key: cord-0765035-9piio754
authors: Zhou, Haixia; Zhang, Shuyuan; Wang, Xinquan
title: Crystallization and Structural Determination of the Receptor-Binding Domain of MERS-CoV Spike Glycoprotein
date: 2019-09-14
journal: MERS Coronavirus
DOI: 10.1007/978-1-0716-0211-9_4
sha: 9dab31b3924e55ae6b46e55f3d0918ec237e20d2
doc_id: 765035
cord_uid: 9piio754

Three-dimensional structures of the receptor-binding domain (RBD) of MERS-CoV spike glycoprotein bound to cellular receptor and monoclonal antibodies (mAbs) have been determined by X-ray crystallography, providing structural information about receptor recognition and neutralizing mechanisms of mAbs at the atomic level. In this chapter, we describe the purification, crystallization, and structure determination of the MERS-CoV RBD.

The first three-dimensional structure of the MERS-CoV spike glycoprotein receptor-binding domain (RBD), providing the molecular basis of viral attachment to host cells, was determined in the complex with it cellular receptor dipeptidyl peptidase 4 (DPP4, also called CD26) by X-ray crystallography [1] . Because of the significance in receptor recognition and specific pathogenesis, RBD became a hot spot in the study of MERS-CoV. A number of structures of RBD bound by monoclonal antibodies (mAbs) have also been determined and deposited in the Protein Data Bank (PDB, http://www.rcsb.org/pdb/) [2] [3] [4] [5] [6] [7] [8] . Our group determined the RBD structures in complex with DPP4 and the mAbs MERS-27, MERS-4 and MERS-GD27, respectively [2] [3] [4] 9] . All the three-dimensional structures of MERS-CoV RBD have been determined by X-ray crystallography, which is a powerful method for determining molecular structures at atomic resolution. Briefly, the ordered and repeated atoms in a single protein crystal can diffract the incident X-ray beam into many specific directions. The angles and intensities of these diffracted X-rays can be collected and measured in an X-ray diffraction experiment. After obtaining the phases of these diffracted X-rays by heavy-atom derivative, anomalous scattering or molecular replacement methods, a protein crystallographer then calculates the density of electrons with the protein crystal and builds a structural model based on the density map. For details on the principles and methodology of protein crystallography, please refer to the range of other excellent textbooks.

In this chapter, an overview of the standard method of protein crystallography is briefly introduced, focusing on crystallization and structural determination of MERS-CoV RBD using the molecular replacement method.

Prepare all solutions using ultrapure water (prepared by purifying deionized water, to attain a resistivity of 18 MΩ cm at 25 C) and analytical grade reagents. When dealing with waste, we strictly follow all waste disposal regulations.

1. pFastBac vector containing the MERS-CoV RBD gene.

3. LB liquid medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, and 1 L ultrapure water; sterilize by high-pressure steam.

4. Liquid LB selection medium: LB liquid medium, 50 μg/mL kanamycin, 7 μg/mL gentamicin, and 10 μg/mL tetracycline.

5. Bacmid selection LB agar plate:10 g tryptone, 5 g yeast extract, 10 g agar powder, 10 g NaCl, and 1 L ultrapure water. Sterilization at high-pressure steam. 50 μg/mL kanamycin, 7 μg/mL gentamicin, 10 μg/mL tetracycline, 100 μg/mL x-gal, 40 μg/ mL IPTG Mix and pout into sterile plates (see Note 1). 

MER-CoV RBD can be expressed using the Bac-to-Bac baculovirus expression system (Fig. 1 ), collected and captured using NTA Sepharose (GE Healthcare) and then further purified by gel filtration chromatography using a Superdex 200 High Performance column (GE Healthcare). Crystallization trials are set up using the hanging-drop or sitting-drop vapor diffusion method in conjunction with the sparse-matrix crystal screening kits. The structure of MERS-CoV RBD in complex with MERS-4scFv was determined using the molecular replacement method. When the total volume remaining is reduced to 50 mL, add 100 mL HBS buffer to collect all the liquid in the system in a beaker. Then dispense into high-speed centrifuge tubes, centrifuge at 3000 Â g for 1 h at 4 C.

4. The supernatant after centrifugation is loaded onto the nickel-NTA beads equilibrated with 30 mL of HBS buffer. MERS-CoV RBD with a His tag could be captured by nickel-NTA beads. Repeat loading the sample once more.

5. Add wash buffer to the beads to remove the nonspecifically bound proteins until the flow-through is not able to discolor the Coomassie Brilliant Blue G250 solution.

6. After adding elution buffer, the target protein will dissociate from the beads; collect it in a 10 kDa Millipore concentrating tube. Similarly, detect protein with Coomassie Brilliant Blue G250. The concentrating tube containing the protein sample is centrifuged at 3000 Â g to concentrate the sample to less than 7. MERS-CoVRBD is further purified by gel filtration chromatography. The sample is loaded onto the Superdex 200 column pre-equilibrated with HBS buffer. Fractions containing RBD are collected and the protein's purity is confirmed by SDS-PAGE (Fig. 2) .

8. Dilute the protein to 1 mg/mL, and digest with endoglycosidase F1 and F3 (F1/F3: RBD at the ratio of 1:100) at 18 C overnight. The digested protein is concentrated and purified by gel filtration chromatography same as above (optional).

9. After preparing the MERS-CoV RBD protein and MERS-4scFv protein (see Note 11) detect the absorption of the protein sample at 280 nm (A280). According to the molecular weight and extinction coefficient, the molar concentration can be calculated. The two proteins were mixed at molar ratio of 1:1, incubated on ice for 1 h, and purified using a Superdex 200 column. Collect the fractions containing the complex and confirm the protein purity by SDS-PAGE.

Sartorius centrifugal concentrator to concentrate to 10-15 mg/mL. After mixing and aspirating, centrifuge at 10,000 Â g for 10 min at 4 C. 3. Use TTP LabTech's mosquito crystallization setup for automated crystallography. Absorb 3 μL protein on the 8-well 5 μL micro-reservoir strip (Fig. 3a) . Then the needles aspirate the protein from the strip onto the SWISSCI plate with 200 nL of protein per well (Fig. 3b) , using the sitting-drop vapor diffusion method by mixing 200 nL reservoir and 200 nL reservoir (Fig. 3c,d) .

4. Seal the plate with tape and gently place it in an 18 C room.

5. Check the sample drops under a microscope at 20-100Â magnifications after 3 and 7 days (and if necessary, after 1 and 2 weeks, and 1, 3, and 6 months). 6 . A week later, we found crystal growth in the PEG/Ion, PEGRX and JCSG+ kits. Specific conditions were as follows (Fig. 4a,b) 2. The diffraction images should be collected on the BL17U beamline (Fig. 4c) . Rotate the mounted crystal and the X-ray diffraction patterns should be recorded at 1 per image, and collected for 360 .

3. The diffraction images in a dataset should be processed with HKL2000 [10] including auto-index, refinement, integration, and scaling steps. After data processing, the crystal unit cell parameters, crystal space group, Miller indexes of reflections, intensities, and error estimates of reflections should be determined and stored in a * .sca file, which provides the dataset applicable to structure determination.

4. Using CCP4 suite solve the structure as follows: Export the * . sca file to a * .mtz file using the program SCALPACK2MTZ. Use the * .mtz file to calculate the solvent via MATTHEWS_-COEF. Run with PHASER MR (see Note 12) with the MERS-CoV RBD structure (PDB ID: 4l72) and the structures of the variable domain of the heavy and light chains available in the PDB with the highest sequence identities as search models (see Note 12) [11] . When the phases are determined, the electron density map can be calculated, from which the molecular model can be constructed. 5. Subsequent model building and refinement were performed using COOT and PHENIX, respectively (see Note 13) [12, 13] .

6. Many validation programs are used to check the structure, until the investigator is satisfied, and then the structure can be deposited in the PDB.

1. Weigh 10 g tryptone, 5 g yeast extract, 10 g agar powder, and 10 g NaCl, and add ultrapure water to 1 L. After sterilization by high-pressure steam, wait until the temperature of the medium drops to about 60 C, add the required antibiotics, inducers etc. (50 μg/mL kanamycin,7 μg/mL gentamicin, 10 μg/mL tetracycline, 100 μg/mL x-gal, 40 μg/mL IPTG). Mix evenly and then pour into sterile plates. When the culture medium has cooled and solidified, store the bacmid selection LB agar plate at 4 C. 6. Endoglycosidase F1 and F3 are expressed and purified from E. coli by our laboratory. The endoglycosidase was added into the reaction system according to the mass ratio of 1:100.

7. The coding sequence of the MERS-CoV RBD (EMC strain, spike residues 367-588) was ligated into the pFastBac-Dual vector (Invitrogen) with a N-terminal gp67 signal peptide to enable the protein secreting outside the cell and a C-terminal His-tag to facilitate further purification processes.

8. Allow the pellet to dissolve for at least 10 min at room temperature. To avoid shearing the DNA, pipet only 1-2 times to resuspend. Store the bacmid at 4 C and use it as soon as possible, usually within 1 week. Aliquot the bacmid DNA into separate tubes and store at À20 C (not in a frost-free fridge). Avoid multiple freeze/thaw cycles as this decreases the transfection efficiency.

9. Characteristics of infected cells: A 25-50% increase in cell diameter can be seen and the size of cell nuclei increases at the early stage. Cells release from the plate and appear lysed, showing signs of clearing in the monolayer. 10 . P0 virus can be stored for years, adding 2% (v/v) FBS at 4 C, protected from light.

11. The expression of MERS-4scFv protein was conducted in 293F cells transiently transfected with plasmid DNA. After 72 h, the supernatant was collected and concentrated. The purified MERS-4scFv protein was obtained by Ni-NTA affinity chromatography and Superdex 200 size-exclusion chromatography. The purification method is the same as that of RBD protein.

12. If the crystal structure of the same protein or a similar protein has been solved, the molecular replacement method can be applied. After obtaining the solutions of the rotation and translation functions, initial phases can be calculated from the reference model, after which the electron density can be calculated.

13. The accuracy of the constructed model is confirmed by the crystallographic R-factor and R-free, which indicate the discrepancy between the calculated and observed amplitudes. The stereo-chemical parameters of the model can also be checked using programs such as MOLPROBITY, PROCHECK, or RAMPAGE.

Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26

Structural basis for the neutralization of MERS-CoV by a human monoclonal antibody MERS-27

Structural definition of a unique neutralization epitope on the receptorbinding domain of MERS-CoV spike glycoprotein

Ultrapotent human neutralizing antibody repertoires against Middle East respiratory syndrome coronavirus from a recovered patient

Evaluation of candidate vaccine approaches for MERS-CoV

Junctional and allelespecific residues are critical for MERS-CoV neutralization by an exceptionally potent germline-like antibody

Introduction of neutralizing immunogenicity index to the rational design of MERS coronavirus subunit vaccines

Importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on MERS-CoV spike to avoid neutralization escape

Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4

Processing of X-ray diffraction data collected in oscillation mode

Phaser crystallographic software

Coot: modelbuilding tools for molecular graphics

PHENIX: building new software for automated crystallographic structure determination