Effects of alanine cluster mutations in the D12 subunit of vaccinia virus mRNA (guanine-N7) methyltransferase.
The (guanine-N7)-methyltransferase domain of the vaccinia virus mRNA capping enzyme is a heterodimer composed of a catalytic subunit D1(498-844) bound to a stimulatory subunit D12. To identify structural elements of the 287-amino-acid D12 subunit that participate in binding and activation of the catalytic subunit, we introduced 12 double-alanine mutations at vicinal residues that are conserved in the D12 homologs of other vertebrate poxviruses. His-tagged D12 mutants were coexpressed in bacteria with the D1(498-544) subunit, and the recombinant D1(498-844)/His-D12 heterodimers were purified. Eight of the mutants (K111A-R112A, N120A-N121A, N126A-N127A, F141A-R142A, K223A-D224A, H260A-S261A, E275A-N276A, and R280A-R281A) had no significant effect on methyltransferase activity. Three of the mutants (L61A-K62A, F176A-K177A, and F245A-L246A) displayed an intermediate level of cap methylation (35-50% of wild-type activity). Only one mutation, N42A-Y43A, elicited a significant loss of the methyltransferase activation function (<20% of the wild-type activity). Nine of the D12-Ala/Ala proteins were produced individually in bacteria and tested for reconstitution of methyltransferase activity in vitro by mixing with the catalytic subunit. K111A-R112A, N120A-N121A, F176A-K177A, F245A-L246A, and L61A-K62A displayed diminished affinity for the D1 catalytic subunit. N42A-Y43A was uniquely defective in its ability to activate cap methylation by the catalytic subunit. Our results suggest that the methyltransferase activation function of D12, though clearly dependent on the physical interaction with D1, also requires constituents of D12 that are engaged specifically in catalysis.
Saha
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Molecular Biology Program, Sloan Kettering Institute, New York, New York 10021, USA.
Shuman
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D000409:Alanine / Q000235:genetics*; D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli; D008780:Methyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000235:genetics*; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics*; D011487:Protein Conformation; D012315:RNA Caps / Q000378:metabolism; D013329:Structure-Activity Relationship; D014764:Viral Proteins
D009097:Multienzyme Complexes; D012315:RNA Caps; D014764:Viral Proteins; D008780:Methyltransferases; D009713:Nucleotidyltransferases; D010744:Phosphoric Monoester Hydrolases; C027731:capping enzyme, vaccinia virus; D000409:Alanine