Functional groups required for the stability of yeast RNA triphosphatase in vitro and in vivo.

Cet1, the RNA triphosphatase component of the yeast mRNA capping apparatus, catalyzes metal-dependent gamma-phosphate hydrolysis within the hydrophilic interior of an eight-strand beta 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 (Ser(373), Thr(375), Gln(405), His(411), Ser(429), Glu(488), Thr(490)), on the tunnel's outer surface (Ser(378), Ser(487), Thr(489), His(491)), at the tunnel-pedestal interface (Ile(304), Met(308)) and in the pedestal (Asp(315), Lys(317), Arg(321), Asp(425)). 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. Arg(321) and Asp(425) interact to form a salt bridge within the pedestal that tethers two of the strands of the tunnel. Mutations R321Q and D411N resulted in ts 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 Ser(429) 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 degrees C, but were inactive when produced at 17 degrees C. Replacement of Ser(429) by threonine partially suppressed the cold sensitivity of the Cet1 phosphohydrolase, but did not suppress the cs growth defect in yeast.

manganese or cobalt. The defining structural elements of the family are two glutamate-rich motifs (strands ␤1 and ␤11 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 (13). Cet1 adopts a novel active site fold whereby an antiparallel eight-strand ␤ 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 ␥-phosphate of the triphosphate-terminated RNA or nucleoside triphosphate substrates (13). 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 ␤1, and to a glutamate in ␤11.
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 (3,4,14). 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 ␤ 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 (4,14). We have grouped the active site residues into three functional classes. Class I residues participate directly in catalysis via coordination of the ␥-phosphate (Arg 393 , Lys 456 , Arg 458 ) or the essential metal (Glu 305 , Glu 307 , Glu 496 ). Class II residues make water-mediated contacts with the ␥-phosphate (Asp 377 , Glu 433 ) or the metal (Asp 471 , Glu 494 ). Class III residues function indirectly in catalysis via their interactions with other essential side chains and/or their stabilization of the tunnel architecture (Lys 409 , Arg 454 , Arg 469 , Thr 473 , Glu 492 ).
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 Arg 393 , Arg 458 , and Lys 456 side chains (emanating from the walls and roof) activate the ␥-phosphate for attack by water and stabilize a pentacoordinate phosphorane transition state in which the attacking water is apical to the ␤-phosphate leaving group (14). We further speculated that the Glu 433 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 ␤ strands of the tunnel (4,15). For example, alanine substitutions for Leu 306 (in ␤1) and Val 493 or Leu 495 (in ␤11) result in temperature-sensitive yeast growth and thermolabil-ity of catalytic activity in vitro (4). 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 (4,15).
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 (3,4,14,15) had identified Cet1 residues at which alanine substitution resulted either in loss of function (!), thermosensitive function (⌬), or no significant effect on function (ϩ). The 17 amino acids of Cet1 that were targeted for mutation in the present study are indicated by dots.
FIG. 2. The 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 (␥-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 Glu 433 , which is posited to act as a general base catalyst. The tunnel rests on a globular pedestal domain (not shown). The hydrophobic side chains (Ile 304 , Leu 306 , Met 308 , Phe 310 , Val 493 , Leu 495 ) on the back side of the ␤ strands of the tunnel floor that comprise the tunnel-pedestal interface are illustrated. The image was prepared using SETOR (17). Ser 429 stabilizes Cet1 when the protein is synthesized at reduced temperature.

Expression and Purification of Mutated Versions of Yeast RNA
Triphosphatase-Missense mutations were introduced into the CET1(201-549) gene by polymerase chain reaction, and the mutated genes were inserted into the bacterial expression vector pET16b as described previously (14). The presence of the desired mutations was confirmed in every case by DNA sequencing; the inserted fragments were sequenced completely to exclude the acquisition of unwanted mutations during amplification and cloning. The pET plasmids were transformed into Escherichia coli BL21(DE3). Single transformants were inoculated into 100 ml of LB medium containing 0.1 mg/ml ampicillin and grown at 37°C until the A 600 reached 0.5. Recombinant protein expression was induced by placing the culture on ice for 30 min, followed by addition of isopropyl-1-thio-␤-D-galactopyranoside (IPTG) 1 to 0.4 mM and ethanol to 2% final concentration. The cultures were then incubated for 24 h at 18°C with constant shaking. Cells were harvested by centrifugation and the pellet was stored at Ϫ80°C. All subsequent procedures were performed at 4°C. The procedures for cell lysis, and recombinant protein purification by nickel-agarose chromatography, were as described previously (17). Protein concentrations were determined by the Bio-Rad dye-binding method with bovine serum albumin as the standard.
Cold-sensitive (cs) Cet1 mutants S429A, S429T, and S429V were expressed in parallel in bacteria at 18 and 37°C. Single E. coli BL21(DE3) transformants containing pET plasmids bearing the WT or cs mutant CET1(201-549) genes were inoculated into 100 ml of LB medium containing 0.1 mg/ml ampicillin and grown at 37°C until the A 600 reached 0.5. The cultures were then split in half. One half-culture was cooled on ice, induced with IPTG and ethanol, and then incubated for 24 h at 18°C as described above. The other half-culture was maintained at 37°C, adjusted to 0.4 mm IPTG, and incubated for 24 h at 37°C with constant shaking. The recombinant proteins were purified from soluble lysates as described above.
ATPase Assay-Reaction mixtures (20 l) containing 50 mM Tris-HCl (pH 7.0), 5 mM dithiothreitol, 2 mM MnCl 2 , 1 mM [␥-32 P]ATP, and enzyme were incubated for 15 min at 30°C. The reactions were quenched by adding 5 l of 5 M formic acid. Aliquots of the mixtures were applied to a polyethyleneimine-cellulose TLC plate, which was developed with 1 M formic acid, 0.5 M LiCl. The extent of 32 P i release was quantitated by scanning the chromatogram with a FUJIX phosphorimager.
Mutational Effects on Cet1 Function in Vivo-NdeI/BamHI fragments encoding mutated versions of Cet1(201-549) were excised from the respective pET16b-CET1 plasmids and inserted into the yeast CEN TRP1 plasmid pCET1-5Ј3Ј (2) so that expression of the inserted gene is under the control of the natural CET1 promoter. The plasmids were then introduced into S. cerevisiae strain YBS20 (MATa trp1 his3 ura3 leu2 ade2 can1 cet1::LEU2 p360-CET1) that is deleted at the chromosomal CET1 locus. Growth of YBS20 depends on maintenance of plasmid p360-CET1 (CEN URA3 CET1). Transformants were selected on SD(-Trp) agar. Individual Trp ϩ isolates were patched to SD(-Trp) agar and then streaked on agar plates containing 0.75 mg/ml of 5-FOA (5-fluoroorotic acid). Individual 5-FOA-resistant colonies were picked and patched on YPD agar. Two isolates of each mutant were then streaked on YPD agar at 16, 22, 30, and 37°C. Growth was assessed as follows: ϩϩ indicates wild-type colony size at all temperatures; ts indicates growth at 16, 22, and 30°C, but no growth at 37°C; cs indicates growth at 22, 30, and 37°C, but no growth at 16°C. The in vivo phenotypes of the mutants were assessed by plasmid shuffle in a yeast cet1⌬ strain as described under "Experimental Procedures." Triphosphatase specific activity of the purified mutant Cet1(201-549) proteins is normalized to that of the wild-type protein (defined as 100%). The atomic contacts made by the wild-type side chains in the Cet1 crystal structure (13) are indicated in the column on the right.

In Vivo Mutational Analysis of Yeast
control of the natural CET1 promoter. The plasmids were transformed into a cet1⌬ strain in which the chromosomal CET1 locus was deleted and replaced by LEU2. Growth of cet1⌬ is contingent on maintenance of a wild-type CET1 allele on a CEN URA3 plasmid. Therefore, cet1⌬ is unable to grow on agar medium containing 5-FOA, a drug that selects against the URA3 plasmid, unless it is transformed with a biologically active CET1 allele.
Effects of Alanine Mutations on Triphosphatase Activity-The Cet1(201-549)-Ala polypeptides were expressed as N-terminal His-tagged derivatives in E. coli at 18°C in parallel with the wild-type Cet1(201-549) protein. The recombinant proteins were purified from soluble bacterial extracts by nickel-agarose chromatography. SDS-polyacrylamide gel electrophoresis analysis showed that the 44-kDa Cet1(201-549) protein was the predominant polypeptide in every case (Fig. 5).
The phosphohydrolase activities of the wild-type and mutant proteins were assayed by the release of 32 P i from 1 mM [␥-32 P]ATP during a 15-min reaction in the presence of 2 mM manganese. Two titration experiments were performed for each protein, and the specific activities were calculated from the average of the slopes of the titration curves in the linear range of enzyme dependence. The wild-type Cet1(201-549) preparation released 210 pmol of 32 P i /ng of protein. The specific activities of the 16 Ala mutants, normalized to the wild-type value (defined as 100%), are listed in Fig. 3. Most of the mutants displayed full activity, and 15/16 had a specific activity within a factor of two of the wild-type enzyme. E488A was 39% as active as the wild-type; this effect does not meet the criterion of a 4-fold decrement in specific activity that we adopted previously as the threshold for a significant mutational effect (14).
Mutants R321A and D425A Are Thermolabile in Vitro-R321A and D425A were fully active in vitro, yet both mutations elicited a ts growth defect in vivo. To evaluate the basis for the ts phenotype, we compared the thermal stability of wild-type Cet1(201-549) to that of the R321A and D425A mutants. This was done by preincubating the purified enzymes for 10 min at 30, 35, 40, 45, or 50°C, followed by quenching on ice. The protein samples were then assayed for ATPase activity at 22°C. The data were expressed as the ratio of ATP hydrolysis by enzyme preincubated at a given test temperature to the activity of the respective unheated control. The thermal inactivation curves are plotted in Fig. 6. The activity of wild-type Cet1(201-549) was stable to preincubation at 30°C and reduced only modestly by treatment at 40°C. The activity fell off more sharply at 45°C (to 40% of the unheated control value) and 50°C (to 18% of the control value). The R321A and D425A proteins, which were temperature-sensitive in vivo, were clearly thermolabile in vitro (Fig. 6). The inactivation curves for both proteins were shifted 15°C to the left relative to the wild-type enzyme.
Structure-Activity Relationships at Arg 321 and Asp 425 -Arg 321 and Asp 425 form a salt bridge within the globular pedestal of Cet1. The salt bridge tethers the inferior portions of the ␤7 and ␤8 strands and the intervening loop, which project deep into the pedestal, to the distal end of ␤1 and the following loop, which comprise the platform-like structure at the entrance of the triphosphate tunnel (13) (Fig. 7). The replacement of only one member of an ion pair by alanine eventuates an unopposed charged residue within the protein core, which may by itself destabilize the structure of the mutant enzyme (16). Thus, the effects of the single R321A and D425A mutations do not indicate whether the salt bridge is inherently stabilizing. To address this issue, we tested the effects of a double-alanine mutation R321A-D425A. The rationale was that if an unopposed buried charge was responsible for destabilization of Cet1, then the double-Ala mutation should restore thermal stability. This is not what was observed. R321A-D425A yeast cells grew at 30°C, but failed to grow at 37°C (Fig. 3 and data not shown), just like the R321A and D425A single mutants. The R321A-D425A protein produced in bacteria at 18°C (Fig. 5) retained phosphohydrolase activity (73% of wild-type specific activity; Fig. 3). Moreover, R321A-D425A displayed the same hypersensitivity to heat inactivation as the singly mutated R321A and D425A proteins (Fig. 6). We conclude that the interaction between Arg 321 and Asp 425 contributes significantly to the stability of Cet1.
To better understand the stabilizing forces, we introduced conservative changes at Arg 321 and Asp 425 . Arg 321 was replaced by lysine and glutamine, and Asp 425 was substituted by asparagine. R321K cells grew at 37°C, whereas R321Q and D425N cells displayed the same ts growth defect as the alanine mutants (Fig. 4). Thus, the ionic interaction between the two residues is essential for Cet1 stability in vivo. The R321K, R321Q, and D425N proteins were produced in E. coli at 18°C and purified from soluble bacterial extracts by nickel-agarose chromatography (Fig. 5). The phosphohydrolase specific activities of the conservative mutants were 86 -91% of the wild-type activity (Fig. 3). R321Q and D425N evinced the same hypersensitivity to heat inactivation as R321A and D425A (Fig. 6). The instructive finding was that replacement of Arg 321 by lysine completely restored the thermal stability of R321K to the level of the wild-type enzyme (Fig. 6). Thus, the conservative Mutations of Ser 429 Elicit Cold-sensitive Phenotypes-The present alanine scan also targeted Ser 429 in strand ␤8 (Fig. 1). The effects of the S429A mutation were initially puzzling, insofar as: (i) yeast cet1⌬ cells transformed with S429A readily formed 5-FOA-resistant colonies, and the resulting S429A cells grew as well as WT cells on YPD agar at 30°C, yet (ii) recombinant S429A protein produced in bacteria at 18°C displayed Յ1% the phosphohydrolase activity of the wild-type enzyme (data not shown). We considered the possibility that the S429A mutation might have resulted in a cold-sensitive folding defect; thus we examined the growth of S429A yeast cells on YPD agar over a broader temperature range. We found that S429A cells grew at 22, 30, and 37°C, but not at 16°C (not shown). Thus, S429A elicited a cold-sensitive (cs) yeast growth defect. Replacement of Ser 429 with either valine or threonine also resulted in cs yeast growth (Fig. 8B).
To better understand this phenotype, we expressed the S429A, S249V, and S429T proteins in bacteria at low and high temperatures. Cultures of bacteria bearing each expression plasmid (or the wild-type Cet1(201-549) plasmid) were ampli-fied initially at 37°C and then split, with one-half induced with IPTG to express the recombinant yeast enzyme at 18°C and the other induced with IPTG at 37°C. The recombinant proteins were purified in parallel from the low versus high temperature-induced bacteria (the purification being conducted at 4°C in both cases). The purity and yield of the recombinant proteins were similar at both induction temperatures ( Fig. 8A and data not shown). The S429A, S429T, and S429V mutants were catalytically active when the proteins were produced at 37°C, with specific activities 75-90% of the wild-type enzyme expressed in parallel at 37°C (Fig. 8B). However, S429A and S429V were grossly defective in ␥-phosphate hydrolysis when the proteins were produced at 18°C (1% and Ͻ0.5% of wildtype activity, respectively), consistent with the cs growth defect in yeast. The introduction of threonine partially restored triphosphatase activity, to one-fifth of the wild-type level, when the S429T protein was produced at 18°C (Fig. 8B). We conclude that the hydroxyl moiety at position 429 is somehow critical for proper folding of the Cet1 when it is synthesized at low temperatures. DISCUSSION 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 (Ser 373 , Thr 375 , Gln 405 , His 411 , Ser 429 , Glu 488 , Thr 490 ), on the tunnel's outer surface (Ser 378 , Ser 487 , Thr 489 , His 491 ), at the tunnel-pedestal interface (Ile 304 , Met 308 ), or in the pedestal itself (Asp 315 , Lys 317 , Arg 321 , Asp 425 ). 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 (Ser 373 and Thr 375 in ␤5 and Glu 488 in ␤11) make no contacts in the crystal structure with the metal cofactor, the ␥-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). Gln 405 (O⑀) engages in a hydrogen bond with Arg 393 (N). Arg 393 is an essential catalytic residue Aliquots (20 l) of wild-type Cet1(201-549) and the indicated mutants were preincubated for 10 min at 30, 35, 40, 45, or 50°C and then quenched on ice. Control aliquots were kept on ice throughout the pretreatment. ATPase reaction mixtures contained 75 ng of control or preheated enzymes. This amount of unheated control WT and mutant enzymes was sufficient to hydrolyze between 34 and 54% of the input ATP during the 15-min ATPase reaction at 22°C. The extent of ATP hydrolysis by preheated enzyme was normalized to that of the unheated control enzyme (defined as 1.0). The normalized activities are plotted as a function of preincubation temperature. Each datum is the average of two separate thermal inactivation experiments. (14) that makes a bidentate interaction with the ␥-phosphate (Fig. 2). The hydrogen bond with Gln 405 is apparently not required to correctly orient Arg 393 for catalysis by Cet1, which is consistent with the lack of conservation of the Gln 405 position in other family members (Fig. 1). His 411 (N␦) forms a hydrogen bond with Asn 431 (O␦), while Asn 431 (N␦) interacts Glu 305 (O⑀) and Glu 307 (O⑀) (Fig. 2). Glu 305 and Glu 307 directly coordinate the metal and are essential for catalysis (3,4). We showed previously that Asn 431 is not important for Cet1 function in vivo or in vitro (14); thus it is sensible that His 411 , which contacts only Asn 431 , is also nonessential. Thr 490 (O␥) forms a hydrogen bond to Glu 492 (O⑀). Glu 492 is an essential side chain that forms a salt bridge with Arg 454 in ␤9 (Fig. 2). Arg 454 is itself essential for Cet1 function, and it has been suggested that Arg 454 contacts the ␣or ␤-phosphate of the substrate (4,14). Apparently, the contact of Thr 490 with Glu 492 is not required for the essential interaction of Glu 492 with Arg 454 .
Ser 378 in ␤5 and Ser 487 , Thr 489 , and His 491 in ␤11 are located on the outer solvent-exposed surface of the tunnel and are noncontributory to Cet1 function. Ser 378 , Ser 487 , and His 491 make no contacts with other residues in the crystal structure (Fig. 3). Thr 489 (O␥) engages in a hydrogen bond with Glu 476 (O⑀), but this interaction is apparently not important.
Studies performed prior to solving the Cet1 structure showed that hydrophobic residues Leu 306 , Phe 310 , Val 493 , and Leu 495 play important roles in Cet1 function and stability (4). 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 ␤ strands of the tunnel floor (Fig. 2) and amino acids in the globular pedestal that supports the tunnel. For example, Phe 310 , which is essential for Cet1 function in vivo and in vitro, makes extensive van der Waals interactions with Val 426 , with Val 493 and Leu 495 in ␤11 (both of which are important for activity in vitro and for thermal stability of Cet1), and with Ile 530 . Val 493 interacts with Ile 343 and Leu 351 in addition to its contact to Phe 310 . Leu 306 (␤1), which is also important for triphosphatase activity and thermal stability (4), interacts with Val 289 . Here we found that two other hydrophobic residues in ␤1, Ile 304 and Met 308 , that comprise part of the tunnel-pedestal interface, are not important for Cet1 stability in vivo or activity in vitro. Ile 304 projects into the hydrophobic core of the pedestal and makes van der Waals interactions with Ile 296 , Leu 430 , and Leu 423 . Met 308 interacts with Val 426 and Phe 523 . The present study highlights the importance of a buried salt bridge between Arg 321 and Asp 425 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. falciparum Prt1 (Fig. 1), yet neither the Arg nor the Asp are found in Chlorella virus RNA triphosphatase cvRtp1. cvRtp1 has an asparagine in lieu of the Arg 321 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) the Chlorella 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 Arg 321 -Asp 425 ion pair is part of a wider local network of interactions within the pedestal (Fig. 7). These include hydrogen bonds between Arg 321 (N) and the main chain carbonyl oxygens of Ser 419 and Asp 422 , which are located in the loop that connects strands ␤7 and ␤8. Also, Asp 425 (O␦) engages in a hydrogen bond to the backbone amide of Ser 419 . We infer that the salt bridge and associated backbone contacts stabilize the inferior portions of the ␤7 and ␤8 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 Ser 429 to Ala and Val and the partial defect of the S429T mutant. Ser 429 projects upward from the tunnel floor into the tunnel cavity, yet it makes no direct contact with other amino acids in the crystal. Ser 429 (O␥) is pointing toward N of Lys 309 (a non-essential 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 Lys 309 is closer in solution. Ser 429 might also make water-mediated contacts, either with other amino acids or with the 5Ј-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. Ser 429 is conserved in all of the other metal-dependent RNA triphosphatases except cvRtp1, which has an alanine instead (Fig. 1).