The Zinc Finger Protein Ynr046w Is Plurifunctional and a Component of the eRF1 Methyltransferase in Yeast
2006; Elsevier BV; Volume: 281; Issue: 47 Linguagem: Inglês
10.1074/jbc.m608571200
ISSN1083-351X
AutoresValérie Heurgué‐Hamard, Marc Graille, Nathalie Scrima, Nathalie Ulryck, Stéphanie Champ, Herman van Tilbeurgh, Richard H. Buckingham,
Tópico(s)Cancer-related gene regulation
ResumoProtein release factor eRF1 in Saccharomyces cerevisiae, in complex with eRF3 and GTP, is methylated on a functionally crucial Gln residue by the S-adenosylmethionine-dependent methyltransferase Ydr140w. Here we show that eRF1 methylation, in addition to these previously characterized components, requires a 15-kDa zinc-binding protein, Ynr046w. Co-expression in Escherichia coli of Ynr046w and Ydr140w allows the latter to be recovered in soluble form rather than as inclusion bodies, and the two proteins co-purify on nickel-nitrilotriacetic acid chromatography when Ydr140w alone carries a His tag. The crystal structure of Ynr046w has been determined to 1.7 Å resolution. It comprises a zinc-binding domain built from both the N- and C-terminal sequences and an inserted domain, absent from bacterial and archaeal orthologs of the protein, composed of three α-helices. The active methyltransferase is the heterodimer Ydr140w·Ynr046w, but when alone, both in solution and in crystals, Ynr046w appears to be a homodimer. The Ynr046w eRF1 methyltransferase subunit is shared by the tRNA methyltransferase Trm11p and probably by two other enzymes containing a Rossman fold. Protein release factor eRF1 in Saccharomyces cerevisiae, in complex with eRF3 and GTP, is methylated on a functionally crucial Gln residue by the S-adenosylmethionine-dependent methyltransferase Ydr140w. Here we show that eRF1 methylation, in addition to these previously characterized components, requires a 15-kDa zinc-binding protein, Ynr046w. Co-expression in Escherichia coli of Ynr046w and Ydr140w allows the latter to be recovered in soluble form rather than as inclusion bodies, and the two proteins co-purify on nickel-nitrilotriacetic acid chromatography when Ydr140w alone carries a His tag. The crystal structure of Ynr046w has been determined to 1.7 Å resolution. It comprises a zinc-binding domain built from both the N- and C-terminal sequences and an inserted domain, absent from bacterial and archaeal orthologs of the protein, composed of three α-helices. The active methyltransferase is the heterodimer Ydr140w·Ynr046w, but when alone, both in solution and in crystals, Ynr046w appears to be a homodimer. The Ynr046w eRF1 methyltransferase subunit is shared by the tRNA methyltransferase Trm11p and probably by two other enzymes containing a Rossman fold. Termination codons in mRNA are recognized on the ribosome by class I protein termination factors (or release factors (RFs)) 5The abbreviations used are: RF, release factor; AdoMet, S-adenosylmethionine; Ni-NTA, nickel-nitrilotriacetic acid; MTase, methyltransferase; IPTG, isopropyl β-d-thiogalactopyranoside; DTT, dithiothreitol; HIV-1, human immunodeficiency virus, type 1; GDPNP, guanine 5′-(β,γ-imido) triphosphate. 5The abbreviations used are: RF, release factor; AdoMet, S-adenosylmethionine; Ni-NTA, nickel-nitrilotriacetic acid; MTase, methyltransferase; IPTG, isopropyl β-d-thiogalactopyranoside; DTT, dithiothreitol; HIV-1, human immunodeficiency virus, type 1; GDPNP, guanine 5′-(β,γ-imido) triphosphate. in eubacteria, archaea, and eukaryotes (1Kisselev L.L. Buckingham R.H. Trends Biochem. Sci. 2000; 25: 561-566Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 2Nakamura Y. Ito K. Trends Biochem. Sci. 2003; 28: 99-105Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 3Kisselev L. Ehrenberg M. Frolova L. EMBO J. 2003; 22: 175-182Crossref PubMed Scopus (198) Google Scholar). Three codons are used as stop signals in most organisms: UAA, UGA, and UAG. In bacteria, two class I RFs are required for termination: RF1, which recognizes UAA and UAG codons, and RF2, which recognizes UAA and UGA codons. In contrast, a single RF, eRF1 or aRF1, is sufficient for termination at all three stop codons in eukaryotes and archaea, respectively. eRF1 and aRF1 form closely related protein families but are evolutionarily distinct from the eubacterial RFs (4Kisselev L. Structure (Camb.). 2002; 10: 8-9Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Thus, despite a similar function, the only sequence element common to all RFs is a tripeptide sequence, GGQ. Structural and mutational analysis shows that this motif is essential for RF activity and is required to interact with the peptidyl transferase center of the large ribosomal subunit and trigger hydrolysis of the ester bond in peptidyl-tRNA (5Petry S. Brodersen D.E. Murphy F.V.t. Dunham C.M. Selmer M. Tarry M.J. Kelley A.C. Ramakrishnan V. Cell. 2005; 123: 1255-1266Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 6Frolova L.Y. Tsivkovskii R.Y. Sivolobova G.F. Oparina N.Y. Serpinsky O.I. Blinov V.M. Tatkov S.I. Kisselev L.L. RNA. 1999; 5: 1014-1020Crossref PubMed Scopus (291) Google Scholar). The Gln residue of the GGQ motif is methylated in both bacteria (7Dinçbas-Renqvist V. Engström Å. Mora L. Heurgué-Hamard V. Buckingham R.H. Ehrenberg M. EMBO J. 2000; 19: 6900-6907Crossref PubMed Scopus (120) Google Scholar) and Saccharomyces cerevisiae (8Heurgué-Hamard V. Champ S. Mora L. Merkulova-Rainon T. Kisselev L.L. Buckingham R.H. J. Biol. Chem. 2005; 280: 2439-2445Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 9Polevoda B. Span L. Sherman F. J. Biol. Chem. 2006; 281: 2562-2571Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), and probably in mammals. Bacterial RF methylation depends on the PrmC methyltransferase (MTase) (10Graille M. Heurgue-Hamard V. Champ S. Mora L. Scrima N. Ulryck N. van Tilbeurgh H. Buckingham R.H. Mol. Cell. 2005; 20: 917-927Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 11Heurgué-Hamard V. Champ S. Engstöm Å. Ehrenberg M. Buckingham R.H. EMBO J. 2002; 21: 769-778Crossref PubMed Scopus (113) Google Scholar), the product of the gene prmC (previously named hemK) situated in Escherichia coli, and most other bacteria immediately downstream of the gene prfA encoding RF1. RF methylation in E. coli strongly stimulates the activity of the factors (7Dinçbas-Renqvist V. Engström Å. Mora L. Heurgué-Hamard V. Buckingham R.H. Ehrenberg M. EMBO J. 2000; 19: 6900-6907Crossref PubMed Scopus (120) Google Scholar, 11Heurgué-Hamard V. Champ S. Engstöm Å. Ehrenberg M. Buckingham R.H. EMBO J. 2002; 21: 769-778Crossref PubMed Scopus (113) Google Scholar). 6L. Mora, unpublished results. 6L. Mora, unpublished results. It is remarkable that the modification of Gln in the GGQ motif is conserved from bacteria to eukaryotes despite the different evolutionary origin of the class I RFs themselves. The S. cerevisiae genome encodes two proteins, Ydr140w and Ynl063w, with significant similarity to bacterial PrmC that goes beyond the motifs known to be involved in AdoMet binding (7Dinçbas-Renqvist V. Engström Å. Mora L. Heurgué-Hamard V. Buckingham R.H. Ehrenberg M. EMBO J. 2000; 19: 6900-6907Crossref PubMed Scopus (120) Google Scholar). Inactivation of Ydr140w was shown to lead to a loss of eRF1 methylation (8Heurgué-Hamard V. Champ S. Mora L. Merkulova-Rainon T. Kisselev L.L. Buckingham R.H. J. Biol. Chem. 2005; 280: 2439-2445Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The gene Ynl063w is required for methylation of the mitochondrial RF, Mrf1p (9Polevoda B. Span L. Sherman F. J. Biol. Chem. 2006; 281: 2562-2571Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Methylation of eRF1 by Ydr140w differs from prokaryotic RF methylation in that the presence of the class II RF (eRF3 in yeast), and GTP was also required. The substrate of the yeast MTase therefore appears to be the ternary complex eRF1·eRF3·GTP rather than eRF1 alone. The role of class II factors in eubacteria is to catalyze the recycling of the class I RFs following peptide release (12Zavialov A.V. Buckingham R.H. Ehrenberg M. Cell. 2001; 107: 115-124Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 13Freistroffer D.V. Pavlov M.Y. MacDougall J. Buckingham R.H. Ehrenberg M. EMBO J. 1997; 16: 4126-4133Crossref PubMed Scopus (242) Google Scholar); in eukaryotic organisms the role of eRF3 seems to be closer to that of EF1α and leaves the ribosome after peptide release (14Alkalaeva E.Z. Pisarev A.V. Frolova L.Y. Kisselev L.L. Pestova T.V. Cell. 2006; 125: 1125-1136Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). Thus, a significant difference between eubacterial and eukaryotic factors is that, in the presence of GTP, the class I and class II factors (eRF1 and eRF3) bind to each other (15Kobayashi T. Funakoshi Y. Hoshino S. Katada T. J. Biol. Chem. 2004; 279: 45693-45700Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), whereas the eubacterial factors do not. Archaea appear to have no class II RF. Some observations made during the characterization of Ydr140w as the eRF1 MTase in S. cerevisiae suggested that at least one further component, in addition to the ternary complex eRF1·eRF3·GTP and Ydr140w, is required for methylation of eRF1 (8Heurgué-Hamard V. Champ S. Mora L. Merkulova-Rainon T. Kisselev L.L. Buckingham R.H. J. Biol. Chem. 2005; 280: 2439-2445Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Only impure preparations of Ydr140w, produced in yeast, were able to methylate RFs in vitro, and with low efficiency. Overproduction of Ydr140w in E. coli led exclusively to insoluble protein, but even when resolubilized by routine renaturation methods, no MTase activity could be detected. Polevoda et al. (9Polevoda B. Span L. Sherman F. J. Biol. Chem. 2006; 281: 2562-2571Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) confirmed the function of Ydr140w in methylation of eRF1, but their assays were also performed with partially purified components and resulted in a low efficiency of methylation. Other studies of proteins interacting with Ydr140w in yeast, either by two-hybrid mapping or by tandem affinity purification-tagged co-purification, followed by mass spectrometry, identified Ynr046w, a 15-kDa protein, as a component interacting with Ydr140w (16Ito T. Chiba T. Ozawa R. Yoshida M. Hattori M. Sakaki Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4569-4574Crossref PubMed Scopus (2912) Google Scholar, 17Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Crossref PubMed Scopus (3950) Google Scholar). Furthermore, affinity studies showed that two S. cerevisiae tRNA MTases, Trm11p and Trm9p, and a further protein, Lys9p, apparently bind to Ynr046w (16Ito T. Chiba T. Ozawa R. Yoshida M. Hattori M. Sakaki Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4569-4574Crossref PubMed Scopus (2912) Google Scholar, 17Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Crossref PubMed Scopus (3950) Google Scholar, 18Kalhor H.R. Clarke S. Mol. Cell. Biol. 2003; 23: 9283-9292Crossref PubMed Scopus (122) Google Scholar), and Purushothaman et al. (19Purushothaman S.K. Bujnicki J.M. Grosjean H. Lapeyre B. Mol. Cell. Biol. 2005; 25: 4359-4370Crossref PubMed Scopus (85) Google Scholar) demonstrated the requirement for both Ynr046w and Trm11p for m2G10 formation in yeast tRNA. Lys9p is a dehydrogenase with a Rossman fold similar to that in most RNA MTases. Sequence analysis has revealed the existence of Ynr046w orthologs within the three kingdoms of life (19Purushothaman S.K. Bujnicki J.M. Grosjean H. Lapeyre B. Mol. Cell. Biol. 2005; 25: 4359-4370Crossref PubMed Scopus (85) Google Scholar). Interestingly, archaeal and bacterial proteins from this family are much shorter (about 55-65 residues) than eukaryotic proteins (about 130 residues; Fig. 1). Hence, Ynr046w is made of a central region specific for eukaryotic members ("insert" domain, residues 39-101) inserted within a region conserved in the three kingdoms of life ("conserved" domain, residues 1-38 and 102-135). Among fungal orthologs, this latter domain harbors a putative zinc finger signature made of the 11CX5C16 and 112CX2C115 motifs (where X can be any residue; from the N- and C-terminal parts, respectively). Here we show that Ynr046w is required as a subunit of the eRF1 MTase in S. cerevisiae. In vitro, purified Ydr140w, Ynr046w, and AdoMet are necessary and sufficient to methylate the ternary complex eRF1·eRF3·GTP. Finally, we present the 1.7 Å resolution crystal structure of this small protein and show that it comprises two domains: a domain built from both N-terminal and C-terminal sequences that contains a zinc-binding site and an inserted domain, absent from bacterial and archaeal orthologs of the protein, composed of three α-helices. Bacterial Growth—LB medium was supplemented according to requirements. Antibiotics were added at the following final concentration: kanamycin, 50 μg/ml; ampicillin, 200 μg/ml; and chloramphenicol, 15 μg/ml. When induction was necessary to overexpress proteins, 1 m IPTG was added to liquid medium to a final concentration of 1 mm. For expression of Ynr046w, alone or as a complex with Ydr140w, ZnCl2 was added to a final concentration of 100 μm. Recombinant DNA Manipulations—General procedures for DNA recombinant techniques, plasmid extraction, etc. were performed as described by Sambrook et al. (20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Yeast eRF1 and Truncated eRF3 Expression Vectors—pYSC1, a derivative of pET11a plasmid encoding eRF1 with a His6 tag on its C terminus, was constructed by gene cloning between the NdeI and BamHI sites. Amplification was performed on chromosomal DNA from yeast strain yIBPC27. The upstream oligonucleotide (5′-AATACTTCACATATGGATAACGAGGT-3′) introduces an NdeI site. The downstream oligonucleotide (5′-CGCGGATCCTTTAGTGGTGGTGGTGGTGGTGAATGAAATCATAGTCGGACCCTTCA-3′) introduces a new BamHI site, eliminating the original one upstream of the stop codon, and encodes the His6 tag. pYSC2, also a derivative of pET11a, encodes yeast eRF3 truncated at the N terminus. Amplification of genomic DNA was done with the following oligonucleotides, introducing, respectively, the NdeI site followed by the His6 tag (5′-GGAATTCCATATGCACCACCACCACCACCACTTTGGTGGTAAAGATCACGTTTC-3′) and the BamHI site (5′-GCGGATCCTTTACTCGGCAATTTTAAC-3′). MTase Ydr140w and Ynr046w Subunit Expression Vectors— pVH450 encodes Ynr046w with a His6 tag at its C terminus and pVH451 encodes the same protein without tag. Both are derivatives of the pET11a vector with a kanamycin cassette and were constructed in the same way after genomic DNA amplification from yeast strain yIBPC27. The upstream oligonucleotide, 5′-TAGCCTAGTCCATATGAAGTTCTT-3′, the same in both cases, introduces an NdeI site. The downstream primers are 5′-ACACAGGGATCCCGCTTGATTTAGTGGTGGTGGTGGTGGTGTACCAGGTGTGGAGGTAACAGCAA-3′ for pVH450 and 5′-ACACAGGGATCCCGCTTGATTTATAC-3′ for pVH451. For co-expression experiments, Ydr140w with a His6 tag on its C terminus was also expressed from plasmid pACYCDuet (ydrH6) compatible with pET plasmids. The gene encoding Ydr140wH6 was cloned in two steps (fragment NdeI-BamHI and fragment NdeI-NdeI) between NdeI and BsrGI in the MCS2 of pACYC-Duet1 (Novagen). pACYCDuet(ydrH6) was constructed from plasmid pTrc(ydr140wH6), a derivative of pVH383 itself derived from pVH371 (8Heurgué-Hamard V. Champ S. Mora L. Merkulova-Rainon T. Kisselev L.L. Buckingham R.H. J. Biol. Chem. 2005; 280: 2439-2445Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In the first step, Ydr140w gene was cloned from pVH371 into pLV1 between NdeI and BamHI sites to yield pVH383. A His6 tag was then introduced by inserting phosphorylated oligonucleotides (5′-GTACAGCTTTACAAGGCACCACCACCACCACCACTGAG-3′ and 5′-GATCCTCAGTGGTGGTGGTGGTGGTGCCTTGTAAAGCT-3′) between the BsrGI and BamHI sites to give pTrc(ydr140wH6). Protein Expression and Purification—After transformation of BL21(DE3) Rosetta by the relevant plasmids, the expression of His-tagged eRF1 or truncated His-tagged eRF3 (eRF3Ct) was induced by IPTG (1 mm) at 16 °C at an optical density (600 nm) of 0.5 in LB medium with appropriate antibiotics, followed by growth overnight. For eRF1, the cells were resuspended in buffer A1 (20 mm Tris-HCl, pH 7.5, 500 mm NaCl, 6 mm β-mercaptoethanol, and 5 mm imidazole) with EDTA-free antiprotease (Roche Applied Science) and broken by passage through a French press. After centrifugation, the supernatant was loaded on Ni-NTA resin (Sigma). The column was washed with buffer A1 and eluted with buffer A1 with 150 mm imidazole but no NaCl. Fractions containing protein were dialyzed against buffer A2 (20 mm Tris-HCl, pH 7.5, 6 mm β-mercaptoethanol) and concentrated by ultrafiltration through Amicon Ultra 30 (Millipore). For eRF3, the cells were resuspended in buffer B1 (50 mm sodium phosphate, pH 7.0, 500 mm NaCl, 6 mm β-mercaptoethanol, and 5 mm imidazole) with EDTA-free antiprotease (Roche Applied Science) and broken by passage through a French press. After centrifugation, the supernatant was loaded on Ni-NTA resin (Sigma). The column was washed with buffer B1 and eluted with buffer B1 with 150 mm imidazole but no NaCl. Fractions containing protein were concentrated by ultrafiltration through Amicon Ultra 30. Imidazole was eliminated by gel filtration on Sephadex G25 in buffer B2 (50 mm sodium phosphate buffer, pH 7.0, 6 mm β-mercaptoethanol). Ynr046wH6 was expressed alone in BL21-Gold (DE3) (Stratagene). Co-expression of Ydr140wH6 and Ynr046w was done in the same strain at 23 °C after induction with 1 mm IPTG at an optical density (600 nm) of 0.5 in LB medium with the appropriate antibiotics, followed by growth overnight. Purification of Ynr046wH6 alone or the complex (Ydr140wH6·Ynr046) was performed as described for eRF1 purification with small modifications. Buffer A1 was modified to buffer C1 (10 mm Tris-HCl, pH 8.0, 500 mm NaCl, 6 mm β-mercaptoethanol, 10 μm ZnCl2). Elution was performed with buffer C1 with 50 mm imidazole and no NaCl. Fractions containing protein were dialyzed against buffer C2 (10 mm Tris-HCl, pH 8.0, 6 mm β-mercaptoethanol, 10 μm ZnCl2). Protein was concentrated by ultrafiltration through Amicon Ultra 30 for the complex and Amicon Ultra 10 for Ynr046wH6 alone. In Vitro Recombinant Ydr140w Purification from Inclusion Bodies—Production of Ydr140w alone in E. coli was achieved as described by Heurgué-Hamard et al. (8Heurgué-Hamard V. Champ S. Mora L. Merkulova-Rainon T. Kisselev L.L. Buckingham R.H. J. Biol. Chem. 2005; 280: 2439-2445Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) from pVH371. Purification was carried out according to Vuillard and Freeman (dwb.unl.edu/Teacher/NSF/C08/C08Links/www.nwfsc.noaa.gov/protocols/inclusion.html). This procedure involves solubilization of proteins from inclusion bodies by guanidinium chloride, followed by rapid dilution in the presence of nondetergent sulfobetaines to limit aggregation. The pellet from 150 ml of bacterial suspension was resuspended in 5 ml of 50 mm HEPES-NaOH, pH 7.5, 0.5 m NaCl, 1 mm phenylmethylsulfonyl fluoride, 5 mm DTT containing 0.35 mg/ml lysozyme and then incubated for 30 min at 20 °C. Triton X-100 was added to a concentration of 1% (v/v), and the cells were broken by passage through a French press. The extract was treated with DNase I (20 μg/l) for 1 h at 37°C and centrifuged at 30,000 × g for 30 min at 4 °C. The pellet (inclusion bodies) was washed twice with phosphate-buffered saline containing 1% Triton X-100, centrifuged at 30,000 × g for 30 min at 4 °C, and then solubilized for 1 h at 4°Cin 2 ml of 50 mm HEPES-NaOH, pH 7.5, 6 m guanidine HCl, 25 mm DTT. After centrifugation at 100,000 × g for 10 min, the insoluble material was removed. The protein concentration in the supernatant was adjusted to 1 mg/ml (from 30 mg/ml) using 50 mm HEPES-NaOH, pH 7.5, 6 m guanidine HCl, 25 mm DTT and then diluted 10-fold as quickly as possible with cold folding buffer (50 mm HEPES, pH 7.5, 0.2 m NaCl, 1 mm DTT, 1 m NDSB201). After 1 h at 4 °C,the protein solution was dialyzed overnight at 4 °C against 10 mm Tris-HCl, pH 7.6, 1 mm β-mercaptoethanol. In Vitro Methylation Assays—Methylation assays were performed in buffer D (100 mm Tris-HCl, pH 7.6, 10 mm MgCl2, 100 mm ammonium acetate, 2 mm DTT, 0.1 mm EDTA, 100 μg/ml bovine serum albumin) and 10 μm [3H]AdoMet (0.86 Ci/mmol). GTP, GDP, or GDPNP were added at a final concentration of 1 mm. The samples were withdrawn at different times, and the reaction was stopped by cold trichloroacetic acid (5%) precipitation, followed by filtration on Whatman GF/C filters and measurement of radioactivity by scintillation counting. Each time point corresponds to 9 pmol of eRF1, 9 pmol of eRF3, and the amount of enzyme as indicated in the figure legends (3 pmol of Ydr140w and 3 pmol of Ynr046w in Fig. 4; 6 pmol of Ydr140w from inclusion bodies and 30 pmol of Ynr046wH6, purified separately, in the experiment of Fig. 3).FIGURE 3Co-elution of His-tagged Ydr140w and nontagged Ynr046w from Ni-NTA resin with imidazole. A cell extract from cells co-expressing the two proteins was applied to a Ni-NTA column, washed, and then eluted with buffer containing 50 mm imidazole. Lane 1, total protein: lane 2, flow-through fractions; lane 3, molecular mass standards (values in kDa shown on the right); lanes 4 and 5, successive fractions containing the bulk of the proteins eluted from the column with imidazole.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Western Blot Analysis—Western blot experiments were performed using rabbit anti-Ydr140w antibodies commercially prepared from pure protein produced as inclusion bodies in E. coli. The proteins were separated on 18% SDS-polyacrylamide gels as described by Laemmli (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205523) Google Scholar). Transfer to nitrocellulose membranes and Western blotting with antibodies (diluted 5000×) were performed as described by Sambrook et al. (20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) using a peroxidase-coupled secondary antibody (diluted 5000×), SuperSignal chemiluminescent substrate (Pierce), and Kodak X-Omat AR. Crystallization and Resolution of the Structure—The protein was stored in 10 mm Tris-HCl, pH 8, 10 μm ZnCl2, 6 mm β-mercaptoethanol. The crystals were grown at 19 °C from a 1:1 μl mixture of 10 mg/ml protein solution with 1.2 m KH2PO4,pH7. For data collection, the crystals were transferred into a cryoprotectant crystallization solution with progressively higher ethylene glycol concentrations up to 30% (v/v). The 2 Å resolution single wavelength anomalous dispersion data at the zinc edge and the high resolution (1.7 Å) native data could be recorded on beamlines ID23-EH1 and ID23-EH2 (ESRF, Grenoble, France), respectively. The structure was determined by the single wavelength anomalous dispersion method using the anomalous signal from the zinc element. The data were processed using the XDS package (23Kabsch W.J. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3200) Google Scholar). The space group was C2 with one molecule/asymmetric unit. One zinc atom site was found with the program SHELXD in the 45-3.5-Å resolution range (24Schneider T.R. Sheldrick G.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1772-1779Crossref PubMed Scopus (1570) Google Scholar). Refinement of the zinc atom, phasing, and density modification were performed with the program SHARP (25Bricogne G. Vonrhein C. Flensburg C. Schiltz M. Paciorek W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 2023-2030Crossref PubMed Scopus (551) Google Scholar). The quality of the final phases allowed automatic building of a partial model with the program RESOLVE (26Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3216) Google Scholar). This model was then refined against the 1.7 Å data set with the Arp/wARP program that allowed automated construction of all the Ynr046w residues (27Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2561) Google Scholar). This model was refined using REFMAC and rebuild with the "TURBO" molecular modeling program (afmb.cnrs-mrs.fr/rubrique113.html). All of the residues from Met1 to His136 (the first residue of the His tag) are well defined in electron density and fall within the allowed regions of the Ramachandran plot as defined by the program Procheck (28Laskowski R.A. Moss D.S. Thornton J.M. J. Mol. Biol. 1993; 231: 1049-1067Crossref PubMed Scopus (1071) Google Scholar). In addition, 150 water molecules and one ethylene glycol molecule (used as cryoprotectant) have been built. Statistics for all of the data collections and refinement of the different structures are summarized in Table 1. The atomic coordinates and structure factors have been deposited into the Brookhaven Protein Data Bank under the accession number 2J6A.TABLE 1Data collection statisticsNativeSADData collectionResolution (Å)20-1.7 (1.75-1.7)45-2.0 (2.2-2.0)Space groupC 2C 2Unit cell parametersa = 92.6 Å, b = 38.5 Å; c = 45.8 Å, β = 102.2°.a = 92.7 Å, b = 38.5 Å; c = 45.8 Å, β = 102.2°.Total number of reflections42,33857,188Total number of unique reflections17,18210,322Rsym (%)aRsym = ΣhΣI|Ihi – | /Σ hΣ iIhi, where Ihi is the ith observation of the reflection h, whereas is the mean intensity of reflection h13.3 (52.4)13.7 (49.2)Completeness (%)97.7 (99)99.4 (99.5)I/s(I)6.2 (2.4)9 (1.3)Redundancy2.55.5RefinementResolution (Å)20-1.7R/Rfree (%)bRfactor = Σ||Fo| – |Fc||/|Fo| · Rfree was calculated with a small fraction (5%) of randomly selected reflections19.8/23.6root mean square deviationBonds (Å)0.011angles (°)1.296Mean B factor (Å2) protein/water16.6/27Ramachandran plotMost favored (%)93.3Allowed (%)6.7a Rsym = ΣhΣI|Ihi – | /Σ hΣ iIhi, where Ihi is the ith observation of the reflection h, whereas is the mean intensity of reflection hb Rfactor = Σ||Fo| – |Fc||/|Fo| · Rfree was calculated with a small fraction (5%) of randomly selected reflections Open table in a new tab Ynr046w/Ydr140w Interaction—Previous observations showed that Ydr140w could be efficiently overproduced in E. coli using the pET expression system, but only as insoluble protein present in inclusion bodies. Attempts to produce soluble protein by reducing the level of induction, growing cells at lower temperatures or co-expressing chaperones were unsuccessful. Denaturation and renaturation of protein from inclusion bodies did yield substantial amounts of soluble Ydr140w. However, the resulting protein did not have methylation activity in vitro and appeared to be unfolded according to circular dichroism measurements. 7V. Heurgué-Hamard, M. Graille, N. Scrima, N. Ulryck, S. Champ, H. van Tilbeurgh, and R. H. Buckingham, unpublished data. On the basis of the data suggesting an interaction between Ydr140w and Ynr046w, we undertook to co-express these two proteins and study their behavior. As shown in Fig. 2, Ynr046w expressed in the absence of Ydr140w was found partly in the supernatant and partly in exclusion bodies. When expressed alone at 23 °C, Ydr140w was found exclusively in inclusion bodies, but when co-expressed with Ynr046w by the use of compatible plasmids, the major part of Ydr140w was found in the soluble fraction, together with Ynr046w (Fig. 2). An interaction between Ydr140w and Ynr046w was previously shown by TAP purification when Ynr046w was tagged (17Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Crossref PubMed Scopus (3950) Google Scholar). After co-expression in E. coli of His-tagged Ydr140w (Ydr140wH6) and Ynr046w, we determined whether Ynr046w could bind efficiently to Ydr140wH6 immobilized on Ni-NTA resin. The data in Fig. 3 show that the two proteins co-elute from the column at an imidazole concentration of 50 mm. Using a similar purification protocol, Ynr046w alone did not bind to Ni-NTA resin (data not shown). Analytical gel filtration experiments realized on the Ynr046w·Ydr140wH6 purified complex shows that it has an apparent molecular mass of 46 kDa, a value slightly higher than that expected for a heterodimer (40.8 kDa, because Ynr046w and Ydr140wH6 are 15- and 25.8-kDa proteins, respectively; data not shown). In comparison, the Ynr046w protein has an apparent molecular mass of 34 kDa, a value close to that expected for a homodimer (30 kDa; data not shown). Ynr046w Restores Ydr140w Activity from Inclusion Bodies Purified in E. coli—The presence of physical intera
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