Stewart Shuman: mRNA Cap Formation - Structure and Mechanism of Yeast RNA Triphosphatase

RNA Triphosphatase Comes in Two Flavors

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 a Promising Antifungal Target

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.

Crystal Structure of Yeast RNA Triphosphatase

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.

Overall fold of the RNA triphosphatase dimer (PDB ID code: 1D8I). Overall fold of the RNA triphosphatase dimer (PDB ID code: 1D8I). One monomer is colored red; the dimer partner is colored blue. (Top) A view of the dimer looking into the tunnel entrance. (Bottom) A view of the dimer looking at the walls of the tunnel.

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.

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.

The Active Site of Yeast RNA Triphosphatase Is within the Tunnel

Surface view of the RNA triphosphatase monomer highlighting conserved residues. 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.

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.

Divalent Cation Binding Site

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. View of a cross section of the triphosphatase tunnel highlighting the network of side-chain interactions that coordinate the sulfate and manganese ions. 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).

Catalytic Mechanism

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.

Pei Y, Hausmann S, Ho CK, Schwer B, Shuman S. 1999 Distinct roles for CTD Ser2 and Ser5 phosphorylation in the recruitment and allosteric activation of mammalian capping enzyme. J Biol Chem. 2001;276:28075-28082.

Schwer B, Saha N, Mao X, Chen HW, Shuman S. 2000 Structure-function analysis of yeast mRNA cap methyltransferase and high-copy suppression of conditional mutants by AdoMet synthase and the ubiquitin conjugating enzyme Cdc34p. Genetics. 2000;155;1561-1576.

Shuman S. 1997 Origins of mRNA identity: capping enzymes bind to the phosphorylated C-terminal domain of RNA polymerase II. Proc Natl Acad Sci USA. 1997;94:12758-12760.

Schwer B, Linder P, Shuman S. (1998) Effects of deletion mutations in the yeast Ces1 protein on cell growth and morphology and on high copy suppresion of mutations in mRNA capping enzyme and translation initiation factor 4A. Nucleic Acids Res. 1998;26:803-809.

Ho CK, Sriskanda V, McCracken S, Bentley D, Schwer B, Shuman S. (1998) The guanylyltransferase domain of mammalian mRNA capping enzyme binds to the phosphorylated carboxyl-terminal domain of RNA polymerase II. J Biol Chem. 1998;273:9577-9585.

Schwer B, Mao X, Shuman S. (1998) Accelerated mRNA decay in conditional mutants of yeast mRNA capping enzyme. Nucleic Acids Res. 1998;26:2050-2057.

Gross CH, Shuman S. (1998) Characterization of a baculovirus-encoded RNA 5'-triphosphatase. J Virol. 1998;72:7057-7063.

Gross CH, Shuman S. (1998) RNA 5'-triphosphatase, nucleoside triphosphatase, and guanylyltransferase activities of baculovirus LEF-4 protein. J Virol. 1998;72:10020-10028.

Yu L, Martins A, Deng L, Shuman S. (1997) Structure-function analysis of the triphosphatase component of vaccinia virus mRNA capping enzyme. J Virol. 1997;71:9837-9843.

Ho CK, Pei Y, Shuman S. (1998) Yeast and viral RNA 5' triphosphatases comprise a new nucleoside triphosphatase family. J Biol Chem. 1998;273:34151-34156.

Wang SP, Shuman S. (1997) Structure-function analysis of the mRNA cap methyltransferase of Saccharomyces cerevisiae. J Biol Chem. 1997;272:14683-14689.

Ho CK, Lehman K, Shuman S. (1999) An essential surface motif (WAQKW) of yeast RNA triphosphatase mediates formation of the mRNA capping enzyme complex with RNA guanylyltransferase. Nucleic Acids Res. 1999;27:4671-4678.

Saha N, Schwer B, Shuman S. (1999) Characterization of human, Schizosaccharomyces pombe and Candida albicans mRNA cap methyltransferases and complete replacement of the yeast capping apparatus by mammalian enzymes. J Biol Chem. 1999;274:16553-16552.

Lehman K, Schwer B, Ho CK, Rouzankina I, Shuman S. (1999) A conserved domain of yeast RNA triphosphatase flanking the catalytic core regulates self-association and interaction with the guanylyltransferase component of the mRNA capping apparatus. J Biol Chem. 1999;274:22668-22678.

Pei Y, Ho CK, Schwer B, Shuman S. (1999) Mutational analyses of yeast RNA triphosphatases highlight a common mechanism of metal-dependent NTP hydrolysis and a means of targeting enzymes to pre-mRNAs in vivo by fusion to the guanylyltransferase component of the capping apparatus. J Biol Chem. 1999;274:28865-28874.

Lima CD, Wang LK, Shuman S. (1999) Structure and mechanism of yeast RNA triphosphatase: an essential component of the mRNA capping apparatus. Cell. 1999;99:533-543.

Pei Y, Lehman K, Tian L, Shuman S. (2000) Characterization of Candida albicans RNA triphosphatase and mutational analysis of its active site. Nucleic Acids Res. 2000;28:1885-18892.

Ho CK, Martins A, Shuman S. (2000) A yeast-based genetic system for functional analysis of viral mRNA capping enzymes. J Virol. 2000;74:5486-5494.

Ho CK, Schwer B, Shuman S. (1998) Genetic, physical, and functional interactions between the triphosphatase and guanylyltransferase components of the yeast mRNA capping apparatus. Mol Cell Biol. 1998;18:5189-5198.

Schwer B, Shuman S. (1996) Multicopy suppressors of temperature-sensitive mutations of yeast mRNA capping enzyme. Gene Expr. 1996;5:331-344.

Cong P, Shuman S. (1993) Covalent catalysis in nucleotidyl transfer: a KTDG motif essential for enzyme-GMP complex formation by mRNA capping enzyme is conserved at the active sites of RNA and DNA ligases. J Biol Chem. 1993;268:7256-7260.

Cong P, Shuman S. (1993) RNA binding properties of vaccinia virus capping enzyme. J Biol Chem. 1993;268:7256-7260.

Schwer B, Shuman S. (1994) Mutational analysis of yeast mRNA capping enzyme. Proc Natl Acad Sci USA. 1994;91:4328-4332.

Mao X, Shuman S. (1994) Intrinsic RNA (guanine-7) methyltransferase activity of the vaccinia virus capping enzyme D1 subunit is stimulated by the D12 subunit: Identification of amino acid residues in the D1 protein required for subunit association and methyl group transfer. J Biol Chem. 1994;269:24472-24479.

Shuman S, Liu Y, Schwer B. (1994) Covalent catalysis in nucleotidyl transfer reactions: essential motifs in S. cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases. Proc Natl Acad Sci USA. 1994;91:12046-12050.

Shuman S, Schwer B. (1995) RNA capping enzyme and DNA ligase - a superfamily of covalent nucleotidyl transferases. Mol Microbiol. 1995;17:405-410.

Mao X, Schwer B, Shuman S. (1995) Yeast mRNA cap methyltransferase is a 50-kDa protein encoded by an essential gene. Mol Cell Biol. 1995;15:4167-4174.

McCracken S, Fong N, Rosonina E, Yankulov K, Brothers G, Siderovski D, Hessel A, Foster S, Shuman S, Bentley DL. (1997) 5'-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II. Genes Dev. 1997;11:3306-3318.

Mao X, Schwer B, Shuman S. (1996) Mutational analysis of the Saccharomyces cerevisiae ABD1 gene: cap methyltransferase activity is essential for cell growth. Mol Cell Biol. 1996;16:475-480.

Hagler J, Shuman S. (1992) A freeze-frame view of eukaryotic transcription during elongation and capping of nascent mRNA. Science. 1992;255:983-986.

Schwer B, Shuman S. (1996) Conditional inactivation of mRNA capping enzyme affects yeast pre-mRNA splicing in vivo. RNA. 1996;2:574-583.

Yu L, Shuman S. (1996) Mutational analysis of the triphosphatase domain of vaccinia virus mRNA capping enzyme. J Virol. 1996;70:6162-6168.

Ho CK, Van Etten JL, Shuman S. (1996) Expression and characterization of an RNA capping enzyme encoded by Chlorella virus PBCV-1. J Virol. 1996;70:6658-6664.

Mao X, Shuman S. (1996) Vaccinia virus mRNA (guanine-7-)methyltransferase: mutational effects on cap methylation and AdoHcy-dependent photo-cross-linking of the cap to the methyl acceptor site. Biochemistry. 1996;35:6900-6910.

Shuman S. (1997) A proposed mechanism of mRNA synthesis and capping by vesicular stomatitis virus. Virology. 1997;227:1-6.

Hakansson K, Doherty AJ, Shuman S, Wigley DB. (1997) X-ray crystallography reveals a large conformational change during guanyl transfer by mRNA capping enzymes. Cell. 1997;89:545-553.

Wang SP, Deng L, Ho CK, Shuman S. (1997) Phylogeny of mRNA capping enzymes. Proc Natl Acad Sci USA. 1997;94:9573-9578.

Cong P, Shuman S. (1995) Mutational analysis of mRNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate complex formation, and GMP transfer to RNA. Mol Cell Biol. 1995;15:6222-6231.