Mutational analysis of the SARS virus Nsp15 endoribonuclease: identification of residues affecting hexamer formation.
The severe acute respiratory syndrome (SARS) coronavirus virus non-structural protein 15 is a Mn2+-dependent endoribonuclease with specificity for cleavage at uridylate residues. To better understand structural and functional characteristics of Nsp15, 22 mutant versions of Nsp15 were produced in Escherichia coli as His-tagged proteins and purified by metal-affinity and ion-exchange chromatography. Nineteen of the mutants were soluble and were analyzed for enzymatic activity. Six mutants, including four at the putative active site, were significantly reduced in endoribonuclease activity. Two of the inactive mutants had unusual secondary structures compared to the wild-type protein, as measured by circular dichroism spectroscopy. Gel-filtration analysis, velocity sedimentation ultracentrifugation, and native gradient pore electrophoresis all showed that the wild-type protein exists in an equilibrium between hexamers and monomers in solution, with hexamers dominating at micromolar protein concentration, while native gradient pore electrophoresis also revealed the presence of trimers. A mutant in the N terminus of Nsp15 was impaired in hexamer formation and had low endoribonuclease activity, suggesting that oligomerization is required for endoribonuclease activity. This idea was supported by titration experiments showing that enzyme activity was strongly concentration-dependent, indicating that oligomeric Nsp15 is the active form. Three-dimensional reconstruction of negatively stained single particles of Nsp15 viewed by transmission electron microscopic analysis suggested that the six subunits were arranged as a dimer of trimers with a number of cavities or channels that may constitute RNA binding sites.
Guarino
Linda A
LA
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA. lguarino@tamu.edu
Bhardwaj
Kanchan
K
Dong
Wen
W
Sun
Jingchuan
J
Holzenburg
Andreas
A
Kao
Cheng
C
D000596:Amino Acids; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D019281:Dimerization; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D046529:Microscopy, Electron, Transmission; D017433:Protein Structure, Secondary; D045473:Severe acute respiratory syndrome-related coronavirus / Q000737:chemistry*; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D017361:Viral Nonstructural Proteins; D004722:Endoribonucleases