Crystallographic snapshots of the Zika virus NS3 helicase help

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Crystallographic snapshots of the Zika virus NS3 helicase help visualize the reactant water replenishment Junnan Fang, Xuping Jing, Guoliang Lu, Yi Xu, and Peng Gong ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00214 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 26, 2019

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ACS Infectious Diseases

Crystallographic snapshots of the Zika virus NS3 helicase help visualize the reactant water replenishment

Junnan Fang1,#, Xuping Jing2,3,#, Guoliang Lu2,3, Yi Xu1,*, Peng Gong1,2,*

1. The Joint Center of Translational Precision Medicine: Guangzhou Institute of Pediatrics, Guangzhou Women and Children’s Medical Center, No. 318 Renminzhonglu, Guangzhou, Guangdong, 510623, China; Wuhan Institute of Virology, Chinese Academy of Sciences, No. 44 Xiao Hong Shan, Wuhan, Hubei, 430071, China; 2. Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, No. 44 Xiao Hong Shan, Wuhan, Hubei, 430071, China; 3. University of Chinese Academy of Sciences, Beijing, 100049, China. # J.F. and X.J. contributed equally to this work. * Correspondence: [email protected] (P.G.); [email protected] (Y.X.).

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Zika virus (ZIKV), a positive-strand RNA virus belonging to the Flavivirus genus, has become an urgent public health concern since recent outbreaks worldwide. Its genome replication is facilitated by the viral NS3 protein bearing helicase function. The NS3 helicase uses energy derived from ATP hydrolysis to unwind RNA duplexed regions. Structural studies of the flavivirus NS3 helicases have suggested a conserved mechanism of ATP hydrolysis. However, the process of the reactant water replenishment, a key part of the hydrolysis cycle, remains elusive. Here we report two high-resolution crystal structures of ZIKV NS3 helicase in complex with ADP and Mn2+, one with the reactant water already loaded as previously observed, and the other with the water molecule still in a loading state. These data suggest that the reactant water replenishment can occurbetween the release of phosphate and the release of ADP and improves the structural basis of NS3 ATP hydrolysis cycle.

Keywords: flavivirus, Zika virus, NS3 helicase, ATP hydrolysis, reactant water


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Zika virus (ZIKV) was initially discovered in 1947 in the blood of a febrile sentinel rhesus monkey in Uganda’s Zika forest and was first identified in human in 1952.1 For many years, ZIKV had not drawn much attention, with a few human cases confined to Africa and Asia, and the ZIKV infection typically causes mild and self-limiting illnesses. Recently, ZIKV drew global attention when it caused outbreaks in Micronesia in 2007 and French Polynesia in 2013, and then led to about 1,300,000 suspected cases in Brazil in late 2015.2 Thereafter, ZIKV was found in infected human brain tissue, presumably accounting for the occurrence of microcephaly in neonates and Guillain–Barré syndrome in adults.3,4 Since then, ZIKV has become an urgent public health concern. ZIKV belongs to the Flavivirus genus of the Flaviviridae family that contains important human pathogens such as dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), etc. Similar to other flaviviruses, ZIKV contains a single-stranded and positive-sense RNA genome of about 10.7 kilobases (kb) in length.5 The single open reading frame in the viral genome is translated into a poly-protein that undergoes proteolytic processing by host and viral proteases to produce three structural proteins (C, prM/M, and E) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).5,6 Among the NS proteins, the NS3 is a multifunctional protein with an N-terminal chymotrypsin-like serine protease and a C-terminal superfamily 2 (SF2) RNA helicase. Together with a cofactor segment in NS2B, the NS3 protease participates in the poly-protein processing. The NS3 helicase also possesses nucleotide 5-triphosphatase (NTPase) and 5-terminal RNA triphosphatase activities, playing essential roles in viral genome replication and 5 capping.7 The RNA unwinding activity of the flavivirus NS3 is coupled to ATP hydrolysis. Crystallographic data, mostly from DENV NS3 and more recently from ZIKV NS3 have nearly completed the structural scheme of the ATP hydrolysis cycle.8-11 The cycle mainly includes ATP substrate binding, hydrolysis, and the release of the ADP and phosphate (Pi) products. Aside from the catalytic motifs in helicase domains 1 and 2 (D1 and D2), a divalent metal ion, naturally being Mg2+ but typically being Mn2+ in



obtaining crystallographic data, and the reactant water (Wr in figure illustrations) play pivotal roles in the hydrolysis cycle. The divalent metal ion coordinates with the and -phosphates of the ATP substrate and stabilizes the chemical transition state. The reactant water forms hydrogen bonds (H-bonds) with two conserved residues, a glutamic acid in D1 (E286 in ZIKV NS3 and E285 in DENV NS3) and a glutamine in D2 (Q455 in ZIKV NS3 and Q456 in DENV NS3), and is believed to be deprotonized and then carries out nucleophilic attack in the hydrolysis reaction.8 In the ATP-bound substrate complex and ADP-bound product complex crystal structures, the reactant water was typically found in place, while in the transition state complex it was absent.9 Although some of the flavivirus NS3 helicase apo structures have a water molecule bound at the equivalent position,8,9 here we tend to focus on ATP/ADP-bound situations that are obviously associated with the ATP hydrolysis when mentioning the reactant water. Theoretically, the reactant water replenishment can occur anytime between the two adjacent hydrolytic chemical steps and the water can be in and out during this period. However, the actual process of the reactant water loading has not been observed. Here we report three crystal structures of the ZIKV NS3 helicase: one in the apo state and two in complex with ADP and Mn2+. Very interestingly, one complex structure has the reactant water in place, while the other complex contains a water molecule at a nearby position, likely representing a partially loaded state. These data suggest that the reactant water replenishment can occur between the release of phosphate and the release of ADP, and may help improve the structural basis of ATP hydrolysis cycle in flavivirus NS3 helicase.

Results and Discussion An N-terminal hexahistidine-tagged ZIKV NS3 helicase (NS3_Hel, residues 181-618) was successively purified by nickel affinity, heparin affinity, and gel filtration chromatography. The purified protein or a mixture of protein, ATP, and MnCl2 was subjected to crystallization condition screening, and well-diffracting plate-like crystals were obtained. Three crystal structures of the ZIKV NS3 helicase,


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one in the apo state (NS3_Hel_apo) and two in complex with ADP and Mn2+ (NS3_Hel-ADP-Mn, forms 1 and 2) were solved by molecular replacement at 2.2 Å, 1.8 Å, and 1.9 Å, respectively (Table 1). All three structures are in the same crystal form with the P21 space group and one NS3 helicase molecule in the asymmetric unit. Likewise, the ATP supplied in the crystallization mixture was hydrolyzed by the NS3_Hel during the co-crystallization process. The overall structure of the ZIKV NS3_Hel_apo is well ordered with 434 out of 437 NS3 residues resolved. The ATP binding P-loop (motif I, residues 193–202), typically disordered in the flavivirus NS3_Hel apo structures,9 is well defined, likely achieved by the binding of a small molecule (modeled as HEPES) from the precipitant solution. The overall structure is highly similar to previously reported ZIKV NS3_Hel apo structures with the root-mean-square deviation (RMSD) values of all superimposable -carbon atoms about 0.7-0.9 Å (ZIKV NS3_Hel_apo as the reference structure, 97-100% coverage).9,10,12,13 The ZIKV NS3_Hel_apo is composed of three domains, with the ATP binding site between D1 and D2 and the RNA binding groove between D1/D2 and domain 3 (D3) (Figure S1A). The conserved motifs of the flavivirus NS3_Hel are within D1 and D2 (Figure S1C).7,14,15 These motifs are important for ATP hydrolysis (motifs I, II, III and VI), RNA binding (motifs I, Ia, Ib, Ic, IV, IVa, and V), or for the coupling of these two activities (motif V).16-19 Both NS3_Hel-ADP-Mn structures are highly similar to our NS3_Hel_apo structure with RMSD values of all superimposable -carbon atoms about 0.2 Å (ZIKV NS3_Hel_apo as the reference structure, 92-93% coverage). Subtle changes within the active site were observed upon the binding of ADP and Mn2+ including residues of motifs I, II, and VI (Figure 1A). In the NS3_Hel_apo structure, the side chain of Lys200 in motif I forms salt bridges with Asp285 and Glu286 in motif II, while in the two NS3_Hel-ADP-Mn structures, the side chain of Lys200 slightly shifts toward the ADP diphosphate moiety and interacts with the ADP -phosphate (Figure 1A). Comparing to that of the apo structure, Arg202 in motif I changes its side chain rotamer and forms pi-pi interactions with the adenine moiety of ADP in the



complex structures. In addition, Glu286 in motif II and Arg459 in motif VI undergo small-scale side chain conformational changes (Figure 1A). These side chain conformations in the two complex structures are consistent with those observed in previously reported NS3_Hel-ADP-Mn structure from DENV and ZIKV. The mechanism of flavivirus ATP hydrolysis is mainly illustrated by a set of DENV NS3_Hel structures with four representative states (Figure S2, A, B, C, and E).8 The first state represents the substrate complex and is obtained using the nonhydrolyzable ATP analog (Figure S2A); the second state mimics the catalytic transition state by using ADP and vanadate in the crystal soaking trial (Figure S2B); the third state represents a form of product complex with both ADP and phosphate (Pi) bound (Figure S2C); the forth state represents a form of product complex with ADP bound (Figure S2E). The reactant water was observed in the DENV AMPPNP-Mn/ADP-Mn structures and the ZIKV NS3_Hel-ADP-Mn structure obtained by ATP-Mn2+ soaking, but not in the ADP-vanadate-Mn structure (10,11). In the latter structure, the site of the reactant water was occupied by the vanadate.8 We should note that in the ADP-Pi-Mn structure, both the Pi and the reactant water were modeled. However, the distance between the reactant water and one of the Pi oxygen atoms is too short (2.0 Å and 2.2 Å in the two NS3_Hel-ADP-Pi-Mn complexes in the crystallographic asymmetric unit), while the electron density of the Pi is relatively weak and may suit well with a water molecule (Figure S2F). Therefore, we propose that this structure may represent an ADP-Mn complex instead of ADP-Pi-Mn complex as reported. The form 1 NS3_Hel-ADP-Mn structure is consistent with previously reported NS3_Hel-ADP-Mn structures from DENV and ZIKV with the reactant water in place (Figure 1B, D and Figure S2). Very interestingly, the form 2 NS3_Hel-ADP-Mn structure rather has a water molecule occupy a position close but not equivalent to the position of the reactant water (Figure 1B, E and Figure S2G). Thus, these two crystallographic snapshots provide the evidence that the reactant water replenishment can occur prior to the release of the ADP. The reactant water carries out an in-line nucleophilic attack of the ATP -phosphate and is consumed in each hydrolysis cycle, playing a pivotal role in the


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ATP hydrolysis. Activation of the reactant water presumably occurs through proton transfer to Glu286 and/or polarization by Gln455. The high resolution of the two NS3_Hel-ADP-Mn structures allows relatively accurate judgment of the subtle differences between the two structures. In the form 1 structure, the reactant water forms two H-bonds with the side chains of Glu286 (motif II) and Gln455 (motif VI) and the H-bond distances are 3.3 Å and 3.1 Å, respectively (Figure 1D). These distances are within regular hydrogen bonding distance range between oxygen and nitrogen

atoms

and

are

consistent

with

the

previously

reported

ZIKV

NS3_Hel-ADP-Mn structure (PDB entry: 5K8U; Figure 1C).20,9 To join the ADP-Mn interaction network, the reactant water forms an H-bond with Glu286, which in turn participates in the Mn2+ coordination. In the form 2 structure, a water molecule (Wr* in Figure 1E) resides at a position near but clearly not equivalent to the reactant water position in the form 1 structure (compare panels D and E in Figure 1). The H-bond distances between this water molecule and the accepter oxygen atoms of Glu286 and Gln455 are 0.4 Å (distance=3.7 Å, beyond regular H-bond distance) and 0.3 Å (distance=3.4 Å) longer than the corresponding distances observed in the form 1 complex, respectively. The correlation of the two forms of our ZIKV NS3_Hel-ADP-Mn structures thus helps visualize the loading of the reactant water, and the animation between these two structures highlights not only the water loading but also the pivotal role of Glu286 for mediating the water molecule movement and the Mn2+ coordination network maintenance through its side chain carboxyl group (supplemental movie S1). To further validate the proposed loading state of the reactant water in the form 2 ZIKV NS3_Hel-ADP-Mn structure, we carried out analyses to examine the spatial distribution of the reactant water and its distance to the two hydrogen bonding partners among ZIKV and DENV NS3 helicase structures in complex with ATP/ADP or their analogs. It turned out that the corresponding distances in the form 1 structure are similar to the average distances in previously reported complexes, while the distances in the form 2 structure are apparently longer (Table 2). Among the complexes analyzed, seven of them have the overall spatial positions of the Glu286



and Gln455 (or Glu285 and Gln466 in DENV NS3) side chains consistent with those of the form 1 and form 2 complexes (Figure 2). We therefore compared the spatial position of the reactant water in these nine complexes and found that the previously reported seven complexes have the reactant water tightly clustered around their average position, while the reactant water in the form 1 and form 2 complexes are 0.8 Å and 1.1 Å away from the average position (Figure 2). Collectively, these observations suggest that the reactant water in the form 2 complex occupies a previously unidentified position and is likely in a loading state. Macromolecule X-ray crystallography, sometimes combined with time-resolved strategy, is effective in understanding the mechanistic details of dynamic processes such as large-scale conformational changes and enzymatic reactions.21,22 The constraints provided by neighboring macromolecules in the crystal lattice increase the opportunity to capture higher-energy intermediate or meta-stable states if compared to the situation in the solution phase.23,24 In this work, the ZIKV NS3_Hel-ADP-Mn structures representing two different states were obtained in regular co-crystallization trials, providing an example of applying batch-to-batch or crystal-to-crystal variation in mechanistic studies of enzymes by crystallography. Building on previous structural work delineating ATP substrate binding, the transition state formation, and the phosphate product release,8-11 our work provides a piece of evidence of the post-chemistry reactant water replenishment, therefore improving the structural view of the ATP hydrolysis cycle in flavivirus NS3 helicase (Figure S2, A-E). This work emphasizes the role of ZIKV NS3 residues Glu286 and Gln455 or their equivalents in other flavivirus NS3 for their pivotal roles in the reactant water replenishment process, and could benefit future development of antivirals targeting the flavivirus NS3 or more specifically the ATP hydrolysis by NS3.

Materials and Methods Plasmid construction and protein production The ZIKV (Strain MR766, GenBank accession code: NC_012532) NS3_Hel gene fragment (correspond to residues 181-617) was cloned into a pET26b vector


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between NdeI and XhoI sites, and the resulting plasmid, pET26b-ZIKV-NS3_Hel, was transformed into Escherichia coli strain BL21-CodonPlus(DE3)-RIL. Cells were grown at 30 C overnight in LB medium with 50 g/mL kanamycin (KAN50) and 20 g/mL chloramphenicol (CHL20). The overnight culture was used to inoculate 0.9 L of LB medium with KAN50 and CHL20, and cells were grown at 37 C to reach an OD600 of 1.0. Isopropyl-β-D-thiogalactopyranoside (IPTG) was then provided at a final concentration of 0.5 mM for protein induction at 25 C for 5 h before harvesting. The NS3_Hel was produced with an N-terminal hexahistidine tag and a Ser-Ser-Ser-Gly linker. Purification of ZIKV NS3_Hel Protein purification was performed following a previously described protocol for the flavivirus NS5 with the following modifications.25 A Heparin HP column (GE Healthcare) was used to replace the cation exchange column in the second chromatography step. The NaCl concentration of pooled Ni-column fractions was reduced to approximately 90 mM, and the sample was then loaded onto the heparin column and eluted with a linear gradient to 1 M NaCl in 25 mM Tris (pH 8.5), 0.1 mM EDTA, 20% (vol./vol.) glycerol, and 0.02% (wt./vol.) NaN3. In the third chromatorgraphy step, a Superdex 75 gel filtration column (GE Healthcare) was used and the column was equilibrated with a GF buffer of 5 mM Tris (pH 7.0), 150 mM NaCl, 10% (vol./vol.) glycerol and 0.02% (wt./vol.) NaN3. Pooled fractions were supplemented with tris-(2-carboxyethyl) phosphine (TCEP) to a final concentration of 5 mM, concentrated to approximately 15 mg/mL, flash frozen with liquid nitrogen, and stored at -80 C in 5-20 μl aliquots. The extinction coefficient was calculated based

on

protein

sequence

using

the

ExPASy

ProtParam

program

(http://www.expasy.ch/tools/protparam.html). The typical yield is 1 mg of pure ZIKV NS3_Hel protein per liter of bacterial culture. Crystallization, data collection, structure determination and structure analysis Crystals of ZIKV NS3_Hel (apo form) were obtained within 1 week by sitting drop vapor diffusion at 16 C. Typically, a volume of 0.5 μL of protein solution at a



concentration of 10 mg/mL in the GF buffer with 5 mM TCEP was mixed with an equal volume of a precipitant solution of 0.1 M HEPES (pH 7.5), 0.2 M Li2SO4, and 25% (wt./vol.) PEG3350. ZIKV NS3_Hel-ADP-Mn crystals were obtained using hanging drop vapor diffusion under the same crystallization condition by mixing 1 μL solution containing 10 mg/mL protein, 1 mM ATP, and 5 mM MnCl2 with 1 μL of the precipitant solution. Crystals were cryo-protected using the precipitant solution supplemented with 10% (vol./vol.) glycerol and stored in liquid nitrogen prior to data collection. All data sets were collected at 100 K at Shanghai Synchrotron Radiation Facility (SSRF) beamline BL17U1. Reflections were integrated, merged, and scaled using HKL2000.26 The initial model of ZIKV NS3_Hel was obtained using the molecular replacement program PHASER using the helicase module of the full-length DENV NS3 structure (PDB entry: 2VBC) as the search model.27 Manual model rebuilding was performed using Coot and refined with the PHENIX software suite.28,29 The 3,500 K composite simulated-annealing omit 2Fo-Fc electron density maps were generated by CNS.30 The data processing and structure refinement statistics are summarized in Table 1. All structure superimpositions were done using the maximum likelihood superpositioning program THESEUS,31 while the RMSD calculations for global structure similarity assessment were based on traditional least-square superpositioning method. The protein alignment uncertainty was estimated using the least-square sigma value (0.1 Å) of the THESEUS superpositioning and was used to estimate the positional uncertainty of the average position of seven previously reported flavivirus NS3 structures in Figure 2. The coordinate error of the reactant water molecules in the form 1 (0.2 Å) and form 2 (0.25 Å) complexes was estimated based on a resolution-based coordinate error estimation,32,33 also considering a fact that the B-factor values of the corresponding water molecules (32.0 and 19.7 Å2 for form 1 and form 2, respectively) are generally consistent with the corresponding average B-factor values reported in Table 1. The distance error reported in Figure 2 is derived from associated coordinate error through standard error propagation.


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Protein structure accession numbers Coordinates and structure factor files have been deposited in the Protein Data Bank, under accession codes 6ADW, 6ADX, and 6ADY.

Acknowledgments We thank Dr. Bo Zhang for providing the DNA clone of the ZIKV Strain MR766 as the cloning material for generating the ZIKV NS3_Hel expression plasmid, and the Shanghai Synchrotron Radiation Facility (SSRF, beamline BL17U1) for access to beamline. This work was supported by the National Key Research and Development Program of China (2016YFC1200400, 2018YFA0507200), the Science and Technology Program of Guangzhou, China (2014Y2-00197), the Natural Science Foundation of Guangdong Province, China (2016A030310249), the Natural Science Foundation of Hubei Province, China (ZRMS2017001469), the “One-Three-Five” Strategic Programs, Wuhan Institute of Virology, Chinese Academy of Sciences, China (Y605191SA1), and the Advanced Customer Cultivation Project of Wuhan National

Biosafety

Laboratory,

Chinese

Academy

of

Sciences,

China

(2018ACCP-MS06).

Author Contributions Conceived and designed the experiments: PG and YX; performed the experiments: JF; analyzed the data: JF, XJ, LG, YX, and PG; wrote the paper: JF, XJ, and PG.

Associated Content Supporting Information: supplemental figures and figure legends for Figures S1-S2 and supplemental movie legend for Movie S1; supplemental Movie S1.

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Figure legends Figure 1. Crystal structures of the ZIKV NS3_Hel help visualize the reactant water replenishment. A) A comparison of crystal structures of apo NS3_Hel (gray) and NS3_Hel-ADP-Mn (form 1 in green; form 2 in pink). In the enlarged view of the ATP binding site, key side chains and the ADP are shown as sticks. Mn2+ in the two complex structures are shown as spheres. For clarity, the HEPES molecule bound in the ATP binding site of the apo structure is not shown. B) A stereo-pair image showing the comparison of the two NS3_Hel-ADP-Mn complexes around the ATP binding site (form 1 in green; form 2 in pink). Mn2+ coordination interactions and the H-bonds formed between the reactant water and residues Gln455 and Glu286 are shown as dashed lines. C-E) A comparison of the previously reported ZIKV NS3_Hel-ADP-Mn structure (C) and the two forms of ZIKV NS3_Hel-ADP-Mn structure in this study (D-E) around the ATP binding site. The Mn2+ and reactant water are shown as cyan and red spheres, respectively. A dashed line between Gln455 and the reactant water in form 2 is shown in addition to the interactions shown in panel B, and the red color of this dashed line indicates a distance beyond regular H-bond distance range. 2Fo-Fc electron density map (C) and 3,500 K composite simulated-annealing omit electron density map (D-E) were overlaid with the density of Mn2+ contoured at 1.5  and the rest of the density contoured at 1.0 . Wr: reactant water. Wr*: reactant water in its loading state. In panels C/D/E, different mesh colors


Page 15 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


are used in each panel to show different components around the active site, and a single map type is used in each panel. Figure 2. The spatial position comparison of the reactant water among selected flavivirus NS3 helicase crystal structures. When superposed with the form 1 (reference structure for superpositioning, orange) and form 2 (brown) complexes, seven crystal structures (PDB entries are 2JLR, 2JLS, 5Y4Z, 5GJC, 5K8I, 5K8T, and 5K8U, protein in gray and reactant water in red) of the flavivirus NS3 helicase in complex with ATP/ADP or their analogs have consistent spatial position of residues Gln455/456 and Glu286/285. The average position of the reactant water molecules from the seven previously reported complexes is shown in black. The estimated values of coordinate error are indicated by the dashed circles (see Materials and methods). Table 1. X-ray diffraction data collection and structure refinement statistics. Structure

ADP-Mn complex

ADP-Mn complex

(form 1)

(form 2)

6ADW

6ADX

6ADY

0.9791

0.9792

0.9792

P21

P21

P21

a, b, c (Å)

53.5, 68.5, 56.9

53.8, 68.6, 56.9

53.7, 68.4, 56.7

α, β, γ (°)

90, 93.3, 90

90, 92.5, 90

90, 93.0, 90

50.0-2.20 (2.28-2.20)

50.0-1.75 (1.8-1.75)

50.0-1.90 (1.97-1.90)

PDB entry Data

Apo

collection1

Wavelength (Å) Space group Cell parameters

Resolution range (Å)2 No. of unique reflections

21,028

41,025

32,052

Completeness (%)

99.8 (98.8)

98.8 (98.5)

99.0 (98.3)

I / σI

13.2 (3.4)

24.9 (5.0)

26.4 (9.2) 3

Rmerge

0.097 (0.42)

0.048 (0.23)

0.046 (0.15)

3.6 (3.6)

3.5 (3.5)

3.4 (3.3)

2.20

1.75

1.90

20,478

41,011

32,019

16.9 / 21.6

17.9 / 21.1

17.8 / 22.6

Redundancy Structure refinement Resolution (Å) No. of unique reflections Rwork / Rfree4 (%)



Page 16 of 20

No. atoms Protein

3,400

3,338

3,338

30 / - / 181

27 / 1 /332

27 / 1 / 271

31.2

20.5

18.8

49.2 / - / 34.3

30 / 24.9 / 28.0

26.5 / 25.6 / 24.5

Bond lengths (Å)

0.007

0.006

0.006

Bond angle (°)

0.936

0.855

0.842

89.7 / 10.3 / 0.0 / 0.1

90.7 / 9.0 / 0.0 / 0.3

90.6 / 9.1 / 0.0 / 0.3

Ligand / Ion / Water B-factors (Å2) Protein Ligands / Ion / Water R.m.s. deviations

Ramachandran stat.5 1

One crystal was used for data collection for each structure.   Values in parentheses are for highest-resolution shell.   3 Higher resolution data were not collected with high completeness due to longer than optimal detector distance used in data collection. 4 5% of data are taken for the R free set. 5 Values are in percentage and are for most favored, additionally allowed, generously allowed, and disallowed regions in Ramachandran plots, respectively.   2

Table 2. A comparison of the distances between the reactant water and the corresponding oxygen atoms in E286/E285 and Q455/Q456 among the flavivirus NS3 helicase complex structures. Structure

Distance 1 (Å)1

Distance 2 (Å)

Number of

classification

(to E286/E285)

(to Q455/Q456)

structures

ZIKV complexes2

3.1 ± 0.2

2.9 ± 0.1

6

DENV complexes3

2.9 ± 0.3

2.8 ± 0.2

6

All complexes

3.0 ± 0.3

2.9 ± 0.2

12

Selected complexes4

3.1 ± 0.2

3.0 ± 0.1

7

Form 1 complex

3.1

3.3

1

Form 2 complex

3.4

3.7

1

Distances are shown as averages and standard deviations for the complex groups. PDB entries: 5Y4Z, 5GJC, 5K8I, 5K8L, 5K8T and 5K8U. 3 PDB entries: 2JLU, 2JLR, 2JLS, 2JLV, 2JLY and 2JLZ. 4 PDB entries: 2JLR, 2JLS, 5Y4Z, 5GJC, 5K8I, 5K8T and 5K8U. These complexes were chosen for the analyses shown in Figure 2. 1

2


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For Table of Contents Use Only



Figure 1


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Figure 2 169x66mm (300 x 300 DPI)




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Crystallographic snapshots of the Zika virus NS3 helicase help

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