mRNA Cap Formation -- Structure and Mechanism of Yeast RNA Triphosphatase

RNA triphosphatase is an essential mRNA processing enzyme that catalyzes the first step in mRNA cap formation. The RNA triphosphatases are not conserved among eukarya and fall into at least 2 mechanistically and structurally distinct families:

• the divalent cation-dependent RNA triphosphatases of DNA viruses and fungi, and
• the divalent cation-independent RNA triphosphatases of nematodes, mammals, and other metazoa.

The metazoan triphosphatases display extensive amino acid sequence similarity to protein tyrosine phosphatases and, by analogy to the protein tyrosine phosphatases, it is proposed that they execute a 2-step phosphoryl transfer reaction involving a covalent enzyme-(cysteinyl-S)-phosphate intermediate.

Saccharomyces cerevisiae Cet1p exemplifies the class of divalent cation-dependent RNA triphosphatase enzymes. Other family members include the RNA triphosphatases encoded by pathogenic fungi (e.g., Candida albicans) and viruses (e.g., vaccinia, smallpox, and molluscum contagiosum). This triphosphatase family is defined by 3 conserved collinear motifs (A, B, and C) that include clusters of acidic and basic amino acids essential for catalytic activity.

The 549-amino acid Cet1p protein contains 3 domains: a 230-amino acid N-terminal segment that makes no discernible contribution to catalysis and is dispensable for Cet1p function in vivo; a protease-sensitive segment from residues 230 to 275 that is essential for Cet1p function in vivo and which mediates both Cet1p self-association and Cet1p binding to the yeast guanylyltransferase Ceg1p; and a catalytic domain from residues 275 to 539 that includes motifs A, B, and C. The amino acid sequence and domain organization of the C. albicans RNA triphosphatase CaCet1p is similar to that of the S. cerevisiae protein.

RNA triphosphatase is an attractive target for antifungal drug development because:

Thus, an inhibitor of fungal RNA triphosphatase should, in principle, have selectivity for the pathogen and minimal effect on the human host.

To facilitate mechanistic and pharmacological studies of the metal-dependent RNA triphosphatases, we have crystallized a biologically active form of yeast Cet1p and determined its structure at 2.05 Å resolution. The structure reveals the architecture of the active site, the Cet1p-Cet1p dimer interface, and the surface peptide domain responsible for Cet1p binding to the yeast guanylyltransferase. The catalytic domain adopts a novel enzyme fold in which an antiparallel 8-strand beta barrel forms a hydrophilic "triphosphate tunnel" in which motifs A and C comprise the metal-binding site. This is the first structure of an mRNA-specific processing enzyme from a eukaryotic cellular source.

Consistent with solution studies, Cet1(241-539)p crystallized as a dimer. The striking feature of the tertiary structure is the formation of a topologically closed tunnel composed of 8 antiparallel beta strands. In the dimer, the 2 tunnels are parallel and oriented in the same direction, i.e., the tunnel "entrances" are on the same face of the dimer.

[Enlarge Image] Surface view of the RNA triphosphatase monomer highlighting conserved residues. (Left) A view looking into the tunnel entrance. (Middle) Side view after a 90° rotation to the right. (Right) A view looking into the tunnel exit. Positions of side chain identity or similarity among fungal RNA triphosphatases are colored in blue.

A surface view of the monomer is shown looking into the tunnel entrance. Rotation of the molecule rightward and leftward about the vertical axis into the plane of the page provides a side view that highlights a platform-like structure in front of the tunnel entrance. A view into the back end of the tunnel is also shown.

Multiple acidic side chains point into the tunnel cavity, including Glu305 and Glu307 in motif A and Glu492, Glu494, and Glu496 in motif C, each of which is essential for triphosphatase activity. The interior of the tunnel contains a single sulfate ion coordinated by the side chains of Arg393, Lys409, Lys456, and Arg458. Insofar as sulfate is a structural analog of phosphate, we posit that the side chain interactions of the sulfate reflect contacts made by the enzyme with the gamma phosphate of the triphosphate-terminated RNA and nucleoside triphosphate substrates. Mutational studies have shown that Lys456, which contacts the sulfate, is important for Cet1p function in vivo and in vitro. Changing Lys456 to alanine or glutamine increases the Km for ATP by an order of magnitude; ATP-binding is restored when arginine is introduced at this position.

	[Enlarge Image] View of a cross section of the triphosphatase tunnel highlighting the network of side-chain interactions that coordinate the sulfate and manganese ions. The manganese (blue sphere) interacts with octahedral geometry with the sulfate, Glu305, Glu307, Glu496, and 2 waters (red spheres).

The location of a metal-binding site on the enzyme was determined by x-ray diffraction of Cet1(241-539)p crystals that had been soaked in manganese chloride. New density corresponding to a manganese ion was discerned within the tunnel cavity. The manganese is coordinated with octahedral geometry to the sulfate, to the side chain carboxylates of essential residues Glu305, Glu307, and Glu496, and to 2 waters. The sulfate is apical to Glu307. Glu496 is apical to a water that is coordinated by Glu307. Glu305 is apical to another water that is coordinated by Asp471 and Glu494. The 3 glutamates that comprise the metal-binding site of yeast RNA triphosphatase are located in motifs A and C, which define the metal-dependent RNA triphosphatase family. Substitution of Glu305, Glu307, or Glu496 by alanine, glutamine, or aspartate inactivates Cet1p. These mutational effects, implying that both the negative charge and the distance of the carboxylate from the main chain are critical for catalysis, are in keeping with the direct contact of these 3 glutamates to the divalent cation observed in the manganese-soaked Cet1(241-539)p crystal. The motif A and C glutamates are also essential for the activities of vaccinia virus RNA triphosphatase, baculovirus RNA triphosphatase, and Candida albicans RNA triphosphatase. Thus, it is likely that motifs A and C comprise the metal binding site in all members of this enzyme family.

The structure of yeast triphosphatase with bound sulfate and manganese is construed to reflect that of the product complex of enzyme with the hydrolyzed gamma phosphate. The structure suggests a catalytic mechanism whereby acidic side chains located on the floor of the tunnel coordinate an essential divalent cation that, in turn, coordinates the gamma phosphate. The metal ion would activate the gamma phosphorus for attack by water and stabilize a pentacoordinate phosphorane transition state in which the attacking water is apical to the beta-phosphate leaving group. Interactions between the sulfate and basic side chains Arg393, Arg458, Lys409, and Lys456 located on the walls and roof of the tunnel would contribute to the coordination of the gamma phosphate in the ground state and the stabilization of the negative charge on the gamma phosphate developed in the transition state. These interactions are illustrated above.