rational drug design of small molecule inhibitors for the treatment of ZIKV infection.

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Figures

Figure 1.
Figure 1.
Overall structure of the ZIKV NS3 helicase domain. (A) Ribbon diagram of the apo structure showing three well-separated domains. Domains 1–3 are shown in raspberry, green and marine blue respectively. Its termini, the NTP-binding site and the NA-binding site are labeled. (B) Side view of the apo structure. (C) Structural comparison of flavivirus family viral NS3 helicases. The ZIKV apo structure is in gray. Helicases from JEV (2Z83), MVEV (2WV9), Kunjin virus (2QEQ), YFV (1YKS) and Kokobera virus (2V6I) are cyan, violet, slate, orange and lime, respectively.
Figure 2.
Figure 2.
Nucleotide-metal tertiary complexes. (A) Post-hydrolysis configuration of the NTP-binding pocket bound with MnADP−. The pocket is formed by the Walker A and Walker B motifs and motif VI. A manganese ion (shown as a purple sphere) is coordinated with β phosphate, T201 of Walker A, E286 of Walker B, and three waters. (B) 2Fo - Fc electron density map of MnADP− ligand calculated at 2σ. (C) Pre-hydrolysis configuration of the NTP-binding pocket bound with MnATP2−. The Walker A motif, Walker B motif and motif VI are shown as green sticks. A manganese ion (shown as a purple sphere) is coordinated with βγ phosphate, T201 of Walker A, E286 of Walker B, and two waters. (D) Structure superposition of apo (gray), MnATP2- (pale green) and MnADP− (yellow) complexes. The NTP-binding pocket bound with an ATP molecule is circled. ATP is colored ruby. (E) Comparison of the MnADP− and MnATP2− complexes. The αβ phosphates and Mn2+ from both structures are in approximately identical positions. Only the hydrogen bonds around the Mn2+ and the attacking water are shown.
Figure 3.
Figure 3.
Activation of the NS3 helicase by divalent metals. (A) Pre-hydrolysis configuration of the NTP-binding pocket bound with MgGTPγS. The nucleotide-binding pocket is presented as light-orange sticks. (B) 2Fo - Fc electron density map of the MgGTPγS bound pocket calculated at 1.5σ. (C) NTP-binding pocket bound with free GTPγS (colored in cyan). (D) 2Fo - Fc electron density map of free GTPγS-bound pocket calculated at 2σ. (E) Structural comparison of the GTPγS-bound (magenta) and MgGTPγS-bound (cyan) complexes, aligned through domain 2. (F) Structural comparison reveals magnesium-induced conformational changes of the Walker A motif. The conformational changes of Walker A motif is indicated by arrows prepared by modevector in pymol.
Figure 4.
Figure 4.
Nucleotide binding and hydrolysis assays. (A) Differential scanning fluorimetry assay shows MgNTP2− increases the thermal stability of ZIKV helicase. (B) Differential scanning fluorimetry assay shows MgNDP− increases the thermal stability of ZIKV helicase. (C) ATPase activity assay shows ATP is hydrolyzed more potently in the presence of Mg2+. (D) GTPase activity assay shows GTP is hydrolyzed more potently in the presence of Mg2+.
Figure 5.
Figure 5.
Snapshot of Walker A motif configuration in an NTP hydrolysis cycle. The actual structure configurations of the Walker A motif determined by our X-ray structures were presented for comparison. (A) In the resting state, Walker A motif may adopt multiple conformations. (B) In the pre-activation state, the Walker A motif binds the NTP that in compact conformation. (C) In the pre-hydrolysis state, the magnesium (purple sphere) binding induces the structural changes of NTP to an extended conformation. (D) In the post-hydrolysis state, the magnesium cation, NDP and the γ phosphate (red sphere) diffuses into the solution and the helicase returns back to the resting state.
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