Mutational Analysis of Encephalitozoon cuniculi mRNA Cap (Guanine-N7) Methyltransferase, Structure of the Enzyme Bound to Sinefungin, and Evidence That Cap Methyltransferase Is the Target of Sinefungin's Antifungal Activity

Abstract

Cap (guanine-N7) methylation is an essential step in eukaryal mRNA synthesis and a potential target for antiviral, antifungal, and antiprotozoal drug discovery. Previous mutational and structural analyses of Encephalitozoon cuniculi Ecm1, a prototypal cellular cap methyltransferase, identified amino acids required for cap methylation in vivo, but also underscored the nonessentiality of many side chains that contact the cap and AdoMet substrates. Here we tested new mutations in residues that comprise the guaninebinding pocket, alone and in combination. The outcomes indicate that the shape of the guanine binding pocket is more crucial than particular base edge interactions, and they highlight the contributions of the aliphatic carbons of Phe-141 and Tyr-145 that engage in multiple van der Waals contacts with guanosine and S-adenosylmethionine (AdoMet), respectively. We purified 45 Ecm1 mutant proteins and assayed them for methylation of GpppA in vitro. Of the 21 mutations that resulted in unconditional lethality in vivo, 14 reduced activity in vitro to <2% of the wild-type level and 5 reduced methyltransferase activity to between 4 and 9% of wild-type Ecm1. The natural product antibiotic sinefungin is an AdoMet analog that inhibits Ecm1 with modest potency. The crystal structure of an Ecm1-sinefungin binary complex reveals sinefungin-specific polar contacts with main-chain and side-chain atoms that can explain the 3-fold higher affinity of Ecm1 for sinefungin versus AdoMet or S-adenosylhomocysteine (AdoHcy). In contrast, sinefungin is an extremely potent inhibitor of the yeast cap methyltransferase Abd1, to which sinefungin binds 900-fold more avidly than AdoHcy or AdoMet. We find that the sensitivity of Saccharomyces cerevisiae to growth inhibition by sinefungin is diminished when Abd1 is overexpressed. These results highlight cap methylation as a principal target of the antifungal activity of sinefungin.The m 7 GpppN cap structure of eukaryotic messenger RNA is formed by three enzymes. RNA triphosphatase hydrolyzes the 5Ј-triphosphate end of the pre-mRNA to a diphosphate, which is capped with GMP by RNA guanylyltransferase. RNA (guanine-N7) methyltransferase then adds a methyl group from AdoMet 2 to GpppRNA to form m 7 GpppRNA and AdoHcy. This pathway is conserved in all eukaryotic organisms and many eukaryotic viruses (1). The capping enzymes are considered attractive targets for antiviral, antifungal, and antiprotozoal drug discovery (2). Inhibition of cap methylation, in particular, has been touted as an anti-infective strategy based on two lines of evidence: (i) raising the cellular levels of AdoHcy by genetic or pharmacological inhibition of AdoHcy hydrolase blocks replication of many viruses (3, 4), and (ii) the AdoMet analog sinefungin (an inhibitor of cap methylation in vitro) inhibits the growth of diverse viruses, fungi, and protozoan parasites (5-11). Indeed, it was shown recently that sinefungin displays selectivity in inhibiting yeast cap methyltransferases versus the human enzyme in vivo (12).The S...

Structure–function analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase

“…To answer these questions, we performed a new round of mutational analysis of the D1-C protein, targeting 21 positions for single-alanine scanning. The choice of residues to mutate was guided by a primary structure alignment of D1-C and Ecm1 and the available Ecm1 structures (Fabrega et al 2004;Hausmann et al 2005;Zheng et al 2006). The targeted residues include: (1) the vaccinia equivalents of the Ecm1 amino acids that contact AdoMet or GTP in the Ecm1 cocrystals; (2) other conserved residues that are predicted to lie on the enzyme surface; and (3) residues unique to the poxvirus protein that are located within putative surface loops and, as such, were candidates to interact with the D12 subunit or mediate D12's activation of D1-C.…”

“…Several lines of evidence point to viral and fungal cap methyltransferases as the targets of sinefungin (Pugh et al 1978;Chrebet et al 2005;Zheng et al 2006Zheng et al , 2007Li et al 2007), a natural product analog of AdoMet with broad anti-infective activities. Poxvirus and yeast cap methyltransferases are exquisitely sensitive to inhibition by sinefungin in vitro, whereas the cap methyltransferase from the microsporidian parasite Encephalitozoon cuniculi is comparatively resistant (Pugh et al 1978;Hausmann et al 2005;Zheng et al 2006;S. Hausmann and S. Shuman, unpubl.).…”

“…A proposed catalytic strategy emphasizes the role of the enzyme in approximating the reactants and orienting the cap guanine-N7 nucleophile for in-line attack on the S-CH 3 of AdoMet (Fabrega et al 2004). The essential functional groups in the methyl donor and methyl acceptor sites have been defined through extensive mutational analyses of Ecm1 and the homologous Saccharomyces cerevisiae cap methyltransferase Abd1 Wang and Shuman 1997;Schwer et al 2000;Fabrega et al 2004;Hausmann et al 2005;Zheng et al 2006).…”

Mutational analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase reveals essential contributions of the N-terminal peptide that closes over the active site

“…Arg632 is conserved among poxvirus and cellular cap methyltransferases and is essential in all cases where its role has been tested (Wang and Shuman 1997;Saha et al 1999;Hausmann et al 2005;Zheng et al 2006). Because the segment corresponding to the D1 lid peptide is disordered in the crystal structures of the E. cuniculi and human cap methyltransferases (Fabrega et al 2004;Wu et al 2007), it is not clear whether the cellular homologs share the three-arginine binding mode for the cap triphosphate bridge.…”

“…The essential functional groups in the methyl donor and methyl acceptor sites have been defined through extensive mutational analyses of Ecm1, the homologous Saccharomyces cerevisiae cap methyltransferase Abd1, and the vaccinia virus cap methyltransferase Wang and Shuman 1997;Schwer et al 2000;Fabrega et al 2004;Hausmann et al 2005;Zheng et al 2006;Zheng and Shuman 2008). Structure-function analysis of the vaccinia protein has been aided by a yeast-based genetic assay in which cell growth depends on catalysis of cap synthesis by the viral enzyme acting in lieu of yeast Abd1 (Saha et al 2003).…”