Computational protocol: A Structural Study of Norovirus 3C Protease Specificity: Binding of a Designed Active Site Directed Peptide Inhibitor†

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[…] For the initial crystallization studies, samples of protease that were stored in glycerol were exchanged using a Sephadex G25 column into 10 mM phosphate buffer, pH 7.45, containing 5 mM β-mercaptoethanol and then concentrated to 3 mg/mL. Crystals of the native enzyme were obtained by the vapor diffusion method in several conditions at room temperature using the Jena Biosciences classic screens, and following further screening the optimum conditions were found to be 7% PEG 8000 and 0.1 M HEPES, pH 7.5, with 8% (v/v) ethylene glycol. Preliminary data collection using station ID14-1 at the ESRF (Grenoble, France) with crystals frozen in 30% glycerol (added stepwise) revealed that they belonged to a hexagonal point group and produced diffraction data to medium resolution.Crystals of vastly improved diffraction quality were obtained by forming a complex of the protease with the irreversible inhibitor MAPI. To obtain these cocrystals, NaCl was added to the protein sample to a final concentration of 300 mM which allowed the protease to be concentrated to 17 mg/mL using a 10 kDa cutoff Centricon concentration vessel. To provide suitable conditions to form a complex of the protease with the essentially insoluble inhibitor, it was necessary to include 10% DMSO in the buffer. An amount of inhibitor giving a 3-fold molar excess over the protein was dissolved in a volume of DMSO that would, once added to the protein sample, result in the final buffer containing 10% DMSO. The inhibitor in DMSO was added to the protein sample in 10 equal volumes at 10 min intervals. The sample was then passed through a Sephadex G25 minispin column to rid the complex of any excess unbound inhibitor and DMSO. Incubation of a small sample of the complex with the chromogenic substrate Ac-EFQLQ-pNA demonstrated that 100% inhibition had been achieved. Further confirmation and accurate assessment of MAPI binding were accomplished by mass spectrometry which revealed a single major protein peak at 20045 Da corresponding to one molecule of SV3CP plus one molecule of the inhibitor. With the complex at a concentration of 15 mg/mL, large crystals were obtained in 25% (w/v) PEG 5000 MME, 100 mM Tris-HCl, pH 8.5, and 200 mM lithium sulfate (Jena Bioscience screen condition JB4 A1). Data collection at station ID23-1 (ESRF, Grenoble) established that the crystals diffracted to high resolution. Data processing in MOSFLM (), SCALA (), and other programs in the CCP4 suite () revealed that the crystals belonged to the space group P212121 with unit cell dimensions a = 49.5 Å, b = 84.1 Å, and c = 121.5 Å; the data set had an overall Rmerge of 5.3% to 1.7 Å resolution.Preparation of selenomethioninyl protease for experimental phasing involved the E. coli cells harboring the expression construct being grown at 37 °C to midlog phase in LB medium with 50 μg/mL ampicillin. The cells were then pelleted by centrifugation prior to resuspension in M9 minimal medium supplemented with 0.4% (w/v) glucose and ampicillin at the above concentration. They were then grown at 37 °C for 30 min to use up remaining amino acids prior to the addition of each of the 20 standard amino acids to final concentrations of 40 mg/L with the exception of methionine, which was replaced by l-selenomethionine at the same final concentration. The cultures were supplemented with the vitamins riboflavin, niacinamide, pyridoxine and thiamin, each at a final concentration of 1 mg/L, and were shaken at 37 °C for another 20 min. The cultures were then induced by addition of IPTG to a final concentration of 1 mM and grown for a further 3 h at 37 °C. Purification of the selenomethioninyl protease was performed as for the native enzyme and yielded approximately 12 mg/L of culture. Analysis of the protein by electrospray mass spectrometry showed that the molecular mass of the Se-Met enzyme was 19518 Da, indicating 100% occupancy of the five Se-Met residues per molecule. Crystals of the selenomethionyl protease complexed with the inhibitor were then grown and frozen as described above. [...] Multiwavelength anomalous diffraction data were collected on station ID23-1 at the ESRF (Grenoble, France) to 1.8 Å resolution using 1° oscillations and an exposure time of 1 s per image. The data at each wavelength were scaled and processed as above in space group P212121 (see Table for details). The Se sites and phases were determined using SOLVE (), which gave a mean figure of merit of 0.56, and density modification using DM () gave an excellent electron density map from which an initial model of the structure was obtained using MAID () and manual building using TURBO-FRODO (BioGraphics, Marseille). The model was refined with the 1.7 Å resolution “native” data set for the inhibitor complex using the programs SHELX () and CNS (). Validation tests were performed with MOLPROBITY (), and buried solvent-accessible areas were calculated using AreaIMol (). The final coordinates and structure factors have been deposited in the Protein Data Bank ( with accession code 2IPH. […]

Pipeline specifications

Software tools iMosflm, CCP4, SHELX, CNS, MolProbity
Databases wwPDB
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Homo sapiens
Diseases Gastroenteritis