Mapping the triphosphatase active site of baculovirus mRNA capping enzyme LEF4 and evidence for a two-metal mechanism

Abstract

The 464-amino acid baculovirus LEF4 protein is a bifunctional mRNA capping enzyme with triphosphatase and guanylyltransferase activities. The N-terminal half of LEF4 constitutes an autonomous triphosphatase catalytic domain. The LEF4 triphosphatase belongs to a family of metal-dependent phosphohydrolases, which includes the RNA triphosphatases of fungi, protozoa, Chlorella virus and poxviruses. The family is defined by two glutamate-containing motifs (A and C), which form a metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight stranded beta barrel (the so-called 'triphosphate tunnel'). Here we probed whether baculovirus LEF4 is a member of the tunnel subfamily, via mutational mapping of amino acids required for triphosphatase activity. We identified four new essential side chains in LEF4 via alanine scanning and illuminated structure-activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight five acidic and four basic amino acids that are likely to comprise the LEF4 triphosphatase active site (Glu9, Glu11, Arg51, Arg53, Glu97, Lys126, Arg179, Glu181 and Glu183). These nine essential residues are conserved in LEF4 orthologs from all strains of baculoviruses. We discerned no pattern of clustering of the catalytic residues of the baculovirus triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). However, there is similarity to the active site of vaccinia RNA triphosphatase. We infer that the baculovirus and poxvirus triphosphatases are a distinct lineage within the metal-dependent RNA triphosphatase family. Synergistic activation of the LEF4 triphosphatase by manganese and magnesium suggests a two-metal mechanism of gamma phosphate hydrolysis.

Figure 1

Conservation of primary structure among baculovirus RNA triphosphatases. The amino acid sequence of AcNPV LEF4 from amino acids 8–186 is aligned to the orthologs encoded by other baculoviruses: Orgyia pseudotsugata NPV, Epiphyas postvittana NPV, Heliocoverpa armigera NPV, Lymantria dispar NPV, Mamestra configurata NPV, Spodoptera litura NPV, Culex nigripalpus NPV, Xestia c-nigrum GV, Plutella xylostella GV, Cydia pomonella GV, Choristoneura fumiferana GV and Phthorimaea operculella GV. Positions of side chain identity or similarity in all the aligned baculovirus proteins are denoted by a circumflex accent below the alignment. The amino acids of AcNPV LEF4 that were newly mutated to alanine in the present study are indicated by a vertical line. Residues defined previously as essential for the triphosphatase activity of LEF4 are denoted by a dot; unimportant residues are indicated by +. The nine essential side chains proposed to comprise the triphosphatase active site are highlighted in shaded boxes. The metal-binding motifs A and C are depicted as probable β strands (horizontal arrows). Two other candidate β strands are indicated by arrows and ?.

Figure 2

Purification and guanylyltransferase activity of wild-type and mutant LEF4 proteins. (A) Purification. Aliquots (4 µg) of the nickel– agarose preparations of wild-type (WT) LEF4 and the indicated mutants were analyzed by SDS–PAGE. Polypeptides were visualized by staining with Coomassie blue dye. The positions and sizes (in kDa) of marker proteins are indicated on the left. The LEF4 polypeptide is denoted by an arrow on the right. (B) Guanylyltransferase activity. Reaction mixtures (10 µl) containing 50 mM Tris–HCl pH 8.0, 5 mM DTT, 20 mM MgCl₂, 2 mM [α-³²P]GTP, and 1 µg of WT or mutant LEF4 as specified were incubated for 15 min at 30°C. The reactions were quenched with SDS and the products were analyzed by SDS–PAGE. Autoradiographs of the dried gels are shown. The positions and sizes of marker polypeptides are indicated on the left. The LEF4–GMP complex is denoted by an arrow on the right.

Figure 3

Synergy between manganese and magnesium suggests two-metal catalysis. Reaction mixtures (10 µl) containing either 50 mM Tris–HCl (A) pH 7.5 or (B) pH 9.0, 1 mM [γ-³²P]ATP, MnCl₂ and/or MgCl₂ as specified, and either (A) 100 ng or (B) 20 ng of LEF4 were incubated for 15 min at 30°C. Each datum is the average of three experiments.

Figure 4

The triphosphate tunnel subfamily of metal-dependent RNA triphosphatases. The amino acid sequence of the catalytic domain of S.cerevisiae RNA triphosphatase Cet1 is aligned to the sequences of Candida albicans CaCet1, S.cerevisiae Cth1, Schizosaccharomyces pombe Pct1, Plasmodium falciparum Prt1, Chlorella virus cvRtp1, Encephalitozoon cuniculi Cet1, Trypanosoma brucei Cet1 and Leishmania major Cet1. Gaps in the alignment are indicated by dashes. Non-conserved inserts in PfPrt1 and LmCet1 are omitted from the alignment and are denoted by a triangle. The β strands that form the triphosphate tunnel of ScCet1 are denoted above the sequence. Peptide segments with the highest degree of conservation in all tunnel subfamily proteins are highlighted by the shaded boxes. Essential hydrophilic amino acids of the Cet1 active site are denoted by dots above the sequence.

Figure 5

Poxvirus RNA triphosphatases. The amino acid sequence of the capping enzyme large subunit of vaccinia virus from amino acid 30–199 is aligned to the related polypeptides encoded by other poxviruses: Yaba-like virus (ya); molluscum contagiosum virus (mc); lumpy skin disease virus (ls); swinepox virus (sp); fowlpox virus (fp); Amsacta moorei entomopoxvirus (Am); and Melanoplus sanguinipes entomopoxvirus (Ms). The nine essential side chains proposed to comprise the triphosphatase active site are highlighted in shaded boxes. The metal-binding motifs A and C are depicted as probable β strands (horizontal arrows). Two other candidate β strands are indicated by arrows and ?.