(Received for publication, November 22, 1995; and in revised form, February 29, 1996)
From the
The arginine-specific carbamoyl phosphate synthetase of Saccharomyces cerevisiae is a heterodimeric enzyme, with a
45-kDa CPA1 subunit binding and cleaving glutamine, and a 124-kDa CPA2
subunit accepting the ammonia moiety cleaved from glutamine, binding
all of the remaining substrates and carrying out all of the other
catalytic events. CPA2 is composed of two apparently duplicated amino
acid sequences involved in binding the two ATP molecules needed for
carbamoyl phosphate synthesis and a carboxyl-terminal domain which
appears to be less tightly folded than the remainder of the protein.
Using deletion mutagenesis, we have established that essentially all of
the carboxyl-terminal domain of CPA2 is required for catalytic function
and that even small truncations lead to significant changes in the CPA2
conformation. In addition, we have demonstrated that the C-terminal
region of CPA2 can be expressed as an autonomously folded unit which is
stabilized by specific interactions with the remainder of CPA2. We also
made the unexpected finding that, even when ammonia is used as the
substrate and there is no catalytic role for CPA1, interaction with
CPA1 led to an increase in the V of CPA2 in
crude extracts.
Formation of carbamoyl phosphate by carbamoyl phosphate
synthetase (CPSase, ()) constitutes the first
step of two biosynthetic pathways, one leading to arginine and/or urea
and the other to pyrimidine nucleotides(1, 2) . The
CPSase reaction consists of four partial reactions (), with the two
molecules of ATP binding at distinct sites and utilized in discrete
reaction
steps(1, 3, 4) .
Although CPSases function to catalyze the same overall reaction (), they differ in various properties as shown in Table 1(1, 2) . However, all of the CPSases thus
far sequenced display marked sequence identity and
similarity(5, 6) . The bacterial CPSases and the
arginine-specific CPSase of yeast are heterodimeric, with the small
subunit (glutamine amidotransferase) binding and cleaving glutamine () whereas the large subunit (synthetase) accepts the
ammonia moiety cleaved from glutamine, binds all of the remaining
substrates and effectors, and carries out all of the other catalytic
events (). In the single subunit
CPSases, the glutamine amidotransferase moiety is fused to the NH terminus of the synthetase moiety. Some of the CPSases are also
fused to additional enzymes involved in pyrimidine biosynthesis; syrian
hamster CAD, with the structure N-CPSase-dihydroorotase-aspartate transcarbamoylase-C, is the
best characterized member of this group(5, 6) . CPSase
I, which cannot utilize glutamine as a substrate and has no detectable
glutaminase activity, also contains the NH
-terminal
glutamine amidotransferase homology region but residues essential for
glutamine amidotransferase activity have been
replaced(7, 8) . Analysis of the CPSase sequences has
also shown that the synthetase moiety consists of two homologous halves
that appear to have resulted from an ancestral gene
duplication(9) . Each synthetase half contains the consensus
primary sequence motifs identified for ATP-utilizing
enzymes(9, 10) . Studies utilizing ATP analog
localization (4, 10, 11, 12) and
site-directed mutagenesis (3) have demonstrated that each
synthetase half contains a functional ATP binding site and that the
NH
-terminal half binds ATP
() while
the COOH-terminal half binds ATP
().
Mild
proteolysis studies with rat liver CPSase I (13, 14, 15) demonstrated the existence of
three independently folded globular domains corresponding to the
NH-terminal glutamine amidotransferase moiety (domain A)
and to the two ATP-utilizing moieties (domains B and C), and also
identified a fourth COOH-terminal structural domain (domain D). The
20-kDa domain D appears to be much less tightly folded than domains A-C
since it undergoes cleavage to small peptides under the mild
proteolysis conditions. The A-D domain organization determined for the
rat liver CPSase I appears to be common to all CPSases since various
features of it have subsequently been observed in the CPSase unit of
CAD (16) and in the Escherichia coli CPSase(17) .
Binding of the allosteric effectors IMP(18) , UMP(17) , UTP(19, 20) , PRPP(19) , and N-acetylglutamate (21) has been localized to domain D. Affinity labeling studies with the rat liver CPSase I (4) have strongly suggested that domain D is also involved in binding of both molecules of ATP. Domain D appears to fold back to complement the ATP-binding regions of globular domains B and C. It is possible that amino acid residues from domain D participate in catalytic processing of the ATP molecules and/or in excluding water from the active site, although they may participate directly in ATP binding. A feasible model for the transmission of allosteric effects within CPSase would be that the binding of effectors to domain D alters the positioning of this domain at the ATP sites and thereby affects the affinity of the CPSase for ATP.
While it is known, from studies on rat liver CPSase I (13, 14, 15) and E. coli CPSase(19, 22) , that elimination of the entire domain D yields inactive CPSase, the effects of less drastic C-terminal truncation of CPSase have not been determined. Several lines of evidence suggest that at least limited COOH-terminal truncation might yield stable and functional CPSase: (a) analysis of amino acid identities among the synthetase moieties of six CPSases (5) revealed 28% identical residues throughout domains B and C, but only 8% identical residues for the 191-residue domain D, with only 5 of the 167 COOH-terminal residues identical; (b) deletion analysis of the E. coli CPSase(22) , aimed at defining the domains necessary for interaction between the small and large subunits, revealed that domain D was not necessary for heterodimer formation; (c) a hybrid construction with domains A-C from E. coli CPSase and domain D from syrian hamster CAD retained catalytic activity(19) ; and (d) deletions would be less likely to exert long-range perturbations of the rather loosely-folded domain D structure, with its multiple sites available for proteolysis, than would be expected for a tightly folded globular domain, with no sites susceptible to proteolysis under nondenaturing conditions. To further define the role of domain D in the structure and function of CPSase, we have carried out deletion mutagenic analysis of CPA2, the large synthetase subunit of the yeast arginine-specific CPSase(9) . Since CPA2 has no known allosteric effectors (Table 1), its use should allow a clearer definition of the relationship between protein structure and catalytic function than would be possible if allosteric effects at domain D were also involved. Furthermore, it is possible to carry out an in vivo assay for the functioning of CPA2 deletion mutants in addition to determining their properties in vitro.
For immunoblot analysis, proteins were separated by
SDS-polyacrylamide gel electrophoresis and then electrophoretically
transferred to a Polyscreen polyvinylidine difluoride membrane (DuPont
NEN) with a Hoefer TE42 unit. Transfer was conducted at 80 V for 30 min
in 25 mM Tris-HCl, 192 mM glycine, pH 8.3, 25%
methanol. The membrane was blocked with 5% dry milk in PBS-T (1.4
mM NaHPO
, 8.1 mM Na
HPO
, 150 mM NaCl, 0.05% Tween
20) for 1 h at room temperature (the temperature for all subsequent
immunoblot steps). After two 5-min PBS-T washes, the membrane was
incubated for 1 h with a 1:10,000 dilution of the primary antibody
(rabbit anti-CPA2) in PBS-T, 1% bovine serum albumin. After one 15-min
and four 5-min PBS-T washes, the membrane was incubated for 1 h with a
1:3,000 dilution of horseradish peroxidase/goat anti-rabbit IgG
conjugate (Bio-Rad) in PBS-T, 1% bovine serum albumin. After one 15-min
and four 5-min PBS-T washes, the membrane was probed for horseradish
peroxidase activity with the Renaissance chemiluminescence reagent,
following the protocol recommended by the supplier (DuPont NEN).
Quantitation of the immunoblots was carried out as described above for
polyacrylamide gels.
Polyclonal rabbit antibody was custom prepared
by Pel-Freez Biologicals, using reduced SDS-treated CPA2 that was
isolated from an SDS-polyacrylamide gel. pET11-d/CPA2 was
utilized to overexpress S. cerevisiae CPA2 (37 °C, LB
medium, 1 mM isopropyl--D-thiogalactoside added
at a cell density where A
is 0.5). The resulting
inclusion bodies were isolated, subjected to electrophoresis, and the
separated 124-kDa CPA2 used as the antigen; there was no detectable
protein of this size present in the host E. coli BL21 (DE3)
cells before induction. CPA2, with a unique BamHI
site at nucleotide 1256, was subcloned into pET11-d as follows: the
first 1299 base pairs of CPA2 was PCR amplified, using
upstream primer P15 (Table 2) that introduced a NcoI
site at the ATG translation start of CPA2 and downstream
primer P16 that did not alter the original BamHI site; the
resulting 1318-base pair PCR fragment was digested with NcoI
and BamHI and ligated into the corresponding sites of the
pET11-d polylinker region; site-directed mutagenesis, using unique site
elimination(23) , was used to reverse the base change (A to G)
at the +4 position of CPA2 that had been necessary to
introduce the NcoI site, yielding the plasmid pET11-d/NB; the BamHI fragment containing the remaining sequence of wild-type CPA2 (1256-3357 base pairs) was then cloned from pAL6
into the BamHI site of pET11-d/NB to yield
pET11-d/CPA2.
Figure 1: Scheme for the construction of CPA2-disrupted protease-deficient strain LPL26. Restriction sites used in constructing the CPA2::hisG-URA3-HisG fragment are: B, BamHI; Bg, BglII; E, EcoRI; and X, XhoI. The filled, hatched, and open boxes represent CPA2, URA3, and hisG sequences, respectively.
Figure 2: Construction of the low copy number CPA2 expression plasmid pAL6. The oligonucleotides, P6 and P9, used for PCR amplification are shown in the upper panel. Open boxes represent CPA2 sequences and hatched boxes represent pUC19 sequences. The restriction sites that were used for cloning are: A, ApaLI; B, BamHI; and S, SalI. BamHI and SalI sites are also in the pRS315 polylinker.
The functional effects of deletion mutations within domain D were analyzed by in vivo screening with the LPL26 and pAL6 system. Since carbamoyl phosphate synthesized by either CPA2 or URA2 enters a common pool in yeast(42) , URA2 of LPL26 was repressed and feedback-inhibited (42) by including uracil in the medium used for screening so that URA2 could not substitute for CPA2 in providing carbamoyl phosphate for arginine synthesis. Under these conditions, LPL26 is dependent for growth on the presence of arginine in the medium or on transformation with a plasmid carrying wild-type CPA2 or a functional cpa2. To determine whether cpa2 deletion constructions could produce carbamoyl phosphate, their ability to functionally complement CPA2-disrupted LPL26 was assessed. LPL26 was transformed with pAL6 or its derived deletion constructions and transformants were selected by ability to grow without added leucine. The transformants were screened for functional pRS315/cpa2 by ability to grow without added arginine, and extent of growth was compared to that of pAL6 transformants.
Figure 3:
Construction of CPA2 domain D
deletions. The positions of the oligonucleotides used in PCR
amplification of domain D sequences are shown in the upper
panel. The restriction sites used for cloning are: A, ApaLI; B, BamHI; S, SalI;
and Sc, ScaI. BamHI and SalI sites
are also in the pRS315 and pRS316 polylinker regions. Hatched boxes represent CPA2 sequences. The sites marked D1 and D2
correspond to the peptide regions which appear to participate in
binding ATP and ATP
,
respectively.
Figure 4: Construction of low and high copy number plasmids for autonomous expression of domain D. The positions of the oligonucleotides used for primary PCR amplifications of the sequences encoding the entire domain D and the regulatory sequence upstream of CPA2 are indicated. The restriction sites that were used for cloning are: B, BamHI and S, SalI. BamHI and SalI sites are also in the pRS424 and pRS315 polylinker regions.
In vivo screening in CPA2-disrupted S.
cerevisiae LPL26 was used to determine whether individually
expressed domain D could associate with individually expressed domains
B+C to form a stable and/or functional CPA2. Transformants of
LPL26 containing pAL6 or pRS315/D were identified by ability to grow in
the absence of exogenous leucine (via the pRS315 LEU selection marker)
whereas transformants of LPL26 containing pRS316/C191 were
identified by ability to grow in the absence of exogenous uracil (via
the pRS316 URA selection marker). Co-transformants of LPL26 containing
both pRS316/
C191 and pRS315/D were identified by ability to grow
in media from which both leucine and uracil were omitted. When tested
for ability to produce carbamoyl phosphate and thus grow in the absence
of exogenous arginine, only pAL6 transformants, expressing wild-type
CPA2, showed cell growth. pRS316/
C191 transformants, expressing
domains B+C, did not show functional complementation, consistent
with previous findings with rat liver CPSase I (13, 14, 15) and E. coli CPSase (19, 22) that elimination of the entire D domain
yields inactive CPSase. Neither pRS316/
C191 plus pRS315/D
co-transformants, expressing domains B+C independently of
expressed domain D, nor pRS315/D transformants, expressing only domain
D, could functionally complement the LPL26 cells.
Western blot
analysis was carried out to confirm that the individual domains D and
B+C were expressed under the conditions of the complementation
assay. Although intact CPA2 was expressed from a pAL6 transformant of
LPL26, and the domain B+C polypeptide was expressed from a
pRS316/C191 transformant and from a pRS316/
C191 plus pRS315/D
co-transformant of LPL26, there was no detectable domain D in the crude
extracts prepared from a pRS315/D transformant or from a
pRS316/
C191 plus pRS315/D co-transformant (data not shown). In
order to increase the potential yield of expressed domain D, the high
copy number plasmids pRS424/D (parallel construction to that of
pRS315/D, Fig. 4) and pRS425/
C191 (parallel construction to
that of pRS316/
C191, Fig. 3) were utilized. When LPL26 was
transformed with these high copy number plasmids and the crude extracts
subjected to Western blot analysis (Fig. 5), intact CPA2 and the
domain B+C polypeptide were again found to be stably expressed
from their corresponding plasmids whereas domain D was not stably
expressed. However, domain D, as well as the domain B+C
polypeptide, was stably expressed in LPL26 co-transformed with high
copy number plasmids pRS424/D and pRS425/
C191. In a control
co-transformation where intact CPA2 and domain D were expressed
independently from high copy number plasmids, there was no domain D
detected in the Western blot, although wild-type CPA2 was present (data
not shown). These findings very strongly suggest that there is a
specific interaction between individually expressed domain D and
domains B+C which can stabilize domain D. In contrast, when domain
D is expressed alone or in the presence of intact CPA2, where the
regions of domain D interaction on domains B+C are already
occupied by the cognate domain D, the conformation of the 20-kDa domain
D must be extremely susceptible to proteolysis. Thus, although a
variety of protease inhibitors were included in the extraction buffer,
no detectable domain D was recovered in the crude extracts unless the
domain B+C polypeptide was also present to interact with domain D.
When the high copy number transformants were tested for ability to
produce carbamoyl phosphate and thus grow in the absence of exogenous
arginine, the findings were identical to the low copy number
transformant findings: only transformants expressing wild-type CPA2
(alone or in the presence of individually expressed domain D) showed
cell growth while functional complementation was not observed with
pRS424/D plus pRS425/
C191 co-transformants or with transformants
carrying either plasmid alone. Thus, the observed structural
interaction between individually expressed domain D and domains
B+C is not sufficient for functional interaction between the
domains.
Figure 5:
Western blot analysis of autonomous domain
D expression. Crude extracts were prepared from LPL26 transformants
grown in media containing arginine, fractionated on an SDS-12%
polyacrylamide gel, and subjected to Western blot analysis, as
described under ``Experimental Procedures.'' Each lane
contained 25 µg of total protein with the exception of the
untransformed host cells (lane 1, 75 µg). The transformed
cells contained the following plasmids: lane 2,
pRS425/CPA2; lane 3, pRS425/C191 CPA2 + pRS424; lane 4, pRS425 + pRS424/D; lane
5, pRS425/
C191 CPA2 +
pRS424/D.
Figure 6: Schematic diagram of domain D deletions. The filled box of the top line corresponds to domain D of CPA2. Each deletion included the 12 nucleotides immediately preceding subsite D1-ATP (open boxes) or subsite D2-ATP (hatched boxes).
All of the COOH-terminal
deletions were constructed in the pRS315 vector as outlined in Fig. 3(and as described above for pRS316/C191): DNA
fragments extending from the unique ApaLI site (nucleotide
2416) to the appropriate COOH terminus were amplified from pAL6
template by PCR, with a stop codon and BamHI site incorporated
in the downstream primer, and the ApaLI- and BamHI-digested PCR products were ligated with the SalI-ApaLI fragment of CPA2 and the SalI + BamHI-digested pRS315 vector. To create
pRS315/
N1-120, where the first 120 amino acids are removed
from domain D, recombinant PCR was used (Fig. 3). In the first
round PCR, the primer pair P6 and P13 was used to amplify a 0.41-kb DNA
fragment complementary to sequences at positions 2380-2781 and
3145-3156 of CPA2, and the primer pair P14 and P9 was
used to amplify a 0.23-kb DNA fragment complementary to sequences at
positions 2770-2781 and 3145-3357 of CPA2. The
first round PCR products were extended and amplified in second round
PCR with the primer pair P6 and P9. The second round PCR product was
digested with ApaLI and BamHI and ligated with the SalI-ApaLI fragment of CPA2 and the SalI + BamHI-digested pRS315 vector to yield
pRS315/
N1-120.
In order to amplify the potentially small activities of
truncated forms of CPA2, all in vitro assays were carried out
on preparations from LPL26 co-transformed with high copy number
plasmids expressing CPA1 and the truncated forms of CPA2. As shown in Table 4, carbamoyl phosphate synthesis activity was observed only
with wild-type CPA2 and with CPA2 from which 5 amino acids had been
deleted. Although the more extensive deletions (20, 25, and 30 amino
acids) could produce sufficient carbamoyl phosphate in vivo to
support the growth of LPL26, their in vitro activity was below
the limits of detection. As expected for the truncations (41 and 191
amino acids) which could not support LPL26 growth, there was no
detectable in vitro activity. Also shown in Table 4are
the activity levels observed when 20 mM glycine was included
in the assay mixture. Previous studies (38) have shown that
glycine increases by 2-3-fold the rate of ammonia-dependent
carbamoyl phosphate synthesis, presumably by occupying the glutamine
site of CPA1 and inducing a more active conformation of CPA2. The
addition of glycine caused an unexpectedly large increase, 12-fold, in
the activity of the C5 CPA2. Thus, although
C5 CPA2 was only
22% as active as wild-type CPA2 in the absence of glycine, their
activities were essentially the same when glycine was added. These
findings strongly suggest that, at least in vitro, the
C5
CPA2 conformation is significantly different from that of wild-type
CPA2, but that interaction with the glycine-CPA1 complex can force
C5 CPA2 into an essentially wild-type conformation. The
conformations of the
C20,
C25, and
C30 deletions of CPA2
appear to be so different from wild-type that they cannot maintain an
interaction with CPA1 when extracted into a necessarily more dilute
solution and/or they are extremely susceptible to proteolysis when the
cell is broken; either situation would result in significant in
vivo activity yet undetectable in vitro activity.
Since in vitro activity determinations had indicated that
interaction with CPA1 yielded an increased V for
CPA2, we carried out Western blot analysis of crude extracts prepared
from LPL26 co-transformed with high copy number plasmids expressing
CPA2 truncations and CPA1 (pRS424 and pJL113/ST4, respectively). As
shown in Fig. 7, under these conditions all of the
constructions, except
C66, could be detected. The mass of the
active
C5 construction present in the crude extract was about half
that of the wild-type CPA2 (Table 4). The mass recoveries of the
C20,
C25, and
C30 constructions, which showed activity in vivo but not in vitro, were 3.6-10.3% of the
wild-type CPA2 recovery. Given these amounts of truncated CPA2 protein
and the detection limits of the in vitro assay, any carbamoyl
phosphate synthesis activity of the truncation mutants would have to be
less than 10% of the wild-type activity. Thus, truncation of even 5
amino acids affected both the activity and the stability of CPA2, and
truncations of 20 and greater amino acids led to very large effects on
activity and stability. It is also noteworthy that the yields (Fig. 7) of the extensively truncated CPA2 forms were not simply
a function of the extent of truncation:
C57
C166 >
C191 >
N1-120
C113. This finding, plus
the fact that the very extensive
C191 and
C166 truncations
could be stably expressed in the absence of CPA1 (data not shown),
strongly suggests that incorrectly-folded domain D truncations can
destabilize the overall CPA2 structure even more than total removal of
domain D does.
Figure 7:
Western blot analysis of CPA2 deletions
expressed from the high copy number plasmid pRS424 in the presence of
CPA1 expressed from the high copy number plasmid pJL113/ST4. Each lane
contained 50 µg of total cell protein. A: lane 1,
LPL26 host cells; lanes 2-7, transformant cells
expressing C5,
C20,
C25,
C30,
C41, and
wild-type CPA2, respectively. B: lanes 1-7,
LPL26 transformed with
C191,
C57,
C166,
C66,
N1-120,
C113, and wild-type CPA2,
respectively.
Our analysis of C-terminal deletions in yeast CPA2 has
demonstrated that essentially all of domain D is required for full
catalytic function. Deletion of only 5 amino acids, out of a total of
1128, led to a decreased ability to synthesize carbamoyl phosphate.
Enzymatic function was further decreased with increased truncation and
was completely abolished with deletion of 41 or more amino acids.
Functional analysis of the deletions also yielded the unexpected
finding that the ability of CPA2 to carry out ammonia-dependent
carbamoyl phosphate synthesis was very strongly influenced by the
presence of CPA1. It thus appears that even though ammonia is being
used as the substrate in the assay and there is no catalytic role for
the amidotransferase moiety of CPA1, interaction with this small
subunit must affect the conformation of CPA2 and thereby greatly
increase the rate of carbamoyl phosphate synthesis. In contrast, it is
known (43, 44) that the large subunit of E. coli CPSase (carB) can function to carry out ammonia-dependent
carbamoyl phosphate synthesis in the absence of its corresponding small
subunit (carA); the V is similar for carB and
the carA/carB holoenzyme although the K
values for
bicarbonate and ATP are increased 2-3-fold in carB. For both the
yeast CPA2/CPA1 system (38) and the E. coli carA/carB
system(1) , occupancy of the glutamine site of the small
subunit by a non-hydrolyzable glutamine analog such as glycine is known
to cause a 2-3-fold increase in the rate of ammonia-dependent
carbamoyl phosphate synthesis by the large subunit. A number of
previous studies have established that there is a functional coupling
between the two subunits so that occupancy of the glutamine site on the
small subunit, by either glutamine or glycine, causes a conformational
change in the large subunit, leading to an increased ability to
synthesize carbamoyl phosphate(1) .
Several lines of
evidence suggested that interaction with CPA1 causes CPA2 to assume a
conformation with much greater catalytic activity and/or stability. The
ability of CPA2 to carry out ammonia-dependent carbamoyl phosphate
synthesis in vitro was greatly enhanced by the presence of
CPA1: high copy number expression of both CPA1 and CPA2 yielded a 3
times greater specific activity than high copy number expression of
CPA2 only. Four of the truncated CPA2 polypeptides (C41,
C57,
C113, and
N1-120) could be detected by Western blot
analysis only when CPA1 was present. Additional support for the
importance of CPA1/CPA2 interaction came from analysis of the
C20-30 mutants, where the inactivity of these truncations in vitro apparently resulted from their inability to maintain
an interaction with CPA1. This proposed activation and/or stabilization
of CPA2 by interaction with CPA1 and the apparently ready dissociation
of the two subunits would explain the previously observed lability of
this enzymatic activity (38, 45) and might also serve
as a basis for regulation of carbamoyl phosphate synthesis in yeast. As
noted in Table 1, all other known CPSases are subject to
allosteric regulation. However, in spite of extensive
screening(38, 45) , no allosteric effectors have been
identified for the S. cerevisiae arginine-specific CPA2.
Transcription of both CPA1 and CPA2 can be induced by
the GCN4 protein involved in the general control of amino acid
biosynthesis system, and expression of CPA1 can be completely repressed
by arginine at a post-transcriptional level(36) . If, as
proposed, CPA2 is only fully active when interacting with CPA1, then
more severe repression of CPA2 would be unnecessary since the
arginine-induced repression of CPA1 would be effectively communicated
to CPA2. Thus, instead of the allosteric control utilized by other
CPSases, the activity of the yeast arginine-specific CPSase could be
regulated by the combination of activation of CPA2 by CPA1 and
repression of CPA1 by arginine.
Prior to the present studies, the CPSase domain structure had been studied only in rat liver CPSase I (13, 14, 15) and syrian hamster CAD (16) . From those studies, using mild proteolysis, domains A, B, and C could be defined as independent folding units. The status of domain D, however, was not clear since it was observed to undergo cleavage to small peptides even under the mild proteolysis conditions where domains A, B, and C were stable. Our present findings allow definition of yeast CPA2 domain D as an autonomous folding unit that is stabilized by interaction with domains B+C. Thus, we found that intact individually expressed domain D was detectable, but only when the polypeptide comprising domains B+C was co-expressed. Although independently folded domain D could interact with independently folded domains B+C to yield a stable structure, this reconstituted CPA2 was not able to synthesize carbamoyl phosphate, indicating that it was not identical in final folded form to the wild-type CPA2 conformation.
The present studies have also indicated that essentially all of
domain D is required for correct folding of CPA2. The amount of C5
CPA2 obtained in crude extracts was about half that obtained for
wild-type CPA2. Western blot analysis of the more extensive truncations
generally revealed much less CPA2 recovery than for
C5 CPA2. It
cannot be ruled out that the lack of expression, or lowered level of
expression, of truncations was due to variable mRNA synthesis and/or
stability. However, since the wild-type CPA2 and truncation mutants
were all expressed from plasmids with identical upstream and downstream
sequences, it is unlikely that significant changes occurred in mRNA
synthesis and/or stability. It therefore seems most likely that the
lower yields of various CPA2 truncated forms resulted from
conformational changes that made the truncated proteins more
susceptible to proteolysis than the wild-type CPA2. This increased
susceptibility to proteolysis was not simply due to smaller size of the
truncated products since the
C113 and
N1-120 deletions
yielded less detectable product than the shorter polypeptides resulting
from the
C166 and
C191 truncations. Functional analysis of
the
C5 mutant further indicated that its conformation was
significantly different from wild-type:
C5 was only 22% as active
as wild-type CPA2 in the absence of glycine, but binding of glycine to
CPA1 resulted in wild-type catalytic behavior of the
C5 mutant;
presumably the glycine-induced CPA1 conformational change was
transmitted to the truncated CPA2 and caused it to assume an
essentially wild-type conformation. The temperature sensitivity of the
C20,
C25, and
C30 constructions was also consistent with
the proposal that their conformations differ significantly from the
wild-type CPA2 conformation. Generally, temperature-sensitive mutations
occur at sites buried within the protein and affect the folding and
stability of the protein(46, 47) .
We have demonstrated that domain D is much more integral to CPA2 structure and function than its lack of sequence conservation might suggest. It thus appears that a common domain D structure can be formed from many different sequences of amino acids, as is known to be the case, for instance, for the dinucleotide-binding fold(48) , where only a relatively few amino acid residues are crucial for maintaining the common structural framework and are therefore conserved, while many different combinations of the remaining amino acid residues can form the bulk of the common structural framework. The significant effects on CPA2 structure and function observed with even small carboxyl-terminal truncations precluded use of domain D deletion analysis to assign specific functions to the specific D1-ATP and D2-ATP structural regions. However, the integral role of domain D in overall CPA2 structure and function would be consistent with its involvement in substrate binding and/or catalysis, as well as in constituting the locus for allosteric effector interaction in the CPSases which undergo allosteric regulation.