Crystal Structure of the C-Terminal Cytoplasmic Domain of Non-Structural Protein 4 from Mouse Hepatitis Virus A59

Xu, Xiaoling; Lou, Zhiyong; Ma, Yanlin; Chen, Xuehui; Yang, Zhangsheng; Tong, Xiaohang; Zhao, Qi; Xu, Yuanyuan; Deng, Hongyu; Bartlam, Mark; Rao, Zihe

The dimerization of nsp4C is also detected by gel filtration (Fig. 4A) . Elution of purified WT nsp4C protein through a Superdex75 column in buffer containing 50 mM Tris-Cl, pH 8.5 and 300 mM NaCl yields two distinct 280 nm absorption peaks: the first peak appears at 13.1 ml and the second at 14.67 ml. According to the profiles of standard marker proteins such as aprotinin (MW: 6,512 Da), RNaseA (MW: 13,700 Da), albumin egg (MW: 45,000 Da) and BSA (MW: 67,000 Da) on the same column and under the same buffer conditions, these two peaks correspond to the dimer and monomer of nsp4C, respectively. While the nsp4C sample in the presence of the reducing agent DTT exhibits a different elution profile under the same conditions, it yields only one 280 nm absorption peak at 14.63 ml, which corresponds to the nsp4C monomer. Furthermore, the elution profile of the C425S point mutant was identical to the profile of nsp4C with DTT, with only a single absorption peak appearing at 14.64 ml and corresponding to the monomer. The reducing agent b-ME also works to reduce this disulfide bond. The dimer exists even in SDS-PAGE analysis under non-reducing conditions (data not shown).