Preliminary X-ray Diffraction Analysis of the Envelope (E) Protein of Far-Eastern Tick-Borne Encephalitis Virus Subtype (Sofjin Strain)

The envelope (E) protein of flaviviruses is an attractive target for the development of antiviral agents because this protein plays an important role in the formation of virus particles and in the virus invasion in host cells. Currently, there is no specific antiviral therapy for tick-borne encephalitis. The goal of this study is to determine the crystal structure of the envelope (E) protein ectodomain of Far-Eastern tick-borne encephalitis virus subtype (Sofjin strain). The knowledge of the three-dimensional structure can serve as the basis for the development of specific inhibitors of conformational rearrangements of the (E) protein, which are essential for the initial stages of infection.


INTRODUCTION
Flaviviruses belong to the family Flaviviridae and can be classified into three groups according to the transmission vectors: tick-borne flaviviruses, mosquitoborne flaviviruses, and flaviviruses with no known vectors [1]. Notorious human pathogens, such as Zika virus, dengue virus, and West Nile virus, are spread by mosquitoes. Tick-borne encephalitis virus and louping ill virus are transmitted by ticks. They can cause encephalitis or encephalomeningitis diseases in the central nervous system. Louping ill virus (LIV) mainly infects small animals, such as sheep and red grouse. Occasionally, this virus can also infect humans. In contrast, the more widespread tick-borne encephalitis virus (TBEV) usually causes encephalitis in humans, although it can infect rodents. Tick-borne encephalitis virus is common in Eurasia, causing about 10000 to 14000 cases of tick-borne encephalitis annually [2,3]. Despite available vaccines, the rate of TBEV infection is still high due to low vaccination coverage. Currently, there are no specific therapeutics for the treatment of TBEV. The drug design is one of the modern approaches to the development of therapeutics based on the knowledge of the three-dimensional structures of the targets [4,5]. In the case of TBEV, the envelope (E) protein serves as the target.
This protein forms the outer virion surface, provides stability of the virus particle, and is involved in the initial stages of infection, such as the binding to cell receptors, the entry of the virus into the cell, and the uncoating of the viral RNA [2].
There are the following three main subtypes of TBEV: the Central Europe subtype, the Siberian subtype, and the Far-Eastern subtype. The Siberian and Far-Eastern subtypes of TBEV are prevalent in Russia. Each subtype contains numerous strains, which have small differences in the amino-acid sequence of the proteins.
The goal of this study was to grow crystals of the E protein ectodomain of Far-Eastern tick-borne encephalitis virus subtype (Sofjin strain), the aminoacid sequence of which is shown in Fig. 1, for the subsequent analysis of the structure of the apo protein and the detailed structural study of interactions between the protein and prototypes of inhibitors.

Isolation and Purification of the E Protein Ectodomain
The strain producing the recombinant E protein ectodomain was constructed by the transformation of the E. coli BL21(DE3) RIPL strain cells with the recombinant pET22 plasmid. The protein was isolated from inclusion bodies by refolding according to a procedure described in [6]. The supernatant was concentrated using VIVAFLOW 200 cassettes with a 10-kDa PES membrane (Sartorius stedim LAB Ltd) and then using a 10-kDa centrifugal filter unit (Millipore, Burlington, MA, USA) followed by the filtration through a filter with a 0.22-μm pore size (PES membrane, Millipore Millex-GP). The target protein was purified by gel filtration on a HiLoad 16/600 Superdex 200prep grade column (GE Healthcare, Sweden).

Crystallization of the E Protein Ectodomain Е(sE)
The crystallization conditions for the monomer and dimer of the E protein ectodomain were screened by the sitting-drop vapor-diffusion method. Drops were composed of 0.7 μL of the protein solution at a concentration of 5 mg/mL and 0.7 μL of the reservoir solution. The volume of the reservoir solution was 200 μL. The initial crystallization conditions were screened in Intelli-Plate 48-3 plates (Hampton Research) using commercially available Hampton Research crystallization screen kits at 20°С. Clearly faceted single crystals were grown in acidic buffer sys-tems. The initial crystallization conditions were as follows: 2.0 M ammonium sulfate in 0.1 M citrate buffer, pH 4.0. Under these conditions, small crystals were obtained (Fig. 2a). The crystallization conditions were then optimized by varying the concentration of ammonium sulfate and the pН value of the buffer and using the following additives: polyethylene glycol, glycerol, and 2-methyl-2,4-pentanediol (MPD). The conditions suitable for obtaining single crystals with a size of at least 0.1 mm were not found as yet.

X-ray Diffraction Data Collection and Preliminary X-ray Diffraction Analysis
Before X-ray diffraction data collection, the crystals were picked up with a cryoloop (Hampton Research), transferred to a cryoprotectant solution containing, apart from the components of the reservoir solution, 20% glycerol, and soaked in this solution for 15 s. Then the crystals in the loop were placed in a metallic storage cassette compatible with an automated sample changer for pre-frozen crystals in liquid nitrogen. The X-ray diffraction data were collected at 100 К at the ID30B beamline of the European Synchrotron Radiation Facility (ESRF, Grenoble,  France) equipped with the PILATUS3 6M detector (Dectris). The X-ray diffraction data were processed using the XDS and XSCALE programs [7]. The X-ray diffraction data collection statistics are given in Table 1. The X-ray diffraction data were collected to 3.2 Å resolution. The structure was solved by the molecularreplacement method with the MOLREP program [8].
The structure of the E protein ectodomain of LIV (Louping ill virus) refined at 3.6 Å resolution (pdb refcode 6J5C) [1] was used as the starting model. This model was refined to an intermediate R factor of 22.6% using the Refmac program [9]. The crystal structure was analyzed with the Coot moleculargraphics program [10], the PyMol molecular viewer [11], and the ССР4 suite of programs [12].
RESULTS AND DISCUSSION The initial crystallization conditions were screened by the sitting-drop vapor-diffusion method using commercially available Hampton Research crystallization screen kits. The crystallization conditions were then optimized to prepare crystals with a size of 0.1-0.15 mm suitable for X-ray diffraction (Fig. 2). To achieve this purpose, the concentration of ammonium sulfate was decreased by 0.2-0.4 M compared to the initial concentration, the pH value was varied, and MPD was added, which made it possible to decrease the number of crystals that grew in the drop and obtain larger single crystals. Since the solubility of the protein is rather low, the protein concentration was not varied. The best crystals were obtained in 0.1 M citrate buffer, pH 4.5, supplemented with 1.6-1.8 M ammonium sulfate and 2% MPD. The crystals with a size of up to 0.1-0.15 mm appeared within two-three days (Fig. 2b).
The X-ray diffraction data were collected from the frozen crystals at 100 К on the ESRF synchrotron radiation source. The data processing demonstrated that the highest spatial resolution was 3.2 Å. The crystals belong to the cubic sp. gr. Р4 1 21 with the unit cell parameters a = b = c = 165.061 Å, α = β = γ = 90°. There is one enzyme molecule per asymmetric unit. The polypeptide chain fold is similar to that in the structure of the E protein ectodomain of TBEV Central European subtype (Neudoerfl strain; pdb refcode 1SVB) [13] and the E protein ectodomain of LIV (pdb refcode 6J5C) (Fig. 3). It should be noted that the conformation of the E protein ectodomain of LIV, the crystals of which suitable for X-ray diffraction were obtained under similar crystallization conditions, is less different from the conformation of the structure under consideration despite the lower homology [1], particularly in the domain III region. The molecular structure of the title enzyme was superimposed with the molecular structure of 6J5C using the Сα atoms with a root-mean-square deviation (rmsd) of 0.546 Å; the superposition with the molecular structure of 1SVB gives the rmsd of 1.519 Å. It is worth noting that the structure 1SVB was determined for the E protein ectodomain of TBEV Western subtype (Neudoerfl strain) cleaved from the surface of virus particles with trypsin. In this study, we determined the structure of the recombinant protein expressed in the bacterial expression system. Nevertheless, the conformational similarity between the recombinant and natural proteins shows that the soluble part of the E protein expressed in E. coli is suitable for the detailed structural study of protein−inhibitor interactions. The structure refinement is currently underway.
It was demonstrated that, despite the utilization of the bacterial expression system for the production of the recombinant E protein, the conformation of the molecule is similar to that observed in the virus particle. Therefore, the crystals of this protein can be used for the X-ray diffraction analysis of the complexes with prototypes of inhibitors of the E protein. However, the found crystallization conditions are not optimal because the crystals of the protein in complexes with inhibitors for the subsequent X-ray diffraction analysis are usually obtained by soaking because of a low solubility of inhibitors. This method generally impairs the spatial resolution of the crystals. The resolution required for the detailed analysis of the binding of inhibitors to the protein should be not lower than 2.5 Å [5]. The soaking of the crystals will apparently lead to a decrease in the resolution from the crystals and, con- sequently, the crystals diffracting to a resolution of 3.2 Å cannot be used for obtaining crystals of the complexes, providing sufficiently detailed data. Therefore, the search for conditions of crystallization of the protein sE, which will allow the structure determination at a resolution of about 2 Å, is currently underway.