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• W2012291245 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 guanine-binding 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. 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 guanine-binding 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 m7GpppN cap structure of eukaryotic messenger RNA is formed by three enzymes. RNA triphosphatase hydrolyzes the 5′-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 2The abbreviations used are: AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; DTT, dithiothreitol. 2The abbreviations used are: AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; DTT, dithiothreitol. to GpppRNA to form m7GpppRNA and AdoHcy. This pathway is conserved in all eukaryotic organisms and many eukaryotic viruses (1Shuman S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 619-625Crossref PubMed Scopus (110) Google Scholar). The capping enzymes are considered attractive targets for antiviral, antifungal, and antiprotozoal drug discovery (2Shuman S. Cold Spring Harbor Symp. Quant. Biol. 2001; 66: 301-312Crossref PubMed Scopus (36) Google Scholar). 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 (3Liu S. Wolfe M.S. Borchardt R.T. Antiviral Res. 1992; 19: 247-265Crossref PubMed Scopus (87) Google Scholar, 4DeClercq E. Nucleosides Nucleotides. 2005; 24: 1295-1415Google Scholar), and (ii) the AdoMet analog sinefungin (an inhibitor of cap methylation in vitro) inhibits the growth of diverse viruses, fungi, and protozoan parasites (5Hamill R.L. Hoehn M.M. J. Antibiot. 1973; 26: 463-465Crossref PubMed Scopus (107) Google Scholar, 6Gordee R.S. Butler T.F. J. Antibiot. 1973; 26: 466-470Crossref PubMed Scopus (39) Google Scholar, 7Pugh C.S.G. Borchardt R.T. Stone H.O. J. Biol. Chem. 1978; 253: 2075-2077Abstract Full Text PDF Google Scholar, 8Dube D.K. Mpimbaza G. Allison A.C. Lederer E. Rovis L. Am. J. Trop. Med. Hyg. 1983; 32: 31-33Crossref PubMed Scopus (41) Google Scholar, 9Ferrante A. Ljungstron I. Huldt G. Lederer E. Trans. R. Soc. Trop. Med. Hyg. 1984; 78: 837-838Abstract Full Text PDF PubMed Scopus (17) Google Scholar, 10Messika E. Golenser J. Abu-Elheiga L. Robert-Gero M. Lederer E. Bachrach U. Trop. Med. Parasitol. 1990; 41: 273-278PubMed Google Scholar, 11Brassuer P. Lemetheil D. Ballet J.J. Antimicrob. Agents Chemother. 1993; 37: 889-892Crossref PubMed Scopus (14) Google Scholar). Indeed, it was shown recently that sinefungin displays selectivity in inhibiting yeast cap methyltransferases versus the human enzyme in vivo (12Chrebet G.L. Wisniewski D. Perkins A.L. Deng Q. Kurtz M.B. Marcy A. Parent S.A. J. Biomol. Screen. 2005; 10: 355-364Crossref PubMed Scopus (22) Google Scholar).The Saccharomyces cerevisiae cap methyltransferase Abd1 has been extensively characterized genetically, but biochemical and structural analyses of the yeast enzyme are not as far advanced (13Mao X. Schwer B. Shuman S. Mol. Cell. Biol. 1995; 15: 4167-4174Crossref PubMed Scopus (90) Google Scholar, 14Mao X. Schwer B. Shuman S. Mol. Cell. Biol. 1996; 16: 475-480Crossref PubMed Scopus (60) Google Scholar, 15Wang S.P. Shuman S. J. Biol. Chem. 1997; 272: 14683-14689Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 16Schwer B. Saha N. Mao X. Chen H.W. Shuman S. Genetics. 2000; 155: 1561-1576Crossref PubMed Google Scholar). Cellular cap methyltransferases from humans, Xenopus laevis, Candida albicans, Schizosaccharomyces pombe, and Trypanosoma brucei have also been characterized (17Saha N. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 16553-16562Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 18Pillutla R.C. Yue Z. Maldonado E. Shatkin A.J. J. Biol. Chem. 1998; 273: 21443-21446Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 19Tsukamoto T. Shibagaki Y. Niikura Y. Mizumoto K. Biochem. Biophys. Res. Commun. 1998; 251: 27-34Crossref PubMed Scopus (33) Google Scholar, 20Yamada-Okabe T. Mio. T. Kashima Y. Matsui M. Arisawa M. Yamada-Okabe H. Microbiology. 1999; 145: 3023-3033Crossref PubMed Scopus (12) Google Scholar, 21Yokoska J. Tsukamoto T. Miura K. Shiokawa K. Mizumoto K. Biochem. Biophys. Res. Commun. 2000; 268: 617-624Crossref PubMed Scopus (12) Google Scholar, 22Hall M.P. Ho C.K. RNA (N. Y.). 2006; 12: 488-497Crossref PubMed Scopus (16) Google Scholar). The cap methyltransferase Ecm1 from the microsporidian parasite Encephalitozoon cuniculi (23Hausmann S. Vivares C.P. Shuman S. J. Biol. Chem. 2002; 277: 96-103Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) is presently the best model for mechanistic studies of cap methylation. Ecm1 is the smallest cap methyltransferase known (298 amino acids), and it suffices for cap methylation in vivo, as gauged by complementation in yeast (23Hausmann S. Vivares C.P. Shuman S. J. Biol. Chem. 2002; 277: 96-103Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Crystal structures of Ecm1 bound to its substrates have been determined (24Fabrega C. Hausmann S. Shen V. Shuman S. Lima C.D. Mol. Cell. 2004; 13: 77-89Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 25Hausmann S. Zheng S. Fabrega C. Schneller S.W. Lima C.D. Shuman S. J. Biol. Chem. 2005; 280: 20404-20412Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Ecm1 contains two ligand-binding pockets, one for the methyl donor AdoMet, and one for the cap guanosine methyl acceptor and the 5′-triphosphate of the cap. Superposition of the structures of Ecm1-ligand complexes suggested a direct in-line mechanism of methyl transfer. It was remarkable that no Ecm1 residues were observed in contact with the guanine-N7 nucleophile, the AdoMet methyl carbon, or the AdoHcy sulfur leaving group, implying that Ecm1 facilitates methyl transfer to cap guanine-N7 by optimizing proximity and geometry of the donor and acceptor. A similar catalytic strategy is used by glycine N-methyltransferase (26Takata Y. Huhang Y. Komoto J. Yamada T. Konishi K. Ogawa H. Gomi T. Fujioka M. Takusagawa F. Biochemistry. 2003; 42: 8394-8402Crossref PubMed Scopus (70) Google Scholar).The availability of a crystal structure has spurred biochemical and structure-function analyses of Ecm1 (24Fabrega C. Hausmann S. Shen V. Shuman S. Lima C.D. Mol. Cell. 2004; 13: 77-89Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 25Hausmann S. Zheng S. Fabrega C. Schneller S.W. Lima C.D. Shuman S. J. Biol. Chem. 2005; 280: 20404-20412Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Purified recombinant Ecm1 is a monomeric protein that catalyzes methyl transfer from AdoMet (Km 25 μm) to GpppA (Km 0.1 mm), GTP (Km 1 mm), or GDP (Km 2.4 mm), but not ATP, CTP, UTP, ITP, or m7GTP. A large collection of alanine and conservative mutants has been generated and tested for activity in yeast by complementation of an abd1Δ strain. This effort has pinpointed critical constituents of the active site that bind to AdoMet (Lys-54, Asp-70, Asp-78, and Asp-94), the cap triphosphate (Arg-106), or the cap guanosine (Phe-141). Tyr-145 is an essential residue that contacts both AdoMet and the cap guanine (Fig. 1). Purification and characterization of recombinant versions of a few Ecm1-Ala mutants verified that Lys-54, Asp-70, Asp-78, Asp-94, and Phe-141 were essential for methylation of GTP in vitro (25Hausmann S. Zheng S. Fabrega C. Schneller S.W. Lima C.D. Shuman S. J. Biol. Chem. 2005; 280: 20404-20412Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar).It was surprising that many of the side chains that contact the substrates in the Ecm1 crystal structures were nonessential for activity in yeast, as surmised from the lack of a growth phenotype when the residue was replaced by alanine. Mutating other residues that contact the substrates resulted in temperature-sensitive growth. It was particularly noteworthy how few of the individual side-chain contacts to the edge of the guanine base were essential for Ecm1 function in vivo, given the stringent specificity for a guanine nucleotide as the methyl acceptor (25Hausmann S. Zheng S. Fabrega C. Schneller S.W. Lima C.D. Shuman S. J. Biol. Chem. 2005; 280: 20404-20412Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). This suggested that there might be functional redundancy among the substrate-binding residues. Here we sought to address this issue by analyzing the effects of new mutations in residues that comprise the guanine-binding pocket, alone and in combination. The outcomes indicate that neither of the polar contacts (from His-144 and Tyr-145) to the O6 atom of guanine are essential in vivo and suggest that the shape of the guanine binding pocket is more crucial than base edge interactions. The results also illuminate the contributions of the aliphatic carbons of Phe-141 and Tyr-145 that engage in multiple van der Waals contacts with guanosine and AdoMet, respectively.To compare the growth phenotypes in yeast with mutational effects on enzyme activity, we purified 45 Ecm1 mutant proteins produced in bacteria and assayed them for methylation of GpppA in vitro. Whereas lethality in vivo generally correlated with ablation of methyltransferase activity, we noted many instances of mutations that were viable or conditional in yeast despite much reduced cap methyltransferase activity in vitro. We surmise that there is a threshold level of methyltransferase required for growth that is exceeded when wild-type Ecm1 is expressed in yeast. The mutational effects on activity in vitro provide a clearer view of the contributions of the active site functional groups.To better understand the mechanism of sinefungin inhibition of cap methylation, we determined the crystal structure of an Ecm1-sinefungin binary complex, which revealed that sinefungin occupied a position similar to that observed for the AdoMet substrate and AdoHcy product in previous structures, although sinefungin makes additional contacts with Ecm1 main-chain and side-chain atoms. We report that S. cerevisiae Abd1 is inhibited potently by sinefungin and that the sensitivity of budding yeast to growth inhibition by the drug is diminished when Abd1 is overexpressed. These results suggest that the cap methylation is a principal target of sinefungin action in vivo.EXPERIMENTAL PROCEDURESMaterials—[3H-CH3]AdoMet was purchased from Perkin-Elmer Life Sciences. GTP, m7GTP, AdoMet, and sinefungin were purchased from Sigma. GpppA and m7GpppA were purchased from New England Biolabs.Mutational Effects on Ecm1 Function in Vivo—Missense mutations were introduced into the ECM1 gene by the PCR-based two-stage overlap extension method, and the mutated genes were inserted into the yeast CEN TRP1 plasmid p358-ECM1, where expression of ECM1 is under the control of the ABD1 promoter (25Hausmann S. Zheng S. Fabrega C. Schneller S.W. Lima C.D. Shuman S. J. Biol. Chem. 2005; 280: 20404-20412Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The inserts were sequenced completely to exclude the acquisition of unwanted mutations during amplification and cloning. The in vivo activity of the mutated ABD1 alleles was tested by plasmid shuffle (24Fabrega C. Hausmann S. Shen V. Shuman S. Lima C.D. Mol. Cell. 2004; 13: 77-89Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 25Hausmann S. Zheng S. Fabrega C. Schneller S.W. Lima C.D. Shuman S. J. Biol. Chem. 2005; 280: 20404-20412Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Yeast strain YBS40 (abd1::hisG p360-ABD1[CEN URA3 ABD1]) was transformed with CEN TRP1 plasmids containing the wild-type and mutant alleles of ECM1. Trp+ isolates were selected and then streaked on agar plates containing 0.75 mg/ml 5-fluoroorotic acid. Growth was scored after 7 days of incubation at 25°, 30°, and 37 °C. Lethal mutants were those that failed to form colonies on 5-fluoroorotic acid at any temperature. Individual colonies of the viable ECM1 mutants were picked from the 5-fluoroorotic acid plate and transferred to YPD (yeast extract, peptone, dextrose) agar medium. Two isolates of each mutant were tested for growth on YPD agar at 25°, 30°, and 37 °C. Growth was assessed as follows: +++ indicates colony size indistinguishable from strains bearing wild-type ECM1; ++ denotes slightly reduced colony size; + indicates that only pinpoint colonies were formed; - indicates no growth.Mutational Effects on Ecm1 Function in Vitro—NdeI/BamHI fragments encoding Ecm1 mutants were excised from the respective p358-ECM1 plasmids and inserted into pET16b. The pET16-Ecm1 plasmids were introduced into Escherichia coli BL21(DE3). The recombinant Ecm1 mutant proteins were produced and purified from soluble bacterial lysates by nickel-agarose chromatography as described previously for wild-type Ecm1 (25Hausmann S. Zheng S. Fabrega C. Schneller S.W. Lima C.D. Shuman S. J. Biol. Chem. 2005; 280: 20404-20412Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The purification was performed at 4 °C. Protein concentrations were determined using the Bio-Rad dye reagent with bovine serum albumin as the standard. Methyltransferase reaction mixtures (20 μl) containing 50 mm Tris-HCl, pH 7.5, 5 mm DTT, 50 μm [3H-CH3]AdoMet, 1 mm GpppA, and 1 μg of Ecm1 were incubated for 60 min at 25 °C. Aliquots (5 μl) were spotted on polyethyleneimine cellulose TLC plates, which were developed with 0.2 m (NH4)2SO4. The AdoMet- and m7GpppA-containing portions of the lanes were cut out, and the radioactivity in each was quantified by liquid scintillation counting. The activity of each protein was determined as the average of three separate experiments (Fig. 2B). The activity of each of the mutants was normalized to that of wild-type Ecm1 (defined as 100%) (Table 1).FIGURE 2Mutational effects on Ecm1 methyltransferase activity. A, Ecm1 purification. Aliquots (5 μg) of the nickel-agarose preparations of wild-type (WT) Ecm1, and the indicated mutants were analyzed by SDS-PAGE. The polypeptides were visualized by staining with Coomassie Blue dye. The positions and sizes (kDa) of marker polypeptides are indicated on the left. B, reaction mixtures (20 μl) containing 50 mm Tris-HCl (pH 7.5), 5 mm DTT, 1 mm GpppA, 50 μm [3H-CH3]AdoMet, and 1 μg of either WT or mutant Ecm1 proteins were incubated for 60 min at 37 °C. The extent of [3H]m7GpppA formation (average of three experiments with standard error bars) is plotted for each enzyme.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Mutational effects on Ecm1 activity in vivo and in vitro The indicated mutant alleles were tested for cap methyltransferase activity in vivo by complementation of the S. cerevisiae abd1Δ strain as described under “Experimental Procedures.” The new alleles constructed for the present study are indicated in boldface type. The complementation data for the other mutants was reported previously (25Hausmann S. Zheng S. Fabrega C. Schneller S.W. Lima C.D. Shuman S. J. Biol. Chem. 2005; 280: 20404-20412Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Cap methyltransferase activity of all of the purified recombinant proteins was determined as described in Fig. 2. The activity of each of the mutants (in boldface type) was normalized to that of wild-type Ecm1 (defined as 100%). The atomic contacts of each mutated side chain are indicated in the rightmost column, on the same line as the alanine mutant.Ecm1abd1Δ complementationGpppA methylationContacts25 °C30 °C37 °C% of WTWT+++++++++100N51A++++++–15Lys81, cap triphosphateN51D–––4K54A–––1AdoMet carboxylK54R–––1K54Q–––<1R59A+++++++++88NoneD70A–––6AdoMet amine (via HOH)D70N–––8D70E+++++++++40K75A++++–8Cap triphosphateD78A–––1Lys-81, AdoMet amineD78N–––<1D78E++++++–24K81A–––19Asp-78, Asn-51K81R–––9K81Q–––12R84A+++++++++110NoneD94A–––<1AdoMet ribose hydroxylsD94N–––<1D94E+++++++++23R106A–––2Cap triphosphateR106K–––4R106Q–––1Y124A++++++–17AdoMet adenine, Ser-142F141A–––<1Cap guanosineF141L+++++++++45F141I+++++++25F141H++++++8F141V++++++16F141N–––1H144A++++++++47Cap guanineY145A–––2AdoMet, cap guanineY145F+++++++++34Y145L+++++++++7Y145S–––2Y145H+++++++++11Y145I+++++2Y145V–––2H144A/Y145F+++++++++12H144A/Y145L+++–1P175A+++++++++18Cap guanineY212A++++++–30Glu-225F214A++++++–10Cap guanineE225A++++–13Cap guanineK267A+++++++++120None Open table in a new tab Crystal Structure of Ecm1 Bound to Sinefungin—Ecm1 was produced for crystallographic analysis as reported previously (24Fabrega C. Hausmann S. Shen V. Shuman S. Lima C.D. Mol. Cell. 2004; 13: 77-89Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Ecm1 (5 mg/ml, 150 μm) was incubated at 4 °C in the presence of 300 μm sinefungin for 30 min prior to crystallization by vapor diffusion against a well solution containing 1.2 m sodium/potassium tartrate, 50 mm bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (pH 6.0 or 6.25), 20 mm DTT. Crystals appeared within 1-3 days at 18 °C and were cryo-protected with well solution containing 18% glycerol prior to freezing them in liquid nitrogen. Crystals diffracted x-rays to 2.6 Å (P3121, a = b = 63.81 Å, c = 112.12 Å, α = β = 90°, γ = 120°). Data were collected using a Rigaku RU200 x-ray generator equipped with confocal Osmic multilayer optics and an R axis-IV imaging plate detector. Data were reduced with DENZO, SCALEPACK, and CCP4 (27Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38368) Google Scholar, 28Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19707) Google Scholar). The Ecm1-sinefungin data set was isomorphous to the previously determined crystal structures of Ecm1 (24Fabrega C. Hausmann S. Shen V. Shuman S. Lima C.D. Mol. Cell. 2004; 13: 77-89Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 25Hausmann S. Zheng S. Fabrega C. Schneller S.W. Lima C.D. Shuman S. J. Biol. Chem. 2005; 280: 20404-20412Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Electron density maps were interpreted using O (29Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-118Crossref PubMed Scopus (13004) Google Scholar), and models were refined using CNS (30Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16930) Google Scholar) to an R of 0.203 and Rfree of 0.265. Waters were added manually into electron density derived from simulated annealing omit maps. The model has excellent geometry with no Ramachandran outliers. The coordinates have been deposited in PDB (accession code 2HV9).Recombinant Abd1—Full-length Abd1 was produced as a His6-Smt3 fusion protein (43Mossessova E. Lima C.D. Molecular Cell. 2000; 5: 865-876Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar) in E. coli BL21(DE3) CodonPlus RIL (Novagen) using a pET-based pSmt3-TOPO vector (Invitrogen). Cultures (10 liters) were grown at 37 °C in a Bioflo 3000 fermentor (New Brunswick Scientific) to an A600 of 3.0, then adjusted to 30 °C, supplemented with 0.75 mm isopropyl 1-thio-β-d-galactopyranoside, and incubated at 30 °C for 4 h. Cells were harvested by centrifugation and resuspended in 20 mm Tris-HCl (pH 8.0), 500 mm NaCl, 20 mm imidazole, 0.1% IGEPAL, 20% sucrose, 1 mm β-mercaptoethanol. Cells were lysed by sonication, and insoluble material was removed by centrifugation. His6-Smt3-Abd1 was purified from the soluble extract by nickel-nitrilotriacetic acid fast flow column chromatography (Qiagen). The His6-Smt3 tag was removed by treatment with the Smt3-specific protease Ulp1 (43Mossessova E. Lima C.D. Molecular Cell. 2000; 5: 865-876Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar). Abd1 was purified free of the tag by gel filtration through a column of Superdex 200. Abd1 appeared homogeneous by SDS-PAGE and Coomassie Blue staining. The protein was concentrated to 10.5 mg/ml in 20 mm Tris-HCl (pH 8.0), 100 mm NaCl, 3 mm DTT, and stored at -80 °C.Inhibition of Yeast Growth by Sinefungin—The CEN URA3 ABD1 plasmid in the S. cerevisiae YBS40 strain was replaced by p358-ABD1 (CEN TRP1 ABD1) or p132-ABD1 (CEN TRP1 ABD1) by plasmid shuffle. In p358-ABD1, methyltransferase expression is directed by the ABD1 promoter, whereas in p132-ABD1, methyltransferase expression is driven by the strong constitutive TPI1 promoter. Wild-type Abd1-expressing and “High-ABD1” yeast cells were grown in YPD medium to mid-log phase (A600 of 0.7 to 0.9) and ∼106 cells were spread on YPD agar plates (15-cm diameter). After incubation of the plates for 1 h at 30°C to allow the cell suspension to dry, 2-μl aliquots of aqueous solutions of sinefungin (125, 250, 500, or 1000 μm) were spotted on the agar plates. Water alone was spotted as a control and resulted in no zone of growth inhibition. The plates were incubated for 2 days at 30 °C, and then photographed.RESULTS AND DISCUSSIONNew Mutations in the Active Site of Ecm1—Amino acids that comprise the GTP binding pocket include Phe-141, His-144, Tyr-145, Pro-175, Phe-214, Glu-225, and Tyr-284 (Fig. 1A). We showed previously that Phe-141, which makes multiple van der Waals contacts with the cap guanine and ribose, is essential for Ecm1 function in yeast, i.e. the F141A mutation was unconditionally lethal (24Fabrega C. Hausmann S. Shen V. Shuman S. Lima C.D. Mol. Cell. 2004; 13: 77-89Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Here we tested the effects of replacing Phe-141 conservatively with leucine, isoleucine, valine, and histidine. The mutant ECM1 alleles were placed under control of the ABD1 promoter on a centromeric plasmid and assayed by plasmid shuffle for abd1Δ complementation (Table 1). The F141L strain grew as well as the wild-type ECM1 strain at all temperatures tested, signifying that an aromatic functional group was not essential for activity in vivo and that the van der Waals contacts of Phe-141 Cz (with the ribose C2′ and C3′ of guanosine), Cϵ1 (with the ribose C3′), and Cϵ2 (with the ribose C2′) observed in the crystal structure are not the critical interactions of this side chain. The isoleucine change resulted in a temperature-sensitive growth defect, whereby the F141I strain grew well at 25 and 30 °C but formed pinpoint colonies at 37 °C. The F141V and F141H strains also formed pinpoint colonies at 37 °C and displayed a slow growth phenotype at 30 °C as well. Thus, we surmise that an aliphatic branched amino acid at position 141 is the minimal requirement for Ecm1 activity and that the van der Waals contacts of Phe-141 Cδ2 (with the N3 atom of the guanine base), Cγ (with guanine C4), and Cβ (with guanine N7, C5, and C6) are functionally most relevant. This inference was reinforced by the finding that replacing Phe-141 with asparagine, which is nearly isosteric with leucine, resulted in unconditional lethality (Table 1). We infer from this result that the Oδ atom of asparagine was the deleterious factor, insofar as a histidine (to which Asn is partially isosteric, and which, like Asn, contains an Nδ atom) was able to support cell growth.Pro-175 makes a van der Waals contact from Cγ to the exocyclic N2 atom of the guanine base (Fig. 1A) and might thereby contribute to methyl acceptor specificity. However, we found presently that changing Pro-175 to Ala had no apparent effect on Ecm1 activity in yeast (Table 1). Given that guanine N2 makes multiple additional contacts to the enzyme (via Tyr-284 Cϵ2 and OH, Glu-225 Oϵ1, and Phe-214 Cδ2 and Cϵ2), it is likely that loss of any one contact will not abolish activity. Indeed, we found previously that the F214A, E225A, and Y284A yeast mutants are viable, albeit unable to grow at 37 °C (Table 1) (24Fabrega C. Hausmann S. Shen V. Shuman S. Lima C.D. Mol. Cell. 2004; 13: 77-89Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar).His-144 donates a hydrogen bond from Nϵ to guanine O6 (Fig. 1A) and was initially thought to be a methyl acceptor specificity determinant. The findings that the H144A mutation had no effect on yeast growth at 25 or 30 °C, although it did slow growth at 37 °C (Table 1), cast doubt on this idea. It was suggested that the guanine O6 contact to His-144 and the water-mediated contact to the Tyr-145 hydroxyl might be functionally redundant. To address this issue presently, we constructed and tested a H144A/Y145F double mutant, which we found to be fully functional in yeast (Table 1). Thus, it seems that neither of the hydrogen-bonding interactions with the guanine O6 seen in the crystal structure are essential for cap methyltransferase activity in vivo. This is not to say that there is no structural basis for the guanine O6 specificity; rather, we suppose that it is the shape of the guanine-binding pocket into which the O6 atom fits that is the defining determinant. Indeed, the O6 atom is sandwiched between the Phe-141 main-chain carbonyl oxygen and the Glu-225 Oϵ2, which are located 3.1 Å from guanine O6 at positions roughly orthogonal to, and on opposite faces of, the plane of the purine base (Fig. 1A). Also, guanine O6 makes a van der Waals contact (4.1 Å) to the Tyr-145 Cϵ1 atom, which is located apical to the guanine 6-carbonyl. The Tyr-145 Cϵ1 contact was not essential per se, insofar as introducing leucine in place of Tyr-145 had no effect on yeast growth (Table 1). However, we found presently that an H144A/Y145L double mutant was defective in vivo, failing to grow at 37 °C, forming microcolonies at 30 °C, and growing slowly at 25 °C (Table 1). The pairwise comparison of the benign H144A/Y145F and severe H144A/Y145L alleles implicates the" @default.
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• W2012291245 title "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" @default.
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• W2012291245 doi "https://doi.org/10.1074/jbc.m607292200" @default.
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