NMR and circular dichroism data for domain 2 of the HCV NS5A protein phosphorylated by the Casein Kinase II

The Hepatitis C Virus (HCV)1 nonstructural 5A protein (NS5A) is a phosphoprotein (Evans et al., 2004; Ross-Thriepland and Harris, 2014) [1], [2] composed of an N-terminal well-structured domain and two C-terminal intrinsically disordered domains (Moradpour et al., 2007; Bartenschlager et al., 2013; Badillo et al., 2017) [3], [4], [5]. So far, no precise molecular function has been identified for this viral protein (Ross-Thriepland and Harris, 2015) [6] which is required for viral replication (Tellinghuisen et al., 2008) [7]. In this article, we present datasets of NMR and circular dichroism analyses of the domain 2 of the HCV NS5A protein (NS5A-D2) phosphorylated in vitro by the Casein Kinase II (CKII) (Dal Pero et al., 2007; Clemens et al., 2015; Masak et al., 2014; Kim et al., 2014) [8], [9], [10], [11]. We describe the in vitro phosphorylation of the serine 288 (pS288) of NS5A-D2 by CKII and report the circular dichroism spectrum of the phosphorylated domain (NS5-D2_CKII). This data article also contains the 1H, 15N and 13C NMR chemical shift assignments (HN, N, Cα, Cβ and C’) for the phosphorylated NS5A-D2 domain, and an assigned 1H,15N-HSQC spectrum is shown. The NMR data have been acquired on an 800 MHz spectrometer. These NMR data have been used to calculate both the 1H,15N combined chemical shift perturbations (CSP) induced by the phosphorylation of pS288 and the secondary structural propensity (SSP) scores that describe the structural tendencies in this intrinsically disordered domain. The circular dichroism spectrum and the SSP scores of NS5A-D2_CKII have been compared with those of unphosphorylated NS5A-D2 [12,13].


a b s t r a c t
The Hepatitis C Virus (HCV) 1 nonstructural 5A protein (NS5A) is a phosphoprotein (Evans et al., 2004; Ross-Thriepland and Harris, 2014) [1,2] composed of an N-terminal well-structured domain and two C-terminal intrinsically disordered domains (Moradpour et al., 2007; Bartenschlager et al., 2013;Badillo et al., 2017) [3][4][5]. So far, no precise molecular function has been identified for this viral protein (Ross-Thriepland and Harris, 2015) [6] which is required for viral replication (Tellinghuisen et al., 2008) [7]. In this article, we present datasets of NMR and circular dichroism analyses of the domain 2 of the HCV NS5A protein (NS5A-D2) phosphorylated in vitro by the Casein Kinase II (CKII) (Dal Pero et al., 2007;Clemens et al., 2015;Masak et al., 2014;Kim et al., 2014) [8][9][10][11]. We describe the in vitro phosphorylation of the serine 288 (pS288) of NS5A-D2 by CKII and report the circular dichroism spectrum of the phosphorylated domain (NS5-D2_CKII). This data article also contains the 1 H, 15 N and 13 C NMR chemical shift assignments (HN, N, Cα, Cβ and C') for the phosphorylated NS5A-D2 domain, and an assigned 1 H, 15 N-HSQC spectrum is shown. The NMR data have been acquired on an 800 MHz spectrometer. These NMR data have been used to calculate both the 1 H, 15 N combined chemical shift perturbations (CSP) induced by the phosphorylation of pS288 and the secondary structural propensity (SSP) scores that describe the structural tendencies in this intrinsically disordered domain. The circular dichroism spectrum and The CD and the 13 Cα and 13 Cβ chemical shifts data may be used to extract structural information, which could then be used in structure-function studies.

Circular dichroism analysis
NS5A-D2 (7.3 µM) and NS5A-D2_CKII (6.4 µM) samples were analyzed at 293 K in a 1 mm-path length quartz cell using a Jobin Yvon-SPEX-Horiba model CD6 spectropolarimeter. Spectra were acquired with a step of 0.5 nm from 190 to 260 nm and an integration time of 2 s. Blank runs (with buffer only) were made before each measurement and were subtracted from the sample runs to obtain the final spectra (Fig. 2). UV intensities were expressed as the specific ellipticity per residue (per decimole of amino acid residue). Protein concentrations were determined by UV absorbance at 280 nm (molar extinction coefficient of 15595 M −1 cm −1 ). The CD spectrum of NS5A-D2_CKII is characteristic of mainly disordered protein with a large negative peak around 200 nm. Compared to the CD spectrum of NS5A-D2 (unphosphorylated), the only significant difference is that the negative contribution around 200 nm is slightly less pronounced.

NMR analyses
NMR data were acquired on a Bruker AvanceIII 800 MHz spectrometer equipped with a 5 mm TXI room-temperature probe using a Shigemi tube (sample volume 350 µL) at 298 K. Proton chemical  Table 1 are shown on the spectrum. shifts were referenced using the methyl signal of sodium 3-trimethylsilyl-[2,2,3,3-d 4 ]propionate at 0 ppm. Data were acquired with Topspin2.1 software (Bruker).
The NMR spectra collected are: -2D 1 H, 15 15 N and 13 C chemical shift assignments of NS5A-D2_CKII were performed with the product plane method developed in-house [15], using the complete NMR dataset mentioned above. The NMR data, analyzed with the software NMRFAM-SPARKY [16], showed that the residue serine 288 (S288) in NS5A-D2 is phosphorylated by CKII in vitro (Table 1 and Fig. 3). Backbone assignments of NS5A-D2_CKII have been deposited in the Biological Magnetic Resonance Data Bank (BMRB ID: 27270).

Chemical shifts perturbation analysis
The chemical shifts of NS5A-D2_CKII reported in this article (BMRB ID: 27270) were compared with those of NS5A-D2 (Biological Magnetic Resonance Data Bank entry 16165) [12]. The 1 H, 15 N combined chemical shift perturbations (CSP) were calculated using the following formula: CSP ¼ √(((ΔδH) 2 þ (ΔδNH/10) 2 )/2) (adapted for IDP from [17]), where ΔδH and ΔδNH correspond to the chemical shift perturbations in the 1 H and 15 N dimension, respectively. The CSP values are illustrated in Fig. 4. The residue with the highest CSP value (0.324 ppm) corresponds to the serine 288 which is phosphorylated in the NS5A-D2_CKII sample. The other residues that exhibit CSP values larger than 0.15 ppm are mainly located around the phosphorylation site and correspond to the NS5A-D2 region encompassing residues 285-291, but also include the T249 and the W312 residues.

Secondary Structure Propensity analysis
Experimental 13 C chemical shifts of 13 Cα and 13 Cβ nuclei of NS5A-D2_CKII were analyzed with the Secondary Structure Propensity (SSP) software [18] to calculate a score illustrating structural propensity for each NS5A-D2_CKII residue. The plot of the SSP data along the NS5A-D2 sequence is shown in Fig. 5. SSP scores close to 0 correspond to fully disordered residues, whereas positive and negative scores represent helical propensities and extended regions, respectively. The low SSP scores * Fig. 5. Secondary structure propensity analysis of 13 Cα and 13 Cβ NMR chemical shifts of NS5A-D2_CKII. Values close to 0 correspond to fully disordered residues, whereas positive and negative scores represent helical propensities and extended regions, respectively [18]. The SSP scores of NS5A-D2_CKII (in red) are compared with those of unphosphorylated NS5A-D2 (in blue). The position of the pS288 residue in NS5A-D2_CKII is highlighted by an asterisk.
in Fig. 5 show that even in the presence of the pS288, NS5A-D2 is still mainly disordered with two main regions exhibiting partial alpha-helical structuration (residues 252-261 and 295-299) and two others showing partial extended conformation (residues 279-288 and 306-308). SSP values of NS5A-D2_CKII were then compared with those obtained from the unphosphorylated NS5A-D2 sample that we have previously reported [13]. Overall, SSP data in the absence and in the presence of the phosphorylation at position 288 are very similar. Only subtle differences were detected in three NS5A-D2 regions: directly around the phosphorylation site (residues 282-287), in the region of residues 271-278, and in residues 301-304 (Fig. 5). Just prior to the pS288 site (residues 282-287) the propensity to adopt an extended conformation is slightly higher than in the absence of phosphorylation. This is probably due to charge repulsion phenomena between the negatively charged phosphate moiety of pS288 and the carboxyl groups in the side chains of E285, E286 and E287. In the region of residues 271-278, NS5A-D2_CKII seems to be more flexible when phosphorylated, as illustrated by SSP scores close to 0. In residues 301-304, the SSP scores shift from low positive to low negative values, showing a slight change in the conformation of this region upon phosphorylation.