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Supplementary material to manuscript JVI02177
“A comprehensive structure-function comparison of hepatitis C virus strains JFH1 and J6 polymerases reveals a key residue stimulating replication in cell culture across genotypes”
Melanie Schmitt, Nathalie Scrima, Danijela Radujkovic, Célia Caillet-Saguy, Philip C. Simister, Peter Friebe, Oliver Wicht, Rahel Klein ,Ralf Bartenschlager, Volker Lohmannand Stéphane Bressanelli
Supplementary text: The high resolution crystal structure of an artificial JFH1_NS5B point mutant further highlights the importance in de novo initiation of a closed thumb conformation
MATERIALS AND METHODS
Crystals of JFH1T385A_NS5B were obtained in conditions almost identical to our previously reported(9)crystals of wildtype JFH1_NS5B. As for the wildtype, crystals of JFH1T385A_NS5B typically grew as clusters of irregular, thin plates after mixing 1:1 protein solution (4-5 mg/ml) and reservoir solution (0.2M NaH2PO4, polyethylene glycol (PEG) 35.000 2-5%) in 2-µl drops. Single plates were separated and swiped through a solution with the same composition as the reservoir supplemented with 30% glycerol before flash-cooling.High resolution X-ray diffraction data were collected at beamline ID14-4 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France). Molecular replacement was carried out with MOLREP(10)using PDB 3I5K(9)as a search model. A first automatic rebuilding was performed with ARP/wARP(7)then manual building was continued with COOT(5). Refinement was done first with Refmac5 (6)of the CCP4suite (1)then with phenix.refine of the PHENIX suite (3).Coordinates and structure factors for JFH1T385A_NS5Bare available from the Protein Data Bank under accession code 2XYM.
RESULTS
In order to further characterize the small conformational differences associated with the JFH1 high de novo synthesis activity, we included in our analysis a mutant JFH1 polymerase, T385A, exhibiting even higher de novo synthesis efficiency than JFH1 wildtype (Fig. S2A). However the increased enzymatic activity was associated with a reduced rather than an increased replication efficiency in cell culture (Fig.S2B). We obtained substantially better crystals of JFH1_NS5BT385A in the same orthorhombic centered crystal form as JFH1_NS5B (Table S1). This O crystal form differs from the M crystal form used in our previous structure of JFH1_NS5B (9). We were therefore in a position to assess both the influence of different crystal packing environments on the same molecule (JFH1_NS5B in crystal forms M and O) and the influence of a single, activity-boosting mutation on the conformation of JFH1_NS5B in the same crystal packing environment (JFH1_NS5B and JFH1_NS5BT385A in crystal form O). Table S2 indicates that each of the three structures is conformationally different from the other two with a cutoff of 2.5 sigmas. The percentages of residues in identical conformation are very similar in the JFH1_M/JFH1_O and the JFH1_M/JFH1T385A_O comparisons, indicating that the JFH1_O/JFH1T385A_O difference is small enough to be nearly invisible from the viewpoint of the lower precision JFH1_M. Close examination of the superpositions output by ESCET indeed indicates that the JFH1_M/JFH1_O difference is ascribable to an extra forward rotation of the thumb in JFH1_O that is only slightly more pronounced in JFH1T385A_O (Fig. S3A).In the point mutant´s case, the structural basis for this displacement is readily understood: Position 385 sits at the base of the thumb (Fig. S3B) at the exit of the 15-16 beta hairpin, a structural element previously recognized as a hinge around which the thumb rotates(2). The strictly conserved T385 is buried between this hinge and the thumb. Both missing groups in the sidechain of the T385A mutant are involved in networks of interactions in the wildtype: The gamma1 hydroxyl makes hydrogen bonds to the sidechains of both E481 at the hydrophilic base of helix S and W420 at the base of the central helix of the thumb (helix Q); The gamma2 methyl participates in van der Waals contacts between the 15-16 beta hinge and the base of helix S. The absence of these interactions in the JFH1T385A construct thus favors the “thumb forward” conformation. All three JFH1 structures were obtained in very similar crystallization conditions (Table 2, table S1, (9)). We conclude that we have captured in crystal form O a slightly different conformation of the JFH1 thumb that exists in solution in equilibrium with the conformation seen in crystal form M. JFH1T385A_O is even more closed than JFH1_O and JFH1_O is even more closed than JFH1_M (Fig. S3A). Because JFH1T385A is even more efficient at de novo synthesis in vitro than the wildtype, we conclude that the “thumb forward” conformation caught in the O crystal form is even closer to the initiation-competent form. Comparing the two conformations should therefore yield further insights into the molecular determinants of de novo initiation.
Two features stand out in this analysis (Table S2, underlined values, Fig.S3B): The first is that the main conformational difference does not involve the whole thumb, but rather a rotation (around the 15-16 beta hinge) of a rigid block (in green) made of the parts involved in the thumb-fingertips interaction (including helix Q and the top of helix S) as well as the thumb-proximal part of the fingertips (including helix A). The second feature is that this rigid block is made of two disconnected parts. The second part includes the short helix at the base of the linker (part of the “C-terminus” segment) on the other side of the thumb. This helix shifts towards the active site as the back of the thumb moves forwards in the series JFH1_M – JFH1_O – JFH1T385A_O. The part of the linker immediately C-terminal displays static disorder, following two alternate paths around the 17-18 beta-flap. We have been able to model these as two alternate conformations in the highest resolution structure (JFH1T385A_O). Further downstream, dynamic disorder occurs: the linker was clearly visible in JFH1_M down to residue 566, folding back upon itself so that residues 565 and 546 make two hydrogen bonds to each other. In contrast, it is now visible only down to residue 563.
DISCUSSION
The HCV (–) strand harbors 4 non-paired bases at its 3’ end (8), whilethe bases at the 3’ end of the (+) strand are buried in RNA secondary structures (4). In this study we used a polyC template with GTP in RdRp assays, mimicking the case of de novo synthesis from the (-) strand. The structure of the artificial JFH1T385A_NS5B therefore confirmsthe correlation betweenthumb closure (Fig. S3A) andde novo RNA synthesis efficiency from the (-) strand (Fig. S2A). It also shows that simply increasing thisde novo synthesis does not necessarily lead to better replication capability in cells (Fig.S2B). Anattractive explanation for this is that the T385A mutation increases de novo RNA synthesis from the (–) strand to the detriment of synthesis from the (+) strand. More detailed biochemical analyses and determination of (+)/ (-) strand ratios of different mutants in cell culture will be required to get a closer insight into the precise roles of thumb closure on RdRp function.
REFERENCES
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Table S1
Statistics of data collection and refinement for JFH1_NS5BT385A
Crystallisation conditions (added for cryocooling) / 0.2M NaH2PO4 ;2 to 5% PEG 35.000 (+ 30% glycerol)
Spacegroup and
Unit cell parameters
(Å ; degrees) / C2221
a = 97.6 b = 110.8
c = 115.3
= 90 = 90 = 90
Resolution rangea (Å) / 25 - 1.8 (1.9 - 1.8)
No. of reflections / 60225
Completeness (%) / 99.3 (95.7)
Rsymb(%) / 7.8 (43.5)
I/SigmaI / 14.1 (2.8)
No. of Molecule in asymmetric unit / 1
Average multiplicity / 5.0 (3.9)
Rfactor c(%) / 17.9 (21.3)
Rfree (%) / 22.2 (27.1)
Rmsd, bond (Å) / 0.006
Rmsd, Angle (degrees) / 0.99
X-ray source / ESRF,
ID14-4
PDB code / 2XYM
a In parentheses, statistics for the highest resolution shell.
b Rsym was determined by the equation
R = ∑ ∑ | Ihkl,j - <Ihkl> | / ∑ ∑ Ihkl,
hklj hkl j
where h, k and l are the unique indices of all reflections measured more than once and j the index for
symmetry-redundant reflections.
c R and Rfree were determined by the equation
R = ∑ | | Fobs| - k | Fcalc| | / ∑ |Fobs|
hkl hkl
where h, k and l are the indices of the reflections used in refinement (R) or of 5% of the reflections set aside and not
used in refinement (Rfree). The same set of reflections was used for Rfree in all structures. Fobs are the structure
factors deduced from the measured intensities and Fcalc the structure factors calculated from the model. k is a scale
factor to put the Fcalc on the same scale as the Fobs.
Table S2:
Pairwise conformational comparisons between the three JFH1_NS5B structures
Parameter / ValueJFH1_M (3I5K) / JFH1_O / JFH1T385A_O
<esd> (Å) / 0.15 / 0.10 / 0.09
JFH1_M (3I5K) / 91.6/97.9 / 91.8/98.9
JFH1_O / 93.8/98.2
Parameters and values are defined as in table 3. Underlined are the values for the
2.5 sigmastolerance ESCET analysis used to determine the rigid groups pictured
in figure xxxB.
Figure S1
Fig. S1: No impact of mutations in the C-terminal segment of NS5B on RdRp activity. JFH1 and J6 wt and mutant enzymes were assayed for enzymatic activity as described in the legend to Fig. 2C and 3B. Note that position 571 is formally not part of deltaC21 constructs, but corresponds to the first position in the linker. In constructs used in this study, this position encoded by chance a serine as present in the JFH1 wildtype sequence. J6-S571L therefore represents the J6 wildtype sequence.
Figure S2
Fig. S2: Comparison of JFH1 wild-type and mutant T385A. A) Enzymatic activity of mutant T385A. B) Replication efficiency of a JFH1 reporter replicon with mutation T385A in the polymerase gene. Enzymatic activity and replication in cell culture was performed as described in the legends of Fig. 2C and Fig. 1B, respectively.
Figure S3
Fig. S3:Comparisons involving structures of subtype 2a NS5B. (A) Schematic of the range of thumb closures seen in various crystal structures of subtype 2a HCV NS5B. (B) Comparison between the three JFH1_NS5B structures now available (JFH1_M, JFH1_O and JFH1T385A_O) with a tolerance threshold for residues in identical positions (ESCET lolim parameter) of 2.5 sigmas. A ribbon representation of the most closed structure (JFH1T385A _O) is shown, colored by rigid groups derived from the three JFH1 structures’ comparisonin order of decreasing size: blue, green, cyan, magenta. Position 385 at the exit of the 15-16 beta hairpin is displayed as spheres.