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W2145070389 abstract "Cet1, the RNA triphosphatase component of the yeast mRNA capping apparatus, catalyzes metal-dependent γ-phosphate hydrolysis within the hydrophilic interior of an eight-strand β barrel (the “triphosphate tunnel”), which rests upon a globular protein core (the “pedestal”). We performed a structure-guided alanine scan of 17 residues located in the tunnel (Ser373, Thr375, Gln405, His411, Ser429, Glu488, Thr490), on the tunnel's outer surface (Ser378, Ser487, Thr489, His491), at the tunnel-pedestal interface (Ile304, Met308) and in the pedestal (Asp315, Lys317, Arg321, Asp425). Alanine mutations at 14 positions had no significant effect on Cet1 phosphohydrolase activity in vitro and had no effect on Cet1 function in vivo. Two of the mutations (R321A and D425A) elicited a thermosensitive (ts) yeast growth phenotype. The R321A and D425A proteins had full phosphohydrolase activity in vitro, but were profoundly thermolabile. Arg321 and Asp425interact to form a salt bridge within the pedestal that tethers two of the strands of the tunnel. Mutations R321Q and D411N resulted ints defects in vivo and in vitro, as did the double-mutant R321A-D435A, whereas the R321K protein was fully stable in vivo and in vitro. These results highlight the critical role of the buried salt bridge in Cet1 stability. Replacement of Ser429 by alanine or valine elicited a cold-sensitive (cs) yeast growth phenotype. The S429A and S429V proteins were fully active when produced in bacteria at 37 °C, but were inactive when produced at 17 °C. Replacement of Ser429 by threonine partially suppressed the cold sensitivity of the Cet1 phosphohydrolase, but did not suppress thecs growth defect in yeast. Cet1, the RNA triphosphatase component of the yeast mRNA capping apparatus, catalyzes metal-dependent γ-phosphate hydrolysis within the hydrophilic interior of an eight-strand β barrel (the “triphosphate tunnel”), which rests upon a globular protein core (the “pedestal”). We performed a structure-guided alanine scan of 17 residues located in the tunnel (Ser373, Thr375, Gln405, His411, Ser429, Glu488, Thr490), on the tunnel's outer surface (Ser378, Ser487, Thr489, His491), at the tunnel-pedestal interface (Ile304, Met308) and in the pedestal (Asp315, Lys317, Arg321, Asp425). Alanine mutations at 14 positions had no significant effect on Cet1 phosphohydrolase activity in vitro and had no effect on Cet1 function in vivo. Two of the mutations (R321A and D425A) elicited a thermosensitive (ts) yeast growth phenotype. The R321A and D425A proteins had full phosphohydrolase activity in vitro, but were profoundly thermolabile. Arg321 and Asp425interact to form a salt bridge within the pedestal that tethers two of the strands of the tunnel. Mutations R321Q and D411N resulted ints defects in vivo and in vitro, as did the double-mutant R321A-D435A, whereas the R321K protein was fully stable in vivo and in vitro. These results highlight the critical role of the buried salt bridge in Cet1 stability. Replacement of Ser429 by alanine or valine elicited a cold-sensitive (cs) yeast growth phenotype. The S429A and S429V proteins were fully active when produced in bacteria at 37 °C, but were inactive when produced at 17 °C. Replacement of Ser429 by threonine partially suppressed the cold sensitivity of the Cet1 phosphohydrolase, but did not suppress thecs growth defect in yeast. isopropyl-1-thio-β-d-galactopyranoside cold-sensitive thermosensitive wild-type 5-fluoroorotic acid Saccharomyces cerevisiae Cet1 is an essential enzyme that performs the first step of mRNA cap formation: the hydrolysis of the γ-phosphate of nascent pre-mRNA to form a 5′-diphosphate end (1Tsukamoto T. Shibagaki Y. Imajoh-Ohmi S. Murakoshi T. Suzuki M. Nakamura A. Gotoh H. Mizumoto K. Biochem. Biophys. Res. Commun. 1997; 239: 116-122Crossref PubMed Scopus (79) Google Scholar, 2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). Cet1 exemplifies a distinctive family of metal-dependent phosphohydrolases that includes other fungal RNA triphosphatases (e.g. Candida albicansCaCet1, S. cerevisiae Cth1, and Schizosaccharomyces pombe Pct1) and the RNA triphosphatase components of the poxvirus, baculovirus, Chlorella virus, and Plasmodium falciparum RNA capping enzymes (3Ho C.K. Pei Y. Shuman S. J. Biol. Chem. 1998; 273: 34151-34156Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 5Rodriguez C.R. Takagi T. Cho E. Buratowski S. Nucleic Acids Res. 1999; 27: 2182-2188Crossref Scopus (28) Google Scholar, 6Pei Y. Lehman K. Tian L. Shuman S. Nucleic Acids Res. 2000; 28: 1885-1892Crossref PubMed Scopus (36) Google Scholar, 7Pei Y. Schwer B. Hausmann S. Shuman S. Nucleic Acids Res. 2001; 29: 387-396Crossref PubMed Google Scholar, 8Jin J. Dong W. Guarino L.A. J. Virol. 1998; 72: 10011-10019Crossref PubMed Google Scholar, 9Gross C.H. Shuman S. J. Virol. 1998; 72: 10020-10028Crossref PubMed Google Scholar, 10Ho C.K. Martins A. Shuman S. J. Virol. 2000; 74: 5486-5494Crossref PubMed Scopus (26) Google Scholar, 11Ho C.K. Gong C. Shuman S. J. Virol. 2001; 75: 1744-1750Crossref PubMed Scopus (27) Google Scholar, 12Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3050-3055Crossref PubMed Scopus (38) Google Scholar). The signature feature of this enzyme family is the ability to hydrolyze nucleoside triphosphates to nucleoside diphosphates and inorganic phosphate in the presence of either manganese or cobalt. The defining structural elements of the family are two glutamate-rich motifs (strands β1 and β11 in Fig.1) that are required for catalysis.The crystal structure of Cet1 illuminates surprising structural complexity for an enzyme that catalyzes a mundane phosphohydrolase reaction (13Lima C.D. Wang L.K. Shuman S. Cell. 1999; 99: 533-543Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Cet1 adopts a novel active site fold whereby an antiparallel eight-strand β barrel forms a topologically closed “triphosphate tunnel” (Fig.2). The hydrophilic tunnel contained a single sulfate coordinated by multiple basic side chains projecting into the cavity. It was proposed that the side chain interactions of the sulfate reflect contacts made by the enzyme with the γ-phosphate of the triphosphate-terminated RNA or nucleoside triphosphate substrates (13Lima C.D. Wang L.K. Shuman S. Cell. 1999; 99: 533-543Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). A manganese ion within the tunnel cavity is coordinated with octahedral geometry to the sulfate, to the side chain carboxylates of the two glutamates in β1, and to a glutamate in β11.Figure 2The triphosphate tunnel and the hydrophobic back surface of the tunnel floor. Stereo view of a cross-section of the tunnel of S. cerevisiae Cet1. The figure highlights the elaborate network of bonding interactions, especially those that coordinate sulfate (γ-phosphate) and manganese. The manganese (blue sphere) interacts with octahedral geometry with the sulfate, three glutamates, and two waters (red spheres). The putative nucleophilic water is coordinated by Glu433, which is posited to act as a general base catalyst. The tunnel rests on a globular pedestal domain (not shown). The hydrophobic side chains (Ile304, Leu306, Met308, Phe310, Val493, Leu495) on the back side of the β strands of the tunnel floor that comprise the tunnel-pedestal interface are illustrated. The image was prepared using SETOR (17Evans S.V. J. Mol. Graph. 1993; 11: 134-138Crossref PubMed Scopus (1249) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The interior of the tunnel has a distinctively baroque architecture supported by an intricate network of hydrogen bonds and electrostatic interactions, of which a surprisingly high proportion are required for enzyme activity (3Ho C.K. Pei Y. Shuman S. J. Biol. Chem. 1998; 273: 34151-34156Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 14Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Alanine scanning mutagenesis has identified 15 individual side chains within the tunnel that are important for Cet1 function in vitro and in vivo (Fig. 1). Moreover, each of the eight strands of the β barrel contributes at least one functional constituent of the active site. The relevant structural features of the 15 key amino acids have been determined through the analysis of conservative mutational effects (4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 14Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We have grouped the active site residues into three functional classes. Class I residues participate directly in catalysis via coordination of the γ-phosphate (Arg393, Lys456, Arg458) or the essential metal (Glu305, Glu307, Glu496). Class II residues make water-mediated contacts with the γ-phosphate (Asp377, Glu433) or the metal (Asp471, Glu494). Class III residues function indirectly in catalysis via their interactions with other essential side chains and/or their stabilization of the tunnel architecture (Lys409, Arg454, Arg469, Thr473, Glu492).Based on the structure of the putative product complex and the mutational results, we have proposed a one-step in-line mechanism whereby the metal ion (coordinated by acidic residues on the tunnel floor) plus the Arg393, Arg458, and Lys456 side chains (emanating from the walls and roof) activate the γ-phosphate for attack by water and stabilize a pentacoordinate phosphorane transition state in which the attacking water is apical to the β-phosphate leaving group (14Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We further speculated that the Glu433 side chain coordinates the nucleophilic water molecule (Fig. 2) and serves as general base catalyst.Mutational studies have also identified several functionally important hydrophobic residues located on the “outward” face of the β strands of the tunnel (4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 15Lehman K. Ho C.K. Shuman S. J. Biol. Chem. 2001; 276: 14996-15002Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). For example, alanine substitutions for Leu306 (in β1) and Val493 or Leu495 (in β11) result in temperature-sensitive yeast growth and thermolability of catalytic activity in vitro(4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). These hydrophobic residues are in no position to participate directly in catalysis (Fig. 2). It is therefore thought that the deleterious effects of mutating these residues reflects the importance of their hydrophobic interactions with the globular protein core that serves as a pedestal upon which the tunnel floor rests (4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 15Lehman K. Ho C.K. Shuman S. J. Biol. Chem. 2001; 276: 14996-15002Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar).To embellish the picture of the enzyme mechanism and the interactions supporting the tunnel structure, we have extended the mutational analysis to 17 new amino acids denoted by dots in Fig. 1. We thereby identified two important side chains (Arg321 and Asp425) that stabilize Cet1 via their interaction to form a buried salt bridge within the pedestal. A third residue Ser429 stabilizes Cet1 when the protein is synthesized at reduced temperature.DISCUSSIONThe unique fold of Cet1 and the complex, delicate architecture of its active site provide the impetus for a comprehensive mutational analysis of both the catalytic mechanism and the interactions that stabilize the fold. Here we have probed the function of 17 individual side chains by alanine scanning. The mutated residues were either located in the triphosphate tunnel (Ser373, Thr375, Gln405, His411, Ser429, Glu488, Thr490), on the tunnel's outer surface (Ser378, Ser487, Thr489, His491), at the tunnel-pedestal interface (Ile304, Met308), or in the pedestal itself (Asp315, Lys317, Arg321, Asp425). Alanine mutations at 14/17 positions had no significant effect on Cet1 phosphohydrolase activity in vitro and had no effect on Cet1 function in vivo. These negative results are instructive when taken together with prior mutational analyses and the crystal structure of Cet1.We have now mutated all of the hydrophilic amino acids that project into the triphosphate tunnel. Three of the six tunnel residues found here to be nonessential for Cet1 function (Ser373 and Thr375 in β5 and Glu488 in β11) make no contacts in the crystal structure with the metal cofactor, the γ-phosphate, or other amino acid side chains in the tunnel. Thus, it is sensible that these three residues are unimportant for Cet1 function; indeed they are not conserved in other family members (Fig.1). However, the four other tunnel residues analyzed presently do participate in the elaborate network of side chain interactions in the tunnel cavity (Fig. 2). Gln405(Oε) engages in a hydrogen bond with Arg393(Nζ). Arg393 is an essential catalytic residue (14Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) that makes a bidentate interaction with the γ-phosphate (Fig. 2). The hydrogen bond with Gln405 is apparently not required to correctly orient Arg393 for catalysis by Cet1, which is consistent with the lack of conservation of the Gln405 position in other family members (Fig. 1). His411(Nδ) forms a hydrogen bond with Asn431(Oδ), while Asn431(Nδ) interacts Glu305(Oε) and Glu307(Oε) (Fig. 2). Glu305 and Glu307 directly coordinate the metal and are essential for catalysis (3Ho C.K. Pei Y. Shuman S. J. Biol. Chem. 1998; 273: 34151-34156Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). We showed previously that Asn431 is not important for Cet1 function in vivo or in vitro (14Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar); thus it is sensible that His411, which contacts only Asn431, is also nonessential. Thr490(Oγ) forms a hydrogen bond to Glu492(Oε). Glu492 is an essential side chain that forms a salt bridge with Arg454 in β9 (Fig. 2). Arg454 is itself essential for Cet1 function, and it has been suggested that Arg454 contacts the α- or β-phosphate of the substrate (4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 14Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Apparently, the contact of Thr490 with Glu492 is not required for the essential interaction of Glu492 with Arg454.Ser378 in β5 and Ser487, Thr489, and His491 in β11 are located on the outer solvent-exposed surface of the tunnel and are noncontributory to Cet1 function. Ser378, Ser487, and His491 make no contacts with other residues in the crystal structure (Fig. 3). Thr489(Oγ) engages in a hydrogen bond with Glu476(Oε), but this interaction is apparently not important.Studies performed prior to solving the Cet1 structure showed that hydrophobic residues Leu306, Phe310, Val493, and Leu495 play important roles in Cet1 function and stability (4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). It is now apparent from the crystal structure that these side chains are part of a rich network of hydrophobic interactions between residues on the “back” sides of the β strands of the tunnel floor (Fig. 2) and amino acids in the globular pedestal that supports the tunnel. For example, Phe310, which is essential for Cet1 function in vivo and in vitro, makes extensive van der Waals interactions with Val426, with Val493 and Leu495 in β11 (both of which are important for activityin vitro and for thermal stability of Cet1), and with Ile530. Val493 interacts with Ile343 and Leu351 in addition to its contact to Phe310. Leu306(β1), which is also important for triphosphatase activity and thermal stability (4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), interacts with Val289. Here we found that two other hydrophobic residues in β1, Ile304 and Met308, that comprise part of the tunnel-pedestal interface, are not important for Cet1 stabilityin vivo or activity in vitro. Ile304projects into the hydrophobic core of the pedestal and makes van der Waals interactions with Ile296, Leu430, and Leu423. Met308 interacts with Val426 and Phe523.The present study highlights the importance of a buried salt bridge between Arg321 and Asp425 for the stability of Cet1 in vivo and in vitro. The Arg and Asp of this ion pair are strictly conserved in other fungal RNA triphosphatases (CaCet1, Cth1, Pct1) and in P. falciparumPrt1 (Fig. 1), yet neither the Arg nor the Asp are found inChlorella virus RNA triphosphatase cvRtp1. cvRtp1 has an asparagine in lieu of the Arg321 side chain and a glycine instead of the Asp (Fig. 1). We surmise that: (i) there is tight co-evolution of both members of the ion pair, and (ii) theChlorella virus enzyme, which is the smallest member of the metal-dependent RNA triphosphatase family (193-amino acids), has developed alternative strategies to stabilize an active conformation.The Arg321-Asp425 ion pair is part of a wider local network of interactions within the pedestal (Fig. 7). These include hydrogen bonds between Arg321(Nζ) and the main chain carbonyl oxygens of Ser419 and Asp422, which are located in the loop that connects strands β7 and β8. Also, Asp425(Oδ) engages in a hydrogen bond to the backbone amide of Ser419. We infer that the salt bridge and associated backbone contacts stabilize the inferior portions of the β7 and β8 strands and the intervening loop as they project down from the wall of the triphosphatase tunnel deep into the pedestal.The crystal structure provides no immediate explanation for the cold-sensitive folding defects elicited by mutations of Ser429 to Ala and Val and the partial defect of the S429T mutant. Ser429 projects upward from the tunnel floor into the tunnel cavity, yet it makes no direct contact with other amino acids in the crystal. Ser429(Oγ) is pointing toward Nζ of Lys309 (a nonessential side chain), but the interatomic distance of 3.9 Å is too long for a standard hydrogen bond. It is conceivable that the distance to Lys309 is closer in solution. Ser429 might also make water-mediated contacts, either with other amino acids or with the 5′-triphosphate substrate, that are not apparent from the crystal structure of the product complex and that assist in the folding of the protein when it is synthesized at low temperatures in vivo. Ser429 is conserved in all of the other metal-dependent RNA triphosphatases except cvRtp1, which has an alanine instead (Fig. 1). Saccharomyces cerevisiae Cet1 is an essential enzyme that performs the first step of mRNA cap formation: the hydrolysis of the γ-phosphate of nascent pre-mRNA to form a 5′-diphosphate end (1Tsukamoto T. Shibagaki Y. Imajoh-Ohmi S. Murakoshi T. Suzuki M. Nakamura A. Gotoh H. Mizumoto K. Biochem. Biophys. Res. Commun. 1997; 239: 116-122Crossref PubMed Scopus (79) Google Scholar, 2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). Cet1 exemplifies a distinctive family of metal-dependent phosphohydrolases that includes other fungal RNA triphosphatases (e.g. Candida albicansCaCet1, S. cerevisiae Cth1, and Schizosaccharomyces pombe Pct1) and the RNA triphosphatase components of the poxvirus, baculovirus, Chlorella virus, and Plasmodium falciparum RNA capping enzymes (3Ho C.K. Pei Y. Shuman S. J. Biol. Chem. 1998; 273: 34151-34156Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 5Rodriguez C.R. Takagi T. Cho E. Buratowski S. Nucleic Acids Res. 1999; 27: 2182-2188Crossref Scopus (28) Google Scholar, 6Pei Y. Lehman K. Tian L. Shuman S. Nucleic Acids Res. 2000; 28: 1885-1892Crossref PubMed Scopus (36) Google Scholar, 7Pei Y. Schwer B. Hausmann S. Shuman S. Nucleic Acids Res. 2001; 29: 387-396Crossref PubMed Google Scholar, 8Jin J. Dong W. Guarino L.A. J. Virol. 1998; 72: 10011-10019Crossref PubMed Google Scholar, 9Gross C.H. Shuman S. J. Virol. 1998; 72: 10020-10028Crossref PubMed Google Scholar, 10Ho C.K. Martins A. Shuman S. J. Virol. 2000; 74: 5486-5494Crossref PubMed Scopus (26) Google Scholar, 11Ho C.K. Gong C. Shuman S. J. Virol. 2001; 75: 1744-1750Crossref PubMed Scopus (27) Google Scholar, 12Ho C.K. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3050-3055Crossref PubMed Scopus (38) Google Scholar). The signature feature of this enzyme family is the ability to hydrolyze nucleoside triphosphates to nucleoside diphosphates and inorganic phosphate in the presence of either manganese or cobalt. The defining structural elements of the family are two glutamate-rich motifs (strands β1 and β11 in Fig.1) that are required for catalysis. The crystal structure of Cet1 illuminates surprising structural complexity for an enzyme that catalyzes a mundane phosphohydrolase reaction (13Lima C.D. Wang L.K. Shuman S. Cell. 1999; 99: 533-543Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Cet1 adopts a novel active site fold whereby an antiparallel eight-strand β barrel forms a topologically closed “triphosphate tunnel” (Fig.2). The hydrophilic tunnel contained a single sulfate coordinated by multiple basic side chains projecting into the cavity. It was proposed that the side chain interactions of the sulfate reflect contacts made by the enzyme with the γ-phosphate of the triphosphate-terminated RNA or nucleoside triphosphate substrates (13Lima C.D. Wang L.K. Shuman S. Cell. 1999; 99: 533-543Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). A manganese ion within the tunnel cavity is coordinated with octahedral geometry to the sulfate, to the side chain carboxylates of the two glutamates in β1, and to a glutamate in β11. The interior of the tunnel has a distinctively baroque architecture supported by an intricate network of hydrogen bonds and electrostatic interactions, of which a surprisingly high proportion are required for enzyme activity (3Ho C.K. Pei Y. Shuman S. J. Biol. Chem. 1998; 273: 34151-34156Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 14Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Alanine scanning mutagenesis has identified 15 individual side chains within the tunnel that are important for Cet1 function in vitro and in vivo (Fig. 1). Moreover, each of the eight strands of the β barrel contributes at least one functional constituent of the active site. The relevant structural features of the 15 key amino acids have been determined through the analysis of conservative mutational effects (4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 14Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We have grouped the active site residues into three functional classes. Class I residues participate directly in catalysis via coordination of the γ-phosphate (Arg393, Lys456, Arg458) or the essential metal (Glu305, Glu307, Glu496). Class II residues make water-mediated contacts with the γ-phosphate (Asp377, Glu433) or the metal (Asp471, Glu494). Class III residues function indirectly in catalysis via their interactions with other essential side chains and/or their stabilization of the tunnel architecture (Lys409, Arg454, Arg469, Thr473, Glu492). Based on the structure of the putative product complex and the mutational results, we have proposed a one-step in-line mechanism whereby the metal ion (coordinated by acidic residues on the tunnel floor) plus the Arg393, Arg458, and Lys456 side chains (emanating from the walls and roof) activate the γ-phosphate for attack by water and stabilize a pentacoordinate phosphorane transition state in which the attacking water is apical to the β-phosphate leaving group (14Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We further speculated that the Glu433 side chain coordinates the nucleophilic water molecule (Fig. 2) and serves as general base catalyst. Mutational studies have also identified several functionally important hydrophobic residues located on the “outward” face of the β strands of the tunnel (4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 15Lehman K. Ho C.K. Shuman S. J. Biol. Chem. 2001; 276: 14996-15002Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). For example, alanine substitutions for Leu306 (in β1) and Val493 or Leu495 (in β11) result in temperature-sensitive yeast growth and thermolability of catalytic activity in vitro(4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). These hydrophobic residues are in no position to participate directly in catalysis (Fig. 2). It is therefore thought that the deleterious effects of mutating these residues reflects the importance of their hydrophobic interactions with the globular protein core that serves as a pedestal upon which the tunnel floor rests (4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 15Lehman K. Ho C.K. Shuman S. J. Biol. Chem. 2001; 276: 14996-15002Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). To embellish the picture of the enzyme mechanism and the interactions supporting the tunnel structure, we have extended the mutational analysis to 17 new amino acids denoted by dots in Fig. 1. We thereby identified two important side chains (Arg321 and Asp425) that stabilize Cet1 via their interaction to form a buried salt bridge within the pedestal. A third residue Ser429 stabilizes Cet1 when the protein is synthesized at reduced temperature. DISCUSSIONThe unique fold of Cet1 and the complex, delicate architecture of its active site provide the impetus for a comprehensive mutational analysis of both the catalytic mechanism and the interactions that stabilize the fold. Here we have probed the function of 17 individual side chains by alanine scanning. The mutated residues were either located in the triphosphate tunnel (Ser373, Thr375, Gln405, His411, Ser429, Glu488, Thr490), on the tunnel's outer surface (Ser378, Ser487, Thr489, His491), at the tunnel-pedestal interface (Ile304, Met308), or in the pedestal itself (Asp315, Lys317, Arg321, Asp425). Alanine mutations at 14/17 positions had no significant effect on Cet1 phosphohydrolase activity in vitro and had no effect on Cet1 function in vivo. These negative results are instructive when taken together with prior mutational analyses and the crystal structure of Cet1.We have now mutated all of the hydrophilic amino acids that project into the triphosphate tunnel. Three of the six tunnel residues found here to be nonessential for Cet1 function (Ser373 and Thr375 in β5 and Glu488 in β11) make no contacts in the crystal structure with the metal cofactor, the γ-phosphate, or other amino acid side chains in the tunnel. Thus, it is sensible that these three residues are unimportant for Cet1 function; indeed they are not conserved in other family members (Fig.1). However, the four other tunnel residues analyzed presently do participate in the elaborate network of side chain interactions in the tunnel cavity (Fig. 2). Gln405(Oε) engages in a hydrogen bond with Arg393(Nζ). Arg393 is an essential catalytic residue (14Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) that makes a bidentate interaction with the γ-phosphate (Fig. 2). The hydrogen bond with Gln405 is apparently not required to correctly orient Arg393 for catalysis by Cet1, which is consistent with the lack of conservation of the Gln405 position in other family members (Fig. 1). His411(Nδ) forms a hydrogen bond with Asn431(Oδ), while Asn431(Nδ) interacts Glu305(Oε) and Glu307(Oε) (Fig. 2). Glu305 and Glu307 directly coordinate the metal and are essential for catalysis (3Ho C.K. Pei Y. Shuman S. J. Biol. Chem. 1998; 273: 34151-34156Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). We showed previously that Asn431 is not important for Cet1 function in vivo or in vitro (14Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar); thus it is sensible that His411, which contacts only Asn431, is also nonessential. Thr490(Oγ) forms a hydrogen bond to Glu492(Oε). Glu492 is an essential side chain that forms a salt bridge with Arg454 in β9 (Fig. 2). Arg454 is itself essential for Cet1 function, and it has been suggested that Arg454 contacts the α- or β-phosphate of the substrate (4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 14Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Apparently, the contact of Thr490 with Glu492 is not required for the essential interaction of Glu492 with Arg454.Ser378 in β5 and Ser487, Thr489, and His491 in β11 are located on the outer solvent-exposed surface of the tunnel and are noncontributory to Cet1 function. Ser378, Ser487, and His491 make no contacts with other residues in the crystal structure (Fig. 3). Thr489(Oγ) engages in a hydrogen bond with Glu476(Oε), but this interaction is apparently not important.Studies performed prior to solving the Cet1 structure showed that hydrophobic residues Leu306, Phe310, Val493, and Leu495 play important roles in Cet1 function and stability (4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). It is now apparent from the crystal structure that these side chains are part of a rich network of hydrophobic interactions between residues on the “back” sides of the β strands of the tunnel floor (Fig. 2) and amino acids in the globular pedestal that supports the tunnel. For example, Phe310, which is essential for Cet1 function in vivo and in vitro, makes extensive van der Waals interactions with Val426, with Val493 and Leu495 in β11 (both of which are important for activityin vitro and for thermal stability of Cet1), and with Ile530. Val493 interacts with Ile343 and Leu351 in addition to its contact to Phe310. Leu306(β1), which is also important for triphosphatase activity and thermal stability (4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), interacts with Val289. Here we found that two other hydrophobic residues in β1, Ile304 and Met308, that comprise part of the tunnel-pedestal interface, are not important for Cet1 stabilityin vivo or activity in vitro. Ile304projects into the hydrophobic core of the pedestal and makes van der Waals interactions with Ile296, Leu430, and Leu423. Met308 interacts with Val426 and Phe523.The present study highlights the importance of a buried salt bridge between Arg321 and Asp425 for the stability of Cet1 in vivo and in vitro. The Arg and Asp of this ion pair are strictly conserved in other fungal RNA triphosphatases (CaCet1, Cth1, Pct1) and in P. falciparumPrt1 (Fig. 1), yet neither the Arg nor the Asp are found inChlorella virus RNA triphosphatase cvRtp1. cvRtp1 has an asparagine in lieu of the Arg321 side chain and a glycine instead of the Asp (Fig. 1). We surmise that: (i) there is tight co-evolution of both members of the ion pair, and (ii) theChlorella virus enzyme, which is the smallest member of the metal-dependent RNA triphosphatase family (193-amino acids), has developed alternative strategies to stabilize an active conformation.The Arg321-Asp425 ion pair is part of a wider local network of interactions within the pedestal (Fig. 7). These include hydrogen bonds between Arg321(Nζ) and the main chain carbonyl oxygens of Ser419 and Asp422, which are located in the loop that connects strands β7 and β8. Also, Asp425(Oδ) engages in a hydrogen bond to the backbone amide of Ser419. We infer that the salt bridge and associated backbone contacts stabilize the inferior portions of the β7 and β8 strands and the intervening loop as they project down from the wall of the triphosphatase tunnel deep into the pedestal.The crystal structure provides no immediate explanation for the cold-sensitive folding defects elicited by mutations of Ser429 to Ala and Val and the partial defect of the S429T mutant. Ser429 projects upward from the tunnel floor into the tunnel cavity, yet it makes no direct contact with other amino acids in the crystal. Ser429(Oγ) is pointing toward Nζ of Lys309 (a nonessential side chain), but the interatomic distance of 3.9 Å is too long for a standard hydrogen bond. It is conceivable that the distance to Lys309 is closer in solution. Ser429 might also make water-mediated contacts, either with other amino acids or with the 5′-triphosphate substrate, that are not apparent from the crystal structure of the product complex and that assist in the folding of the protein when it is synthesized at low temperatures in vivo. Ser429 is conserved in all of the other metal-dependent RNA triphosphatases except cvRtp1, which has an alanine instead (Fig. 1). The unique fold of Cet1 and the complex, delicate architecture of its active site provide the impetus for a comprehensive mutational analysis of both the catalytic mechanism and the interactions that stabilize the fold. Here we have probed the function of 17 individual side chains by alanine scanning. The mutated residues were either located in the triphosphate tunnel (Ser373, Thr375, Gln405, His411, Ser429, Glu488, Thr490), on the tunnel's outer surface (Ser378, Ser487, Thr489, His491), at the tunnel-pedestal interface (Ile304, Met308), or in the pedestal itself (Asp315, Lys317, Arg321, Asp425). Alanine mutations at 14/17 positions had no significant effect on Cet1 phosphohydrolase activity in vitro and had no effect on Cet1 function in vivo. These negative results are instructive when taken together with prior mutational analyses and the crystal structure of Cet1. We have now mutated all of the hydrophilic amino acids that project into the triphosphate tunnel. Three of the six tunnel residues found here to be nonessential for Cet1 function (Ser373 and Thr375 in β5 and Glu488 in β11) make no contacts in the crystal structure with the metal cofactor, the γ-phosphate, or other amino acid side chains in the tunnel. Thus, it is sensible that these three residues are unimportant for Cet1 function; indeed they are not conserved in other family members (Fig.1). However, the four other tunnel residues analyzed presently do participate in the elaborate network of side chain interactions in the tunnel cavity (Fig. 2). Gln405(Oε) engages in a hydrogen bond with Arg393(Nζ). Arg393 is an essential catalytic residue (14Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) that makes a bidentate interaction with the γ-phosphate (Fig. 2). The hydrogen bond with Gln405 is apparently not required to correctly orient Arg393 for catalysis by Cet1, which is consistent with the lack of conservation of the Gln405 position in other family members (Fig. 1). His411(Nδ) forms a hydrogen bond with Asn431(Oδ), while Asn431(Nδ) interacts Glu305(Oε) and Glu307(Oε) (Fig. 2). Glu305 and Glu307 directly coordinate the metal and are essential for catalysis (3Ho C.K. Pei Y. Shuman S. J. Biol. Chem. 1998; 273: 34151-34156Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). We showed previously that Asn431 is not important for Cet1 function in vivo or in vitro (14Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar); thus it is sensible that His411, which contacts only Asn431, is also nonessential. Thr490(Oγ) forms a hydrogen bond to Glu492(Oε). Glu492 is an essential side chain that forms a salt bridge with Arg454 in β9 (Fig. 2). Arg454 is itself essential for Cet1 function, and it has been suggested that Arg454 contacts the α- or β-phosphate of the substrate (4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 14Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Apparently, the contact of Thr490 with Glu492 is not required for the essential interaction of Glu492 with Arg454. Ser378 in β5 and Ser487, Thr489, and His491 in β11 are located on the outer solvent-exposed surface of the tunnel and are noncontributory to Cet1 function. Ser378, Ser487, and His491 make no contacts with other residues in the crystal structure (Fig. 3). Thr489(Oγ) engages in a hydrogen bond with Glu476(Oε), but this interaction is apparently not important. Studies performed prior to solving the Cet1 structure showed that hydrophobic residues Leu306, Phe310, Val493, and Leu495 play important roles in Cet1 function and stability (4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). It is now apparent from the crystal structure that these side chains are part of a rich network of hydrophobic interactions between residues on the “back” sides of the β strands of the tunnel floor (Fig. 2) and amino acids in the globular pedestal that supports the tunnel. For example, Phe310, which is essential for Cet1 function in vivo and in vitro, makes extensive van der Waals interactions with Val426, with Val493 and Leu495 in β11 (both of which are important for activityin vitro and for thermal stability of Cet1), and with Ile530. Val493 interacts with Ile343 and Leu351 in addition to its contact to Phe310. Leu306(β1), which is also important for triphosphatase activity and thermal stability (4Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), interacts with Val289. Here we found that two other hydrophobic residues in β1, Ile304 and Met308, that comprise part of the tunnel-pedestal interface, are not important for Cet1 stabilityin vivo or activity in vitro. Ile304projects into the hydrophobic core of the pedestal and makes van der Waals interactions with Ile296, Leu430, and Leu423. Met308 interacts with Val426 and Phe523. The present study highlights the importance of a buried salt bridge between Arg321 and Asp425 for the stability of Cet1 in vivo and in vitro. The Arg and Asp of this ion pair are strictly conserved in other fungal RNA triphosphatases (CaCet1, Cth1, Pct1) and in P. falciparumPrt1 (Fig. 1), yet neither the Arg nor the Asp are found inChlorella virus RNA triphosphatase cvRtp1. cvRtp1 has an asparagine in lieu of the Arg321 side chain and a glycine instead of the Asp (Fig. 1). We surmise that: (i) there is tight co-evolution of both members of the ion pair, and (ii) theChlorella virus enzyme, which is the smallest member of the metal-dependent RNA triphosphatase family (193-amino acids), has developed alternative strategies to stabilize an active conformation. The Arg321-Asp425 ion pair is part of a wider local network of interactions within the pedestal (Fig. 7). These include hydrogen bonds between Arg321(Nζ) and the main chain carbonyl oxygens of Ser419 and Asp422, which are located in the loop that connects strands β7 and β8. Also, Asp425(Oδ) engages in a hydrogen bond to the backbone amide of Ser419. We infer that the salt bridge and associated backbone contacts stabilize the inferior portions of the β7 and β8 strands and the intervening loop as they project down from the wall of the triphosphatase tunnel deep into the pedestal. The crystal structure provides no immediate explanation for the cold-sensitive folding defects elicited by mutations of Ser429 to Ala and Val and the partial defect of the S429T mutant. Ser429 projects upward from the tunnel floor into the tunnel cavity, yet it makes no direct contact with other amino acids in the crystal. Ser429(Oγ) is pointing toward Nζ of Lys309 (a nonessential side chain), but the interatomic distance of 3.9 Å is too long for a standard hydrogen bond. It is conceivable that the distance to Lys309 is closer in solution. Ser429 might also make water-mediated contacts, either with other amino acids or with the 5′-triphosphate substrate, that are not apparent from the crystal structure of the product complex and that assist in the folding of the protein when it is synthesized at low temperatures in vivo. Ser429 is conserved in all of the other metal-dependent RNA triphosphatases except cvRtp1, which has an alanine instead (Fig. 1)." @default.
W2145070389 created "2016-06-24" @default.
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W2145070389 date "2001-08-01" @default.
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W2145070389 title "Functional Groups Required for the Stability of Yeast RNA Triphosphatase in Vitro and in Vivo" @default.
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