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

Topology studies of MHV nsp4 indicate that the N-terminal of nsp4C is connected to the ER membrane by about 8 amino acids, Figure 3 . The molecular surface model of the monomer from nsp4C (T408-Q496) WT dimer and mutant C425S. Electrostatic potential is mapped on the surface, with positive charged region colored in blue and negative charged region in red. Both molecules in one asymmetric unit of the WT nsp4C dimer and mutant C425S are shown in three orientations. Fig. 3A and 3B represents molecule A and molecule B of the WT nsp4C dimer respectively; while Fig. 3C represents the monomer of the C425S mutant. Fig. 3D represents the superposition of molecule A (gold) and B (magenta) from WT and the monomer of the C425S mutant (green), Cys425 is shown in stick representation, with the carbon, nitrogen, oxygen, and sulfur atoms colored yellow, blue, red, and green, respectively; and also the crystal packing of WT nsp4C (magenta), symmetry-related molecules are colored in green, and the molecule forming the equivalent of the C425S mutant dimer is colored in blue. Selected amino acids are labeled, and the figure is drawn by PyMol [47] . doi:10.1371/journal.pone.0006217.g003 Residues highlighted in red are identical among the compared proteins; residues highlighted in yellow are conserved. The alignment was generated using the program ClustalX [48] and drawn with ESPript [49] . doi:10.1371/journal.pone.0006217.g004 -TEVRSDG-, when nsp4 is initially processed from the p150 precursor; the region spanning residues T408 to Q496 is located in the cellular cytosol. The cytosol has long been regarded as a reducing cellular compartment in which the reduced state of cysteine in proteins is largely favored; the disulfide bond is generally formed in the more oxidizing ER lumen rather than the reducing cytosol [35, 36] . Furthermore, our results confirm that the nsp4C dimer can readily switch to a monomer in a reducing environment, so it is evident that nsp4C may exist as monomer in the reducing environment of the cytosol when it is expressed alone in uninfected cells. Our crystal structure further revealed that the N-termini of the two monomers in the WT nsp4C dimer are not suitably aligned to bind to the ER membrane. And also the dimer is formed in a non-reducing and highly condensed protein solution, and is therefore not representative of the physiological state. Moreover, from the multiple sequence alignment in Fig. 4B , the Cys425 is not a conserved amino acids among the coronavirus nsp4 proteins, thus the dimer is a specific case to MHV nsp4C. Mutation of Cys425 to Ser prevents formation of the disulfide bond, which does not influence the conformation of monomer, so the conformation of monomer is stable and conserved. Thus, the monomer of nsp4C may be physiologically functional when it is bound to the ER membrane in the cytosol.