Identification of a Ca2+-binding domain in the rubella virus nonstructural protease.
The rubella virus (RUB) nonstructural protein (NS) open reading frame (ORF) encodes a polypeptide precursor that is proteolytically self cleaved into two replicase components involved in viral RNA replication. A putative EF-hand Ca(2+)-binding motif that was conserved across different genotypes of RUB was predicted within the nonstructural protease that cleaves the precursor by using bioinformatics tools. To probe the metal-binding properties of this motif, we used an established grafting approach and engineered the 12-residue Ca(2+)-coordinating loop into a non-Ca(2+)-binding scaffold protein, CD2. The grafted EF-loop bound to Ca(2+) and its trivalent analogs Tb(3+) and La(3+) with K(d)s of 214, 47, and 14 microM, respectively. Mutations (D1210A and D1217A) of two of the potential Ca(2+)-coordinating ligands in the EF-loop led to the elimination of Tb(3+) binding. Inductive coupled plasma mass spectrometry was used to confirm the presence of Ca(2+) ([Ca(2+)]/[protein] = 0.7 +/- 0.2) in an NS protease minimal metal-binding domain, RUBCa, that spans the EF-hand motif. Conformational studies on RUBCa revealed that Ca(2+) binding induced local conformational changes and increased thermal stability (Delta T(m) = 4.1 degrees C). The infectivity of an RUB infectious cDNA clone containing the mutations D1210A/D1217A was decreased by approximately 20-fold in comparison to the wild-type (wt) clone, and these mutations rapidly reverted to the wt sequence. The NS protease containing these mutations was less efficient at precursor cleavage than the wt NS protease at 35 degrees C, and the mutant NS protease was temperature sensitive at 39 degrees C, confirming that the Ca(2+)-binding loop played a structural role in the NS protease and was specifically required for optimal stability under physiological conditions.
Zhou
Yubin
Y
Department of Chemistry, Georgia State University, 50 Decatur St., Atlanta, GA 30303, USA.
Tzeng
Wen-Pin
WP
Yang
Wei
W
Zhou
Yumei
Y
Ye
Yiming
Y
Lee
Hsiau-wei
HW
Frey
Teryl K
TK
Yang
Jenny
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D018076:DNA, Complementary; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism*; D012412:Rubella virus / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D018076:DNA, Complementary; D010447:Peptide Hydrolases; D002118:Calcium