Mechanistic insight into the RNA-stimulated ATPase activity of tick-borne encephalitis virus helicase

The helicase domain of nonstructural protein 3 (NS3H) unwinds the double-stranded RNA replication intermediate in an ATP-dependent manner during the flavivirus life cycle. While the ATP hydrolysis mechanism of Dengue and Zika viruses NS3H has been extensively studied, little is known in the case of the tick-borne encephalitis virus NS3H. We demonstrate that ssRNA binds with nanomolar affinity to NS3H and strongly stimulates the ATP hydrolysis cycle, whereas ssDNA binds only weakly and inhibits ATPase activity in a noncompetitive manner. Thus, NS3H is an RNA-specific helicase, whereas DNA might act as an allosteric inhibitor. Using modeling, we explored plausible allosteric mechanisms by which ssDNA inhibits the ATPase via nonspecific binding in the vicinity of the active site and ATP repositioning. We captured several structural snapshots of key ATP hydrolysis stages using X-ray crystallography. One intermediate, in which the inorganic phosphate and ADP remained trapped inside the ATPase site after hydrolysis, suggests that inorganic phosphate release is the rate-limiting step. Using structure-guided modeling and molecular dynamics simulation, we identified putative RNA-binding residues and observed that the opening and closing of the ATP-binding site modulates RNA affinity. Site-directed mutagenesis of the conserved RNA-binding residues revealed that the allosteric activation of ATPase activity is primarily communicated via an arginine residue in domain 1. In summary, we characterized conformational changes associated with modulating RNA affinity and mapped allosteric communication between RNA-binding groove and ATPase site of tick-borne encephalitis virus helicase. The helicase domain of nonstructural protein 3 (NS3H) unwinds the double-stranded RNA replication intermediate in an ATP-dependent manner during the flavivirus life cycle. While the ATP hydrolysis mechanism of Dengue and Zika viruses NS3H has been extensively studied, little is known in the case of the tick-borne encephalitis virus NS3H. We demonstrate that ssRNA binds with nanomolar affinity to NS3H and strongly stimulates the ATP hydrolysis cycle, whereas ssDNA binds only weakly and inhibits ATPase activity in a noncompetitive manner. Thus, NS3H is an RNA-specific helicase, whereas DNA might act as an allosteric inhibitor. Using modeling, we explored plausible allosteric mechanisms by which ssDNA inhibits the ATPase via nonspecific binding in the vicinity of the active site and ATP repositioning. We captured several structural snapshots of key ATP hydrolysis stages using X-ray crystallography. One intermediate, in which the inorganic phosphate and ADP remained trapped inside the ATPase site after hydrolysis, suggests that inorganic phosphate release is the rate-limiting step. Using structure-guided modeling and molecular dynamics simulation, we identified putative RNA-binding residues and observed that the opening and closing of the ATP-binding site modulates RNA affinity. Site-directed mutagenesis of the conserved RNA-binding residues revealed that the allosteric activation of ATPase activity is primarily communicated via an arginine residue in domain 1. In summary, we characterized conformational changes associated with modulating RNA affinity and mapped allosteric communication between RNA-binding groove and ATPase site of tick-borne encephalitis virus helicase. Tick-borne encephalitis virus (TBEV) is an enveloped single-stranded positive-sense RNA virus from the family Flaviviridae, genus Flavivirus. The virus is neurotropic and causes tick-borne encephalitis, which affects primarily adult population within European and North-Eastern Asian countries (1Beauté J. Spiteri G. Warns-Petit E. Zeller H. Tick-borne encephalitis in Europe, 2012 to 2016.Euro. Surveill. 2018; 23: 1800201Crossref Scopus (89) Google Scholar). While TBEV vaccines are available, because of the lack of targeted campaigns, the actual vaccination coverage is low even in high-risk areas. There is currently no specific treatment available (2Füzik T. Formanová P. Růžek D. Yoshii K. Niedrig M. Plevka P. Structure of tick-borne encephalitis virus and its neutralization by a monoclonal antibody.Nat. Commun. 2018; 9: 436Crossref PubMed Scopus (74) Google Scholar, 3Ruzek D. Avsic Zupanc T. Borde J. Chrdle A. Eyer L. Karganova G. et al.Tick-borne encephalitis in Europe and Russia: review of pathogenesis, clinical features, therapy, and vaccines.Antivir. Res. 2019; 164: 23-51Crossref PubMed Scopus (131) Google Scholar). Therefore, to develop antivirals against TBEV, it is essential to understand the structure and function of the key viral enzymes that are involved in the replication.TBEV encodes three structural and seven nonstructural (NS) proteins. Among the NS proteins, NS3 is a multifunctional protein that comprises two functional domains: a protease and a helicase. The chymotrypsin-like serine protease is located with the N-terminal region (172 amino acids) and is responsible for viral polyprotein processing. This domain is connected by a short flexible linker to the C-terminal helicase domain (NS3H, 434 amino acids), which exhibits several activities: NTPase, RNA helicase, and RNA 5′-triphosphatase. The RNA helicase activity uses energy from NTP hydrolysis for unwinding of dsRNA replication intermediates, whereas RNA 5′-triphosphatase removes the terminal γ-phosphate from the 5′-triphosphate end of the positive-sense ssRNA before mRNA capping by the methyl transferase domain of NS5 protein (4Brand C. Bisaillon M. Geiss B.J. Organization of the Flavivirus RNA replicase complex.Wiley Interdiscip. Rev. RNA. 2017; 8https://doi.org/10.1002/wrna.1437Crossref PubMed Scopus (32) Google Scholar, 5Bollati M. Alvarez K. Assenberg R. Baronti C. Canard B. Cook S. et al.Structure and functionality in flavivirus NS-proteins: perspectives for drug design.Antivir. Res. 2010; 87: 125-148Crossref PubMed Scopus (225) Google Scholar).In this study, we focus on NS3H, which is a monomeric enzyme belonging to the DEAD/H box subfamily within the helicase superfamily 2 (SF2) (6Fairman-Williams M.E. Guenther U.P. Jankowsky E. SF1 and SF2 helicases: family matters.Curr. Opin. Struc Biol. 2010; 20: 313-324Crossref PubMed Scopus (598) Google Scholar, 7Chen C. Han X. Wang F. Huang J. Zhang L. Wang Z. et al.Crystal structure of the NS3 helicase of tick-borne encephalitis virus.Biochem. Biophys. Res. Commun. 2020; 528: 601-606Crossref PubMed Scopus (2) Google Scholar) and consists of three subdomains. Subdomains 1 and 2 both exhibit the highly conserved RecA-like fold typical of P-loop NTPases. The NTPase active site is located in a cleft between the subdomains 1 and 2 and involves motifs I (Walker A), II (Walker B), and VI. A groove between subdomain 3 and subdomain 1 and 2, respectively, forms the RNA-binding site. The coupling between NTPase-binding and RNA-binding sites is essential for the helicase activity (8Du Pont K.E. Davidson R.B. McCullagh M. Geiss B.J. Motif V regulates energy transduction between the flavivirus NS3 ATPase and RNA-binding cleft.J. Biol. Chem. 2020; 295: 1551-1564Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 9Davidson R.B. Hendrix J. Geiss B.J. McCullagh M. Allostery in the dengue virus NS3 helicase: insights into the NTPase cycle from molecular simulations.PLoS Comput. Biol. 2018; 14e1006103Crossref Scopus (23) Google Scholar).Several structural studies of flavivirus helicases from Dengue virus (DENV), Zika virus (ZIKV), yellow fever virus, Kunjin virus, and Japanese encephalitis virus revealed high structural conservation among them (10Fang J. Jing X. Lu G. Xu Y. Gong P. Crystallographic snapshots of the Zika virus NS3 helicase help visualize the reactant water replenishment.ACS Infect. Dis. 2019; 5: 177-183Crossref PubMed Scopus (5) Google Scholar, 11Luo D. Xu T. Watson R.P. Scherer-Becker D. Sampath A. Jahnke W. et al.Insights into RNA unwinding and ATP hydrolysis by the flavivirus NS3 protein.EMBO J. 2008; 27: 3209-3219Crossref PubMed Scopus (192) Google Scholar, 12Yamashita T. Unno H. Mori Y. Tani H. Moriishi K. Takamizawa A. et al.Crystal structure of the catalytic domain of Japanese encephalitis virus NS3 helicase/nucleoside triphosphatase at a resolution of 1.8 angstrom.Virology. 2008; 373: 426-436Crossref PubMed Scopus (56) Google Scholar, 13Wu J. Bera A.K. Kuhn R.J. Smith J.L. Structure of the flavivirus helicase: implications for catalytic activity, protein interactions, and proteolytic processing.J. Virol. 2005; 79: 10268-10277Crossref PubMed Scopus (134) Google Scholar, 14Mastrangelo E. Milani M. Bollati M. Selisko B. Peyrane F. Pandini V. et al.Crystal structure and activity of Kunjin virus NS3 helicase; protease and helicase domain assembly in the full length NS3 protein.J. Mol. Biol. 2007; 372: 444-455Crossref PubMed Scopus (71) Google Scholar). In addition, previous biochemical and structural studies have provided insight into substrate binding and revealed structural changes associated with the ATP hydrolysis cycle and identified RNA-interacting residues for the DENV and ZIKV helicases (11Luo D. Xu T. Watson R.P. Scherer-Becker D. Sampath A. Jahnke W. et al.Insights into RNA unwinding and ATP hydrolysis by the flavivirus NS3 protein.EMBO J. 2008; 27: 3209-3219Crossref PubMed Scopus (192) Google Scholar, 15Swarbrick C.M.D. Basavannacharya C. Chan K.W.K. Chan S.A. Singh D. Wei N. et al.NS3 helicase from dengue virus specifically recognizes viral RNA sequence to ensure optimal replication.Nucl. Acids Res. 2017; 45: 12904-12920Crossref PubMed Scopus (40) Google Scholar, 16Yang X. Chen C. Tian H. Chi H. Mu Z. Zhang T. et al.Mechanism of ATP hydrolysis by the Zika virus helicase.FASEB J. 2018; 32: 5250-5257Crossref PubMed Scopus (14) Google Scholar, 17Tian H.L. Ji X.Y. Yang X.Y. Zhang Z.X. Lu Z.K. Yang K.L. et al.Structural basis of Zika virus helicase in recognizing its substrates.Protein Cell. 2016; 7: 562-570Crossref PubMed Scopus (53) Google Scholar). Recent molecular dynamics (MD) simulations (9Davidson R.B. Hendrix J. Geiss B.J. McCullagh M. Allostery in the dengue virus NS3 helicase: insights into the NTPase cycle from molecular simulations.PLoS Comput. Biol. 2018; 14e1006103Crossref Scopus (23) Google Scholar, 18Sarto C. Kaufman S.B. Estrin D.A. Arrar M. Nucleotide-dependent dynamics of the Dengue NS3 helicase.Biochim. Biophys. Acta Proteins Proteom. 2020; 1868140441Crossref PubMed Scopus (3) Google Scholar) provided further insight into conformational changes associated with RNA binding and stimulation of the ATP hydrolysis, which was considered the rate-limiting step. In contrast, inorganic phosphate (Pi) release has been established as the rate-limiting step for hepatitis C virus (HCV) NS3 helicase that is considered a mechanistic model system for viral SF2 (19Wang Q. Arnold J.J. Uchida A. Raney K.D. Cameron C.E. Phosphate release contributes to the rate-limiting step for unwinding by an RNA helicase.Nucl. Acids Res. 2010; 38: 1312-1324Crossref PubMed Scopus (27) Google Scholar). Hence, it remains to be seen whether Pi release or the hydrolysis is the rate-limiting step and whether the coupling and the associated allosteric changes are conserved among all SF2 helicases and between helicases from different flaviviruses.Here, we biochemically characterized NS3H from TBEV and obtained structures of key intermediates along the ATPase cycle (nucleotide-free, apo; ADP-; adenylyl-imidodiphosphate, AMPPNP-; and hydrolysis product, ADP–Pi-bound NS3H). While the overall structure of the TBEV NS3H is closely related to that of DENV and ZIKV, structural variation is observed in different nucleotide states. Trapping of ATP hydrolysis products within the crystal and the negligible basal activity (i.e., in the absence of RNA) suggest that the ATPase rate-limiting step is phosphate release, which is allosterically stimulated by RNA. Indeed, ssRNA binds to NS3H with nanomolar affinity and stimulates its ATPase activity, whereas ssDNA inhibits the ATPase activity without directly competing with RNA. We explored ssRNA binding by modeling and MD simulations and identified conserved RNA-binding residues. We examined the allosteric roles of these residues by site-directed mutagenesis and functional assays. These results and simulations suggest that RNA binding is relayed to the ATPase active site via a conserved residue within domain 1, whereas RNA affinity is modulated by conformational changes associated with opening and closing of the ATP-binding cleft.Results and discussionRNA-stimulated NTPase activity of recombinant NS3H is inhibited by DNAThe recombinant NS3H protein fused with N-terminal 10X-histidine tag was purified to homogeneity (Fig. S1). To assess the ATPase activity of NS3H, a phosphate release assay was performed in the absence and presence of nucleic acids, ssRNA or ssDNA. ATPase activity of NS3H was strongly dependent on the presence of RNA substrate, poly(A), whereas the basal ATPase activity was negligible at the same protein concentration (Fig. 1A). NS3H hydrolyzed all four NTP substrates with comparable turnover (Fig. 1B). This demonstrated that NS3H exhibits little specificity for different NTP substrates, as shown previously for DENV helicase (20Benarroch D. Selisko B. Locatelli G.A. Maga G. Romette J.L. Canard B. The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core.Virology. 2004; 328: 208-218Crossref PubMed Scopus (143) Google Scholar). Basic steady-state kinetic parameters for ATP were determined in the presence of poly(A) (Fig. 1C). The Michaelis–Menten constant Km = 125 ± 15 μM and turnover kcat = 8.8 ± 0.2 s−1 of NS3H are similar to those of DENV helicase in the presence of short 5′-UTR RNA fragment (15Swarbrick C.M.D. Basavannacharya C. Chan K.W.K. Chan S.A. Singh D. Wei N. et al.NS3 helicase from dengue virus specifically recognizes viral RNA sequence to ensure optimal replication.Nucl. Acids Res. 2017; 45: 12904-12920Crossref PubMed Scopus (40) Google Scholar).While ssDNA fails to stimulate ATPase activity significantly (Fig. 1A), it can partially inhibit it in a dose-dependent manner at micromolar concentrations (Fig. 1D). However, these micromolar concentrations are much higher than the affinity for ssRNA (apparent KD = 49.7 ± 9.3 nM), which was determined by fluorescence anisotropy–based binding assay (Fig. 1E). Using the same approach, we observed that ssDNA partially competes with RNA binding at ∼20-fold molar excess of apparent KD for ssRNA substrate. Longer ssDNAs inhibited RNA binding more effectively (Fig. 1F), suggesting that the longer DNA might bind to several weak nonspecific binding sites. Altogether, these results indicate that the binding of the helicase to RNA substrate stimulates ATP hydrolysis, and DNA may act as a noncompetitive inhibitor via nonspecific binding. This specificity of NS3H toward RNA substrate is different from HCV helicase (21Gwack Y. Kim D.W. Han J.H. Choe J. DNA helicase activity of the hepatitis C virus nonstructural protein 3.Eur. J. Biochem. 1997; 250: 47-54Crossref PubMed Scopus (64) Google Scholar) and DENV helicase (22Wang C.C. Huang Z.S. Chiang P.L. Chen C.T. Wu H.N. Analysis of the nucleoside triphosphatase, RNA triphosphatase, and unwinding activities of the helicase domain of dengue virus NS3 protein.FEBS Lett. 2009; 583: 691-696Crossref PubMed Scopus (59) Google Scholar), which, in addition to RNA binding, also exhibit DNA binding with affinities ranging from nanomolar to micromolar range.TBEV NS3H structure is similar to that of other flavivirus helicasesThe apoNS3H protein crystallizes in space group P41212 at 1.83 Å (Table 1). The refined model of apoNS3H (residues 173–621) is complete except for an N-terminal 10X-histidine tag region derived from the pET-19b vector together with a short flexible linker region between protease and helicase domains of NS3 (residues E173–Q181) and two nonconserved disordered loop regions (residues P251–G259 and T502–P506). Nonprotein positive 2Fo–Fc electron density was attributed to water molecules. The resulting 3D protein structure shows overall similarity with already published helicase structures of other flaviviruses (10Fang J. Jing X. Lu G. Xu Y. Gong P. Crystallographic snapshots of the Zika virus NS3 helicase help visualize the reactant water replenishment.ACS Infect. Dis. 2019; 5: 177-183Crossref PubMed Scopus (5) Google Scholar, 11Luo D. Xu T. Watson R.P. Scherer-Becker D. Sampath A. Jahnke W. et al.Insights into RNA unwinding and ATP hydrolysis by the flavivirus NS3 protein.EMBO J. 2008; 27: 3209-3219Crossref PubMed Scopus (192) Google Scholar, 12Yamashita T. Unno H. Mori Y. Tani H. Moriishi K. Takamizawa A. et al.Crystal structure of the catalytic domain of Japanese encephalitis virus NS3 helicase/nucleoside triphosphatase at a resolution of 1.8 angstrom.Virology. 2008; 373: 426-436Crossref PubMed Scopus (56) Google Scholar, 13Wu J. Bera A.K. Kuhn R.J. Smith J.L. Structure of the flavivirus helicase: implications for catalytic activity, protein interactions, and proteolytic processing.J. Virol. 2005; 79: 10268-10277Crossref PubMed Scopus (134) Google Scholar, 14Mastrangelo E. Milani M. Bollati M. Selisko B. Peyrane F. Pandini V. et al.Crystal structure and activity of Kunjin virus NS3 helicase; protease and helicase domain assembly in the full length NS3 protein.J. Mol. Biol. 2007; 372: 444-455Crossref PubMed Scopus (71) Google Scholar, 16Yang X. Chen C. Tian H. Chi H. Mu Z. Zhang T. et al.Mechanism of ATP hydrolysis by the Zika virus helicase.FASEB J. 2018; 32: 5250-5257Crossref PubMed Scopus (14) Google Scholar, 17Tian H.L. Ji X.Y. Yang X.Y. Zhang Z.X. Lu Z.K. Yang K.L. et al.Structural basis of Zika virus helicase in recognizing its substrates.Protein Cell. 2016; 7: 562-570Crossref PubMed Scopus (53) Google Scholar, 23Li L. Wang J. Jia Z. Shaw N. Structural view of the helicase reveals that Zika virus uses a conserved mechanism for unwinding RNA.Acta Crystallogr. F Struct. Biol. Commun. 2018; 74: 205-213Crossref PubMed Scopus (5) Google Scholar). NS3H has a clover-shaped architecture divided into three domains containing seven conserved motifs of SF2 helicase family (Figs. 2A and S2). The Rec-A-like domains 1 and 2 adopt the α/β open sheet topology (Rossman fold) (5Bollati M. Alvarez K. Assenberg R. Baronti C. Canard B. Cook S. et al.Structure and functionality in flavivirus NS-proteins: perspectives for drug design.Antivir. Res. 2010; 87: 125-148Crossref PubMed Scopus (225) Google Scholar). Domain 1 (residues W188–E329) consists of six β-strands surrounded by four α-helices, whereas domain 2 (residues P330–G486) consists of three α-helices and six β-strands with one antiparallel β hairpin passing close to domain 3. Domain 3 (residues L487–R621) consists of four α-helices and one β-hairpin. To our knowledge, the apoNS3H crystal structure derived from the TBEV MucAr-HB-171/11 virus strain has also been reported elsewhere (7Chen C. Han X. Wang F. Huang J. Zhang L. Wang Z. et al.Crystal structure of the NS3 helicase of tick-borne encephalitis virus.Biochem. Biophys. Res. Commun. 2020; 528: 601-606Crossref PubMed Scopus (2) Google Scholar); however, the coordinates have not been deposited in Protein Data Bank (PDB). A similar crystal structure of apoNS3H from TBEV strain HYPR is also available in the PDB (code: 7JNO) with the overall RMSD of 0.8 Å against our apoNS3H calculated using DALI server (GNU General Public License) (24Holm L. DALI and the persistence of protein shape.Protein Sci. 2020; 29: 128-140Crossref PubMed Scopus (288) Google Scholar).Table 1Crystallographic data collection and refinement statisticsDatasetApoADP–Mn2+AMPPNP–Mn2+ADP–Pi–Mn2+PDB code7OJ47BLV7BM07NXUData collection Space groupP 41 21 2P 41 21 2P 41 21 2P 41 21 2Unit cell parameters a = b, c (Å), α = β = γ (°)73.30, 196.05, 90.0073.12, 196.13, 90.0072.95, 196.45, 90.0072.98, 196.59, 90.00 Resolution range (Å)aValues in parentheses are for the highest resolution shell.49.06–1.83 (1.94–1.83)45.78–2.10 (2.23–2.10)45.71–1.90 (2.00–1.90)48.81–2.10 (2.22–2.10) Unique reflectionsaValues in parentheses are for the highest resolution shell.48,205 (7581)32,056 (5054)43,006 (6796)31,603 (5033) MultiplicityaValues in parentheses are for the highest resolution shell.24.54 (17.03)14.31 (13.77)14.25 (14.64)13.57 (13.42) Completeness (%)aValues in parentheses are for the highest resolution shell.99.9 (99.3)99.9 (99.7)99.9 (99.7)98.4 (99.5) I/δa28.87 (2.22)24.38 (2.53)19.10 (2.03)23.02 (3.21) Rmeas (%)aValues in parentheses are for the highest resolution shell.7.5 (126.6)6.7 (113.3)8.6 (118.1)9.9 (83.7) Wilson B-factor (Å2)34.347.135.634.2Refinement Rwork/RfreebRfree was calculated using 5% of the data excluded from refinement. (%)18.1/22.923.3/25.921.6/22.921.4/25.1 Average B-factor (Å2)Overall46574242Protein46574241.3Ligands—100.8 (ADP)84 (AMPPNP)90.7 (ADP), 31.8 (PO4)No. of non-H atomsProtein3375341433893357Water20975230200Metal ions—1 (Mn2+)1 (Mn2+), 1 (Na+)1 (Mn2+), 1 (Na+)Ligands—27 (ADP)31 (AMPPNP)27 (ADP), 5 (PO4)RMSDs Bond length (Å)0.01460.01070.01550.00091 Bond angles (°)1.9291.6191.7651.540Ramachandran plotFavored (%)979597.3797Allowed (%)352.6312Outliers (%)0000a Values in parentheses are for the highest resolution shell.b Rfree was calculated using 5% of the data excluded from refinement. Open table in a new tab Figure 2Overall structure of apoNS3H and structural comparison with DENV and ZIKV helicases. A, a 3-dimensional structure of apoNS3H is shown in cartoon representation. SF2 helicase motifs are highlighted in colors. The N and C termini are labeled. B, superposition of NS3H (wheat) with DENV (light blue) and ZIKV (pale yellow) helicases showing ATPase site. NS3H adapts similar P-loop conformation as ZIKV helicase. DENV, Dengue virus; NS3, nonstructural protein 3; SF2, superfamily 2; ZIKV, Zika virus.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The NTPase site is situated at the interface between domains 1 and 2. It encompasses Walker A and Walker B motifs identified in domain 1 and motif VI in domain 2. Walker A motif consists of G202SGKT206 sequence forming the P-loop region of NTPase site with residue K205 known to recognize the β- or γ-phosphate of ATP in homologous helicases (12Yamashita T. Unno H. Mori Y. Tani H. Moriishi K. Takamizawa A. et al.Crystal structure of the catalytic domain of Japanese encephalitis virus NS3 helicase/nucleoside triphosphatase at a resolution of 1.8 angstrom.Virology. 2008; 373: 426-436Crossref PubMed Scopus (56) Google Scholar). Walker B motif contains a conserved D290EAH293 sequence, which is common among flavivirus helicases (reviewed in Ref. (4Brand C. Bisaillon M. Geiss B.J. Organization of the Flavivirus RNA replicase complex.Wiley Interdiscip. Rev. RNA. 2017; 8https://doi.org/10.1002/wrna.1437Crossref PubMed Scopus (32) Google Scholar)). Motif VI (Q459RRGRVGR466) includes the arginine fingers (R463 and R466), which are important in the energy coupling during the NTP hydrolysis (12Yamashita T. Unno H. Mori Y. Tani H. Moriishi K. Takamizawa A. et al.Crystal structure of the catalytic domain of Japanese encephalitis virus NS3 helicase/nucleoside triphosphatase at a resolution of 1.8 angstrom.Virology. 2008; 373: 426-436Crossref PubMed Scopus (56) Google Scholar, 25Crampton D.J. Guo S.Y. Johnson D.E. Richardson C.C. The arginine finger of bacteriophage T7 gene 4 helicase: role in energy coupling.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4373-4378Crossref PubMed Scopus (45) Google Scholar) and in recognizing the γ-phosphate of ATP (13Wu J. Bera A.K. Kuhn R.J. Smith J.L. Structure of the flavivirus helicase: implications for catalytic activity, protein interactions, and proteolytic processing.J. Virol. 2005; 79: 10268-10277Crossref PubMed Scopus (134) Google Scholar). The empty NTPase site is filled with solvent molecules, and the P-loop adopts a “relaxed” conformation as found in ZIKV structure (10Fang J. Jing X. Lu G. Xu Y. Gong P. Crystallographic snapshots of the Zika virus NS3 helicase help visualize the reactant water replenishment.ACS Infect. Dis. 2019; 5: 177-183Crossref PubMed Scopus (5) Google Scholar), whereas DENV helicase exhibits yet more open conformation (11Luo D. Xu T. Watson R.P. Scherer-Becker D. Sampath A. Jahnke W. et al.Insights into RNA unwinding and ATP hydrolysis by the flavivirus NS3 protein.EMBO J. 2008; 27: 3209-3219Crossref PubMed Scopus (192) Google Scholar) (Fig. 2B).Structural snapshots of ATP hydrolysis cycleThree ternary complexes of NS3H with AMPPNP–Mn2+, ADP–Pi–Mn2+, or ADP–Mn2+, respectively, were captured in crystallographic structures. Based on these structures, NS3H seems to follow a common mechanism of ATP hydrolysis cycle in flaviviruses (11Luo D. Xu T. Watson R.P. Scherer-Becker D. Sampath A. Jahnke W. et al.Insights into RNA unwinding and ATP hydrolysis by the flavivirus NS3 protein.EMBO J. 2008; 27: 3209-3219Crossref PubMed Scopus (192) Google Scholar, 16Yang X. Chen C. Tian H. Chi H. Mu Z. Zhang T. et al.Mechanism of ATP hydrolysis by the Zika virus helicase.FASEB J. 2018; 32: 5250-5257Crossref PubMed Scopus (14) Google Scholar). Here, the hydrolysis cycle is represented by four states: (i) a prehydrolysis, AMPPNP–Mn2+-bound state representing the binding of ATP molecule to the helicase; (ii) a post-hydrolysis intermediate, ADP–Pi–Mn2+-bound state; (iii) a product dissociation intermediate with Pi released and ADP–Mn2+ are still bound to the protein; and (iv) nucleotide-free (apo) product release state, which is ready to bind ATP.The AMPPNP–Mn2+- and ADP–Mn2+-bound NS3H structures were obtained via cocrystallization of apoNS3H with corresponding nucleotides, whereas the ADP–Pi–Mn2+ complex was obtained as a result of ATP hydrolysis during crystallization, that is, Pi remained trapped within the structure. The nucleotide complexes crystallize in P41212 space group with one molecule in the asymmetric unit similar to the apoNS3H (Table 1). Hence, structural changes seen in the ternary complexes are not likely because of differences in crystal contacts. Several localized conformational changes within individual domains were observed (Fig. 3A) when the nucleotide complexes were compared with apoNS3H. Upon nucleotide and divalent ion (Mn2+) binding, the P-loop swings toward the bound nucleotide (Fig. 3B) with an inward orientation of K205 side chain to coordinate the phosphate group (Fig. 4). In AMPPNP–Mn2+ and ADP–Pi–Mn2+ complexes, the tip of α7 helix moves away from the relative position in apoNS3H (Fig. 3B) to accommodate γ-phosphate. This motion is absent in the ADP–Mn2+ structure.Figure 3Nucleotide-bound complexes. A, superposition of apo and ternary NS3H structures showing global protein conformation. B, close-up view of ATPase site corresponding to (A) where α2, α7, and P-loop are highlighted. A conformational change in the tip of α7 and P-loop is observed with respect to the bound-ligand. AMPPNP (stick representation) and manganese ion (purple sphere) are shown. NS3, nonstructural protein 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Interactions within nucleotide-binding site in apo and nucleotide-bound complexes. Important residues in the NTPase site are shown in the stick representation. (A) apoNS3H, (B) AMPPNP–Mn2+ ternary complex, (C) the ADP–Pi–Mn2+ ternary complex, and (D) the ADP–Mn2+ ternary complex. The manganese ion and water molecules are represented as purple spheres and red spheres, respectively. Hydrogen bonds and metal ion coordination are displayed as black dashed lines. The difference Fourier map shows nucleotide ligands at level of 1σ and manganese ions at level of 2σ (all in gray mesh). NS3, nonstructural protein 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the prehydrolysis state, NS3H–AMPPNP–Mn2+ complex (Fig. 4B), the triphosphate moiety of AMPPNP interacts with P-loop residues K205 and T206 via hydrogen bonds and with residues D290 and E291 of Walker B motif and Q459 of motif VI via water-mediated coordination. One of the arginine fingers, R463, and residue G420 (motif V), also interacts with γ-phosphate of AMPPNP. The 3′-OH group of the ATP ribose C3′ endo ring pucker is hydrogen-bonded with the main chain amide nitrogen in between residues T206 and H207. One potentially catalytic water molecule coordinates with the side chains of E291 (Walker B), Q459 (motif VI), and γ-phosphorus atom in location suggested by AMPPNP-bound DENV helicase (11Luo D. Xu T. Watson R.P. Scherer-Becker D. Sampath A. Jahnke W. et al.Insights into RNA unwinding and ATP hydrolysis by the flavivirus NS3 protein.EMBO J. 2008; 27: 3209-3219Crossref PubMed Scopus (192) Google Scholar) and ATP-bound ZIKV helicase structures (16Yang X. Chen C. Tian H. Chi H. Mu Z. Zhang T. et al.Mechanism of ATP hydrolysis by the Zika virus helicase.FASEB J. 2018; 32: 5250-5257Crossref PubMed Scopus (14) Google Scholar). The triphosphate moiety of AMPPNP is in a staggered conformation (as defined in Ref. (26Hornak V. Abel R. Okur A. Strockbine B. Roitberg A. Simmerling C. Comparison of multiple amber force fields and development of improved protein backbone parameters.Proteins-Struct. Funct. Bioinform. 2006; 65: 712-725Crossref PubMed Sc