Crystallization and preliminary X-ray diffraction analysis of the N-terminal domain of human coronavirus OC43 nucleocapsid protein

Abstract

The N-terminal domain of nucleocapsid protein from human coronavirus OC43 (HCoV-OC43 N-NTD) mostly contains positively charged residues and has been identified as being responsible for RNA binding during ribonucleocapsid formation in the coronavirus. In this study, the crystallization and preliminary crystallographic analysis of HCoV-OC43 N-NTD (amino acids 58-195) with a molecular weight of 20 kDa are reported. HCoV-OC43 N-NTD was crystallized at 293 K using PEG 1500 as a precipitant and a 99.9% complete native data set was collected to 1.7 A resolution at 100 K with an overall R(merge) of 5.0%. The crystals belonged to the hexagonal space group P6(5), with unit-cell parameters a = 81.57, c = 42.87 A. Solvent-content calculations suggest that there is likely to be one subunit of N-NTD in the asymmetric unit.

Figure 1

SDS–PAGE analysis of HCoV-OC43 N-NTD stained with Coomassie Brilliant Blue. Lane M, protein markers (kDa); lane 1, purified HCoV-OC43 N-NTD; lane 2, concentrated HCoV-OC43 N-NTD after dialysis.

Figure 2

Crystals of HCoV-OC43 N-NTD obtained with 25% PEG 1500 as a precipitant at pH 6.0 by the sitting-drop vapour-diffusion method. The approximate dimensions of the crystal are 200 × 100 × 100 µm.

Figure 3

Typical X-ray diffraction pattern of HCoV-OC43 N-NTD. The arrow shows the data at the resolution limit (1.7 Å).

Figure 4

Multiple sequence alignment of CoV N-NTDs; T-Coffee (Notredame et al., 2000 ▶) was used to define conserved residues. The numbering above the sequence is for the amino-acid sequence from OC43. Conserved residues are shaded. Fully conserved residues are shaded red. Residues that were partially conserved at levels of 75% and 50% are shaded orange and yellow, respectively. The amino-acid sequences of OC43 (HCoV-OC43; NP_937954), SARS (SARS-CoV; ABI96968), 229E (HCoV-229E; AAG48597) and IBV (infectious bronchitis virus; AAB24054) N-NTD were obtained from GenBank.