From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021
Received for publication, February 1, 2001
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ABSTRACT |
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Cet1, the RNA triphosphatase component of
the yeast mRNA capping apparatus, catalyzes
metal-dependent RNA 5' triphosphatase executes the first step of mRNA cap
formation, the hydrolysis of the The S. cerevisiae RNA triphosphatase (Cet1) is essential for
yeast cell growth (5, 16). The crystal structure of Cet1 illuminates a
surprising structural complexity for an enzyme that catalyzes a
seemingly mundane phosphohydrolase reaction (17). Cet1 adopts a novel
enzyme fold whereby an antiparallel 8-strand Initial efforts to identify the Cet1 active site were made without the
benefit of an atomic structure and entailed alanine scanning of strands
The observed mutational effects are interpretable in light of the Cet1
crystal structure (17). For example, four of the essential glutamates
(306, 307, 494, and 496) coordinate directly, or via water, to the
essential metal ion, and the essential Lys456 coordinates
the sulfate (i.e. the The Cet1 crystal structure now provides a blueprint for further
mutagenesis aimed at identifying all of the functionally important side
chains within the triphosphate tunnel. Here we analyzed the contributions of 14 individual charged or polar amino acids in seven of
the Expression and Purification of Mutated Versions of Yeast RNA
Triphosphatase--
Missense mutations were introduced into the
CET1(201-549) gene by polymerase chain
reaction by using the twostage overlap extension method
(18). The mutated genes were digested with NdeI and
BamHI and then inserted into the bacterial expression vector
pET16b. 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 of ampicillin
and grown at 37 °C until the A600 reached
0.5. Recombinant protein production was induced by placing the culture
on ice for 30 min, followed by addition of
isopropyl-1-thio- ATPase Assay--
Reaction mixtures (20 µl) containing 50 mM Tris-HCl, pH 7.0, 5 mM DTT, 2 mM
MnCl2, 1 mM [ RNA Triphosphatase Assay--
Reaction mixtures (10 µl)
containing 50 mM Tris-HCl, pH 7.5, 5 mM DTT, 1 mM MgCl2, 20 pmol of 5'
[ Mutational Effects on RNA Triphosphatase 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
pCET1-5'3' (5) 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( Structure-based Mutational Analysis of Yeast RNA
Triphosphatase--
Many of the charged and polar amino acids in the
Effects of Alanine Mutations on Nucleoside Triphosphatase
Activity--
The nucleoside triphosphatase activities of the
wild-type and mutant proteins were assayed by the release of
32Pi from 1 mM
[ Effects of Alanine Mutations on RNA Triphosphatase
Activity--
The RNA triphosphatase activities of the wild-type and
mutant proteins were assayed by the release of
32Pi from 2 µM
[ Effects of Alanine Mutations on Cet1 Function in Vivo--
The
CET1(201-549)-Ala genes were cloned into a CEN
TRP1 vector so as to place them under the transcriptional control
of the natural CET1 promoter. The plasmids were transformed
into the cet1
In contrast, the eight other CET1(201-549)-Ala mutants did
support growth of cet1
The in vivo phenotypes of the alanine mutants correlated
with their in vitro activities. The most severe catalytic
defects resulted in lethal phenotypes in vivo, whereas
catalytically benign mutations had no effect on cell growth. Even the
D397A mutation, which reduced RNA triphosphatase activity to
one-fourth the wild-type level in vitro, had no apparent
effect in vivo (Table I). The marginally functional mutants
D377A and K409A, with 7 and 5% of wild-type RNA
triphosphatase activity, respectively, were nonetheless capable of
sustaining growth at 25 and 30 °C. These findings underscore the
suggestion from earlier mutational analyses (7) that yeast cells
require a threshold level of RNA triphosphatase activity for growth and
can tolerate at least a 5-fold reduction before growth is overtly affected.
Mutants D377A and K409A Are Thermolabile in Vitro--
The thermal
stability of wild-type Cet1(201-549) and the D377A and K409A mutants
was tested by preincubation of the purified enzyme preparations 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
levels of input WT, D377A, and K409A enzyme in the assay mixtures were
adjusted to achieve similar extents of ATP hydrolysis in the control
reaction mixtures containing unheated enzyme. 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.
4. The activity of WT Cet1(201-549) was
stable to preincubation at 30 °C and reduced only modestly by
treatment at 35 and 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). D377A and K409A were clearly thermolabile. The
inactivation curve for K409A was shifted almost 15 °C to the left
relative to the WT enzyme, and a shift to the left of 10 °C was
observed for D377A.
Structure-Activity Relationships at Essential Amino Acids--
Two
conservative substitutions were introduced at each of the 6 residues
defined by alanine scanning as essential for function in
vitro and in vivo (Arg393,
Glu433, Arg458, Arg469,
Asp471, and Thr473). The 12 recombinant
proteins were purified from soluble bacterial extracts by
nickel-agarose chromatography (Fig. 3). The
manganese-dependent ATPase and
magnesium-dependent RNA triphosphatase activities of the
conservative mutants were determined by protein titration (Table I).
The mutant alleles were also cloned into a CEN TRP1 vector
under the transcriptional control of the natural CET1
promoter and tested by plasmid shuffle for functional complementation
of the cet1
Substitution of any of the three essential arginines (393, 458, and
469) by lysine or glutamine failed to restore the NTPase and RNA
triphosphatase activities above the levels seen for the respective
alanine-substituted proteins. Moreover, the R393K, R393Q, R458K, R458Q, R469K,
and R469Q mutations were lethal in vivo. We
surmise that Cet1 function requires a bidentate arginine side chain at
each position and not merely positive charge.
Replacing the essential Glu433 side chain by either
aspartate or glutamine had no salutary effect on the NTPase or RNA
triphosphatase activities in vitro, and the E433D
and E433Q alleles were lethal in vivo. Similarly,
changing the essential Asp471 residue to either glutamate
or asparagine yielded catalytically defective proteins that were
nonfunctional in vivo. Thus, an acidic moiety is strictly
essential at both positions 433 and 471, and the distance of the
carboxylate from the main chain is also critical for Cet1 activity.
The essential Thr473 residue was replaced by serine and
valine. Whereas the T473V protein was as defective in Structure-Activity Relationships at Asp377 and
Lys409--
Conservative changes were also introduced at
positions Asp377 and Lys409, where alanine
mutations had resulted in partial loss of function in vitro
and a ts growth defect. The D477A, D477E, K409R, and K409Q proteins
were purified from soluble bacterial extracts by nickel-agarose
chromatography (Fig. 3). The ATPase and RNA triphosphatase activities
were restored to WT levels when Asp377 was replaced by
asparagine or glutamate, and the D377N and D377E strains grew normally at all temperatures (Table I). The sufficiency of
asparagine suggests that a polar side chain with hydrogen bonding potential is the relevant functional group. The noteworthy findings at
position Lys409 were that the conservative changes to
arginine and glutamine were at least an order of magnitude more
deleterious that the alanine mutation (Table I). Consequently,
K409R and K409Q were lethal in
vivo.
Using the crystal structure of Cet1 as a guide for mutational
analysis, we have identified 6 side chains within the triphosphate tunnel that are essential for RNA triphosphatase activity in
vitro and in vivo. Five of the six critical amino acids
(Arg393 in The present findings, together with earlier mutational analyses (6, 7,
9), reveal an unusually complex active site for the yeast RNA
triphosphatase, in which a total of 15 individual side chains in the
tunnel cavity are essential or important for catalysis, and each of the
8 strands of the First, we can group the tunnel residues into three functional classes
(Fig. 5). Class I residues participate
directly in catalysis via coordination of the phosphate hydrolysis within the
hydrophilic interior of a topologically closed 8-strand
barrel (the
"triphosphate tunnel"). We used structure-guided alanine scanning
to identify 6 side chains within the triphosphate tunnel that are
essential for phosphohydrolase activity in vitro and
in vivo: Arg393, Glu433,
Arg458, Arg469, Asp471 and
Thr473. Alanine substitutions at two positions,
Asp377 and Lys409, resulted in partial
catalytic defects and a thermosensitive growth phenotype.
Structure-function relationships were clarified by introducing
conservative substitutions. Five residues were found to be
nonessential: Lys309, Ser395,
Asp397, Lys427, Asn431, and
Lys474. The present findings, together with earlier
mutational analyses, reveal an unusually complex active site in which
15 individual side chains in the tunnel cavity are important for
catalysis, and each of the 8 strands of the
barrel contributes at
least one functional constituent. The active site residues fall into three classes: (i) those that participate directly in catalysis via
coordination of the
phosphate or the metal; (ii) those that make
critical water-mediated contacts with the
phosphate or the metal;
and (iii) those that function indirectly via interactions with other
essential side chains or by stabilization of the tunnel structure.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phosphate of nascent pre-mRNA to form a 5' diphosphate end. Two classes of eukaryotic RNA
triphosphatases can be distinguished on the basis of their cofactor
requirements, structures, and catalytic mechanisms (1). The RNA
triphosphatases of metazoans and higher plants belong to the cysteine
phosphatase enzyme superfamily (2). Metazoan RNA triphosphatases cleave the
-
phosphoanhydride bond via the formation and hydrolysis of a
covalent enzyme-(cysteinyl-S)-phosphate intermediate
(3).1 The active site
cysteine resides within the signature phosphate-loop motif
HCXXXXXR(S/T). The cysteine phosphatase enzymes do not
require a metal cofactor and are characteristically inhibited by
divalent cations. In contrast, the RNA triphosphatases of fungal
species such as Saccharomyces cerevisiae, Candida
albicans, and Schizosaccharomyces pombe are strictly
dependent on a divalent cation (5-10). The fungal enzymes belong to a
new family of metal-dependent phosphohydrolases that
embraces the poxvirus, baculovirus, phycodnavirus, and
Plasmodium falciparum RNA capping enzymes (6, 11-15). The
signature biochemical property 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 features of the metal-dependent RNA
triphosphatases are two glutamate-rich motifs (motifs A and C; see Fig.
1) that are required for catalysis by every family member.
barrel forms a
hydrophilic "triphosphate tunnel" (see Fig. 2). Multiple acidic
side chains point into the tunnel cavity, including the essential
glutamates of motifs A and C. The interior of the tunnel contains a
single sulfate ion coordinated by basic side chains projecting into the
tunnel. Insofar as sulfate is a structural analog of phosphate, it is
proposed that the side chain interactions of the sulfate reflect
contacts made by the enzyme with the
phosphate of the
triphosphate-terminated RNA and nucleoside triphosphate substrates
(17). 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 motif A, and to a glutamate in motif C.
1 and
11, strand
9, and the connecting loop between strands
10 and
11 (see Fig. 1 and Refs. 6 and 7). Eight of the side
chains analyzed, Glu305, Glu307, and
Phe310 in
1, Arg494 and Lys496
in
9, and Glu492, Glu494, and
Glu496 in
11, were deemed to be important for Cet1
function in vitro and in vivo (indicated by ! in
Fig. 1). Alanine substitutions for these residues resulted in a
significant decrement in the hydrolysis of RNA or NTP substrates by
purified recombinant mutant proteins, and the mutant alleles were
unable to complement the growth of a yeast cet1
strain.
Alanine mutations of aliphatic residues Leu306 in
1 and
Val493 and Leu495 in
11 resulted in
temperature-sensitive (ts)2
growth and thermolability of enzyme activity in vitro
(indicated by
in Fig. 1). Mutations of Thr455,
Ser460, His463, Asn481,
Lys483, Ser484, and Arg485 (denoted
by + in Fig. 1) had no apparent effect on cell growth and either little
effect or only a modest effect (2- to 5-fold) on triphosphatase
activity in vitro.
phosphate; see Fig. 2).
Essential side chains Arg454 and Glu492 form a
salt bridge that likely stabilizes the side wall and roof of the tunnel
(see Fig. 2). Leu305, Phe310,
Val493, and Leu495 are all located on the
"outward" face of the
strands and are in no position to
directly participate in catalysis. Thus, the lethal or conditionally
deleterious effects of mutating these residues reflect the importance
of their hydrophobic interactions with the globular protein core upon
which the tunnel floor rests.
strands of the tunnel. The positions chosen for mutation,
denoted by dots (·) in Fig. 1, are conserved in the three other known
fungal RNA triphosphatases: C. albicans CaCet1, S. cerevisiae Cth1, and S. pombe Pct1. The
results of this analysis, together with previous data, identify a total
of 15 important side chains in the tunnel cavity. Thus, Cet1 has an
unusually complex active site. The active site residues fall into three functional classes: (i) those that participate directly in catalysis via coordination of the
phosphate or the metal; (ii) those that make critical water-mediated contacts with the
phosphate or the
metal; and (iii) those that function indirectly in catalysis by
interaction with other essential side chains and/or stabilization of
the tunnel architecture. We propose a reaction mechanism based on these results.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside to 0.4 mM and ethanol to 2% (v/v) final concentrations. 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
80 °C. All subsequent procedures were performed at
4 °C. Thawed bacteria were resuspended in 5 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10%
sucrose). Cell lysis was achieved by addition of lysozyme and Triton
X-100 to final concentrations of 50 µg/ml and 0.1%, respectively.
The lysates were sonicated to reduce viscosity, and insoluble material
was removed by centrifugation in a Sorvall SS34 rotor at 18,000 rpm for
45 min. The supernatants were applied to 1-ml columns of
nickel-nitrilotriacetic acid-agarose (Qiagen, Valencia, CA) that had
been equilibrated with lysis buffer containing 0.1% Triton X-100. The
columns were washed with 5 ml of lysis buffer containing 0.1% Triton
X-100 and then eluted stepwise with a buffer solution (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2 mM DTT, 10% glycerol, 0.05% Triton X-100) containing 50, 100, 200, 500, and 1000 mM imidazole. SDS polyacrylamide
gel electrophoresis analysis showed that the recombinant Cet1(201-549)
proteins were recovered predominantly in the 200 mM
imidazole eluate fractions. The peak fractions were pooled, adjusted to
5 mM EDTA, and dialyzed against buffer C (50 mM
Tris-HCl, pH 8.0, 2 mM DTT, 5 mM EDTA, 10%
glycerol, 0.05% Triton X-100) containing 50 mM NaCl.
Protein concentrations were determined by the Bio-Rad dye binding
method with bovine serum albumin as the standard. The enzyme
preparations were stored at
80 °C.
-32P]ATP, and
enzyme were incubated for 15 min at 30 °C. The reactions were
quenched by adding 5 µl 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 32Pi release was
quantitated by scanning the chromatogram with a FUJIX phosphorimager.
-32P]poly(A), and enzyme were incubated for 15 min at
30 °C. The reactions were quenched by adding 2 µl of 5 M formic acid. Aliquots of the mixtures were applied to a
polyethyleneimine-cellulose TLC plate, which was developed with 0.75 M potassium phosphate, pH 4.3. The release of
32Pi was quantitated by scanning the TLC plate
with a phosphorimager.
Trp) agar. Individual Trp+
isolates were patched to SD(
Trp) agar and then streaked on agar
plates containing 0.75 mg/ml of 5-fluoroorotic acid (5-FOA). Growth was
scored after 7 days of incubation at 25 and 30 °C. Lethal alleles
were those that failed to form colonies on 5-FOA after 7 days at either
temperature. For the viable alleles, individual colonies were picked
from the 5-FOA plates and patched on YPD agar. Two isolates of each
mutant were streaked on YPD agar at 16, 22, 30, and 37 °C. Growth
was assessed as follows: ++ indicates wild-type colony size at all
temperatures, and ts indicates growth at 16, 22, and 30 °C but no
growth at 37 °C (Table I).
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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strands of the Cet1 tunnel are conserved in the three other known
fungal RNA triphosphatases (Fig. 1). The
Cet1 crystal structure shows that these hydrophilic side chains point
into the tunnel cavity (Fig. 2) and are
thus plausible candidates to participate in substrate binding and
reaction chemistry. Here we tested the effects of single alanine
mutations at the 14 conserved side chains indicated by dots in Fig. 1.
These were as follows: Lys309 in
1; Asp377
in
5; Arg393, Ser395, and Asp397
in
6; Lys409 in
7; Lys427,
Asn431, and Glu433 in
8; Arg458
in
9; and Arg469, Asp471,
Thr473, and Lys474 in
10. The Ala mutations
were introduced into the biologically active domain Cet1(201-549), and
the mutant polypeptides were expressed as N-terminal His-tagged
derivatives in E. coli 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. 3).
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Fig. 1.
Sequence conservation within the triphosphate
tunnel of fungal RNA triphosphatases. The sequence of S. cerevisiae Cet1 from residue 304 to 539 is aligned to the
homologous segments of C. albicans CaCet1, S. cerevisiae Cth1, and S. pombe Pct1. Gaps in the
alignment are indicated by dashes (-). The strands that comprise
the triphosphate tunnel of Cet1 are shown above the amino
acid sequence. Conserved motifs A (
1) and C (
11) that define the
metal-dependent RNA triphosphatase family are highlighted
in shaded boxes. Previous studies (6, 7) had identified Cet1
residues at which alanine substitution resulted in loss of function
(!), thermosensitive function (
), or no significant effect on
function (+). The 14 amino acids of Cet1 that were targeted for
mutation in the present study are indicated by dots (·).
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Fig. 2.
The triphosphate tunnel and the metal-binding
site. 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 the
sulfate and manganese ions. The manganese (blue sphere)
interacts with octahedral geometry with the sulfate, three glutamates,
and two waters (red spheres). The image was prepared using
SETOR (4).
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Fig. 3.
Expression and purification of mutated
versions of Cet1(201-549). Aliquots (5 µg) of the
nickel-agarose preparations of WT Cet1(201-549) and the indicated
mutants were analyzed by electrophoresis through a 12.5%
polyacrylamide gel containing 0.1% SDS. Polypeptides were visualized
by staining with Coomassie Blue dye. The positions and sizes (in kDa)
of marker proteins are indicated on the left.
-32P]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 32Pi per ng of protein. The
specific activities of the 14 Ala mutants, normalized to the wild-type
value (defined as 100%), are listed in Table
I. Six of the mutations resulted in a
severe (at least a 20-fold) decrement in catalytic activity: R393A,
E433A, R458A, R469A, D471A, and T473A. Three mutant enzymes displayed
partial defects: D377A (8%), D397A (21%), and K409A (11%). Five of
the mutations had little or no effect on ATP hydrolysis: K309A, S395A, K427A, N431A, and K474A.
Mutational effects on Cet1 function in vitro and in vivo
-32P]poly(A) during a 15-min reaction in the presence
of 1 mM magnesium. 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 17 pmol of 32Pi per ng of protein. The specific
activities of the mutants were normalized to the wild-type value and
are listed in Table I. Seven of the mutations resulted in at least a
20-fold activity decrement: R393A, K409A, E433A, R458A, R469A, D471A,
and T473A. Two mutants displayed partial defects, D377A (7%) and D397A
(24%). Five of the mutations had little or no effect on RNA
triphosphatase activity: K309A, S395A, K427A, N431A, and K474A. Note
that there was an excellent correlation between the effects of
each mutation on the ATPase and RNA triphosphatase activities.
strain YBS20, in which the chromosomal
CET1 locus has been deleted and replaced by LEU2.
Growth of YBS20 is contingent upon maintenance of a wild-type
CET1 allele on a CEN URA3 plasmid. Therefore,
YBS20 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. Expression of
CET1(201-549) in cet1
cells permitted their
growth on 5-FOA, whereas expression of the catalytically defective
mutants R393A, E433A, R458A,
R469A, D471A, and T473A did not. Thus,
we conclude that these six mutations were lethal in vivo
(Table I).
cells on 5-FOA during selection at
25 or 30 °C. The viable CET1(201-549)-Ala strains were
then tested for growth on rich medium (YPD) at 16, 25, 30, and
37 °C. K309A, S395A, D397A,
K427A, N431A, and K474A cells grew at
all temperatures, and their colony sizes were similar to that of
wild-type CET1(201-549) cells (scored as ++ growth in Table
I). D377A and K409A cells displayed a ts
phenotype; they grew well at 25 and 30 °C but failed to grow at
37 °C.
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Fig. 4.
D377A and K409A are thermolabile in
vitro. Aliquots (20 µl) of WT Cet1(201-549), D377A,
and K409A 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 control or
pre-heated enzymes as follows: WT, 75 ng; D377A, 500 ng; and K409A, 500 ng. The amounts of unheated control WT and mutant enzymes were
sufficient to hydrolyze between 30 and 60% of the input ATP during the
15-min ATPase reaction at 22 °C. The extent of ATP hydrolysis by
pre-heated 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.
mutant (Table I).
phosphate
hydrolysis as T473A (1% of WT activity) and was nonfunctional in
vivo, the introduction of serine restored ATPase activity to 25%
of the WT level and RNA triphosphatase to 17% of the WT value (Table I). The T473S allele complemented the cet1
mutation at all temperatures. We conclude that the hydroxyl moiety at
position 473 is required for Cet1 function.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6, Glu433 in
8,
Arg458 in
9, and Asp471 and
Thr473 in
10) have identical counterparts in C. albicans CaCet1, S. cerevisiae Cth1, and S. pombe Pct1 (Fig. 1). The sixth essential position,
Arg469 in
10, is occupied by lysine or histidine in the
other fungal enzymes. We also identified Asp377 in
5 and
Lys409 in
7 as being important for function, albeit not
essential, insofar as their replacement by alanine resulted in partial
loss of function in vitro and a ts growth defect in
vivo. Lys409 has an identical counterpart in all three
other fungal triphosphatases, whereas Asp377 is either an
aspartate or a glutamine (Fig. 1). Several other residues were
nonessential for Cet1 function in vitro and in
vivo, including Lys309, Ser397,
Lys427, and Lys474, which are conserved in the
other fungal RNA triphosphatases.
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 (see Table I and Ref. 7).
Interpretation of the mutational results in light of the Cet1 crystal
structure (17) engenders a plausible model for catalysis by the fungal
RNA triphosphatase family.
phosphate
(Arg393, Lys456, and Arg458) or the
essential metal (Glu305, Glu307, and
Glu494). Class II residues make water-mediated contacts
with the
phosphate (Asp377 and Glu433) or
the metal (Asp471 and Glu496). 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, and Glu492).
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Fig. 5.
Three functional classes of active site
residues. Class I amino acids directly coordinate the phosphate or the essential metal. Class II amino acids make
water-mediated contacts with the
phosphate or the metal. Class III
residues function indirectly via their interactions with other
essential side chains and/or stabilization of the tunnel
architecture.
How do these functional groups contribute to catalysis? We postulate a
one-step in-line mechanism whereby the metal ion (coordinated by
residues on the tunnel floor) plus the Arg393,
Arg458, and Lys456 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.
We speculate further that the substrate is bound within the tunnel such
that the
and
phosphates are located on the entrance side of the
tunnel (i.e. anterior to the sulfate in Fig. 2), in which
case the water molecule situated posterior to the
phosphate (Fig.
2) would be poised to act as the attacking nucleophile, and the
Glu433 side chain coordinating this water molecule would
serve as a general base catalyst.
How does the proposed mechanism fit to the available mutational data?
The critical role of the enzyme-bound metal ion is clearly underscored
by that fact that Cet1 is intolerant of virtually any perturbations of
the metal coordination sphere. None of the three glutamates that
contact the metal directly (Glu305, Glu307, or
Glu496) can be functionally substituted by either glutamine
or aspartic acid (7). In addition to their contacts with the metal,
Glu305 and Glu307 are engaged in a network of
interactions with other side chains projecting from the tunnel floor
and lateral wall (Fig. 2). Of these, only the
Glu305-Lys409 ion pair is functionally
relevant, as surmised from the deleterious effects of
Lys409 mutations. Conceivably, Lys409 helps
position the Glu305 side chain with respect to the metal
whereas Glu305 helps tether the 7 strand to the tunnel
wall via Lys409. Glu307 interacts with the
conserved Lys309 in strand
1, but the elimination of
Lys309 has no apparent functional consequences, nor does
the loss of Asn431, which makes a bifurcated hydrogen bond
to both Glu305 and Glu307 (Fig. 2).
A most remarkable feature of the octahedral metal complex is the
requirement for two residues, Asp471 and
Glu494, to position the same metal-bound water (Fig. 2).
RNA triphosphatase and NTPase activity in vitro and Cet1
function in vivo are abolished when Asp471 is
replaced by either asparagine or glutamate. In addition to its
water-mediated metal interaction, Asp471 forms a hydrogen
bond with its essential neighbor Arg469 in 10.
Arg469 is located on the exit side of the tunnel and is not
in proximity to the
phosphate. Thus, we suspect that
Arg469 is not a direct catalyst and that its essentiality
reflects its role in positioning Asp471. Note that although
Arg469 also engages in a hydrogen bond to
Ser460 in strand
9 (Fig. 2), the fact that the S460A
mutation has only a modest effect on activity in vitro and
no effect on yeast cell growth (7) (i.e.
S460A does not phenocopy R469A) would argue against an important role for the hydrogen bond to serine. Instructive mutational effects occur at position Glu494, which
interacts exclusively with the metal-bound water in the crystal
structure. The E494Q mutation abolishes phosphohydrolase activity
in vitro and is lethal in vivo (7). However,
although the E494D mutation also abrogates the
magnesium-dependent RNA triphosphatase function and is
accordingly lethal in vivo, the E494D change spares the
manganese-dependent ATPase activity, E494D being one-fourth
as active as the wild-type enzyme (7). We speculate that the larger
atomic radius of manganese versus magnesium compensates for
the presumed retraction of the carboxylate functional group in the
E394D mutant.
Arg393 and Arg458 make bidentate
contacts via their terminal guanidinium nitrogens with the phosphate oxygens (Fig. 2). The mutational analysis highlights a strict
requirement for these bidentate interactions, because neither
Arg393 nor Arg458 can be functionally
substituted by lysine. Arg458 also forms a hydrogen bond
via N
to the essential hydroxyl group of Thr473
(Fig. 2). Thr473 may be required to correctly position the
Arg458 side chain with respect to the
phosphate; in
turn, Arg458 may help tether the
10 strand to the tunnel
wall via Thr473. Lys456 interacts with one of
the
phosphate oxygens (Fig. 2). The positive charge is required for
the function of this side chain, insofar as the growth defect incurred
by substitution with alanine or glutamine is reversed by introduction
of an arginine (7).
The mutational and structural data are consistent with the hypothesis
that Glu433 serves as a general acid to promote the attack
of water on the phosphorus. Glu433 coordinates a water
in the crystal structure, which is in turn coordinated by the sulfate
at a distance of 3.7 Å from the sulfur center. Cet1 activity is
reduced by 2 orders of magnitude by the E433Q mutation, which suggests
that the side chain must be able to accept a proton from the water, not
merely serve as a hydrogen bonding partner. Although Asp377
also coordinates a water in the tunnel cavity, the loss of this side
chain is less deleterious than the loss of Glu433, and the
function of Asp377 is restored fully by asparagine, which
would not be able to abstract a proton from the water. Consequently,
Asp377 and its associated water are not attractive
candidates for the roles of general acid and nucleophile, respectively.
There are likely to be additional important interactions of active site
amino acids with the and
phosphates of the substrate, and
perhaps with a second metal ion bound to the 5' triphosphate, that
cannot be appreciated from the available crystal structure. Lys409 and Arg454 are candidates to interact
with the
or
phosphates based on their location anterior to the
sulfate in the product complex. The observation that mutations of
Arg454 result in a 30-fold increase in the
Km for ATP is consistent with such a role (7),
although it remains to be shown that the effect is a consequence of a
direct contact between Arg454 and the substrate.
Arg454 forms a salt bridge to Glu492, and this
interaction appears to be important in stabilizing the tunnel structure
(6, 7).
In summary, the interior of the Cet1 triphosphate tunnel has a
distinctive baroque architecture supported by an intricate network of
hydrogen bonds and electrostatic interactions, of which a surprisingly
high proportion are required for phosphohydrolase activity. A more
complete picture of the enzyme mechanism and the interactions
supporting the tunnel structure should emerge as the mutational
analysis is extended to the remaining residues of the component strands
and as Cet1 is crystallized with a 5' triphosphate bound in the active site.
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FOOTNOTES |
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* This research was supported in part by National Institutes of Health Grant GM52470 (to S. S.) and a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada (to M. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Fax: 212-717-3623;
E-mail: s-shuman@ski.mskcc.org.
Published, JBC Papers in Press, February 13, 2000, DOI 10.1074/jbc.M100980200
1 Changela, A., Ho, C. K., Martins, A., Shuman, S., and Mondragon, A. (2001) Embo J., in press.
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ABBREVIATIONS |
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The abbreviations used are: ts, temperature-sensitive; DTT, dithiothreitol; FOA, fluoroorotic acid; WT, wild-type.
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