(Received for publication, October 24, 1996)
From the Department of Cell Biology, Institute for Virus Research, Kyoto University, Kyoto 606-01, Japan
Alkaline phosphatase of Escherichia coli (a homodimeric protein found in the periplasmic space) contains two intramolecular disulfide bonds (Cys-168-Cys-178 and Cys-286-Cys-336) that are formed after export to the periplasmic space. The location-specific folding character of this enzyme allowed its wide usage as a reporter of protein localization in prokaryotic cells. To study the roles of disulfide bonds in alkaline phosphatase, we eliminated each of them by Cys to Ser mutations. Intracellular stability of alkaline phosphatase decreased in the absence of either one or both of the disulfide bonds. The mutant proteins were stabilized in a DegP protease-deficient strain, allowing accumulation at significant levels and subsequent characterization. A mutant protein that lacked the N-terminally located disulfide bond (Cys-168-Cys-178) was found to have Cys-286 and Cys-336 residues disulfide-bonded, to have a dimeric structure, and to have almost full enzymatic activity. Nevertheless, the mutant protein lost the trypsin-resistant conformation that is characteristically observed for the wild-type enzyme. In contrast, mutants lacking Cys-286 and Cys-336 were monomeric and inactive. These results indicate that the Cys-286-Cys-336 disulfide bond is required and is sufficient for correctly positioning the active site region of this enzyme, but such an active conformation is still insufficient for the conformational stability of the enzyme. Thus, a fully active state of this enzyme can be formed without full protein stability, and the two disulfide bonds differentially contribute to these properties.
Alkaline phosphatase (PhoA) is a periplasmic enzyme of Escherichia coli. It is a zinc- and magnesium-containing enzyme consisting of two identical subunits encoded by the phoA gene (1). Each of the subunits contains two intramolecular disulfide bridges, Cys-168-Cys-178 and Cys-286-Cys-336 (2). PhoA is initially synthesized as a precursor with an N-terminal 20-residue signal sequence that is removed during translocation across the cytoplasmic membrane. The mature 450-residue (or 449 when the N-terminal arginine residue is removed; Ref. 2) part of PhoA is then oxidized for its cysteine residues to form the disulfide bridges and is folded into a partially protease-resistant conformation (3). The subunit folding process is then followed by dimerization into the active metalloenzyme. This pathway of enzyme formation is completed within 2-3 min at 15 °C in the living cell (3).
The processes of folding/assembly of PhoA occur only after export to the periplasmic space (4-6). This property of PhoA has enabled its wide usage as a reporter of protein localization and membrane protein topology in prokaryotic cells (7, 8). The formation of disulfide bonds is a crucial event that triggers the correct folding of PhoA. In the cytoplasm, thioredoxin reductase and other factors seem to prevent disulfide bond formation on proteins (9). In the periplasm, the Dsb system facilitates disulfide bond formation (10, 11). It has been established that a periplasmic factor (DsbA) directly catalyzes oxidation of cysteines (12, 13). The membrane-bound factor DsbB is believed to reoxidize the reduced form of DsbA to allow it to function catalytically (14-16). Although biochemical studies on denaturation-renaturation as well as subunit association processes of PhoA were conducted previously (3, 17-22), and the structure of the enzyme was determined at a resolution of 2.8 Å (23) or 2.0 Å (24), the roles of the two disulfide bonds have not been precisely defined. In view of the importance of the disulfide bond formation for the formation of active PhoA enzyme, it is important to assign roles to each disulfide bond in the activity and stability of the enzyme. Thus, we have undertaken site-directed mutagenesis of cysteine residues of PhoA and examined the properties of the resulting mutant forms of the enzyme. The results show that the two disulfide bonds have different roles. Cys-286-Cys-336 was found to be required and was sufficient for the formation of the enzymatically active conformation, but was insufficient for supporting the protease-resistant structure, which proved to require Cys-168-Cys-178 as well. Thus, a fully active state of this enzyme can be formed without having the full stability of the protein.
E. coli strains KS272
(phoA) and KS474 (
phoA
degP41::Tn5) were described by Strauch and
Beckwith (25). Strains MS3 and MS5 were derivatives of KS272 and KS474,
respectively, into which F
lacIQ lacPL8
lacZ+Y+A+
pro+ plasmid had been introduced from strain KI267
(26). Strains BW313 (27) and MV1184 (28) were used for site-directed
mutagenesis.
pMS002 was a derivative of pBluescript-SK()
carrying the phoA gene under the control of the
lac promoter. It was constructed as follows. A polymerase
chain reaction product was donated by N. Kusukawa of Takara Shuzo in
which the phoA region of the chromosome was amplified using
primers 5
GAAACAAAGCACTATTGC (starting at the third nucleotide of
phoA) and 5
GTGATCTGCCATTAAGTC (starting at the 75th
nucleotide after the end of the phoA reading frame). It was
treated with the Klenow enzyme and cloned into the EcoRV site of pBluescript-SK(
). To eliminate any possible mutations introduced by polymerase chain reaction, a 1.4-kilobase pair
BclI-KpnI fragment (representing most of the
mature PhoA and some of the vector sequence) was replaced with the
corresponding BclI-KpnI fragment that was
prepared from a plasmid carrying the phoA gene of the
confirmed sequence1 in which the
phoA mature sequence had been similarly cloned at the
EcoRV site of pT7Blue-Vector (Novagen). The remaining 5
part of the phoA sequence was confirmed by sequencing. In
the resulting plasmid (pMS001), the initiation codon of phoA
was recreated but was in the same frame as the lacZ
sequence of the vector. Therefore, the PstI site at the
upstream region was subjected to cleavage, filling-in by the Klenow
enzyme and religation. The final product (pMS002) encodes the wild-type
PhoA precursor.
Cys to Ser substitution mutations of PhoA were introduced into plasmid
pMS002 by the procedures described by Kunkel (27), using the Muta-Gene
Phagemid In Vitro Mutagenesis Kit (Bio-Rad). Synthetic
oligonucleotides used as mutagenic primers were as follows: 5GCGCAAA
TACGGTC3
for Cys-168
Ser mutation,
5
AGTGAAAAA
CCGGGTA3
for Cys-178
Ser mutation,
5
AGTCACC
ACGCCAA3
for Cys-286
Ser mutation, and
5
GAATCCT
GGGCAAA3
for Cys-336
Ser mutation (codons for Ser are underlined). Coding for
PhoA(SCCC),2 pMS003 was first constructed
using the Cys-168
Ser primer. pMS004 (for PhoA(SSCC)), pMS006 (for
PhoA(SSSC)), and pMS013 (for PhoA(SSCS)) were then constructed
successively using appropriate primers. Similarly, pMS012 (for
PhoA(CCSC)), pMS014 (for PhoA(CCCS)), and pMS015 (for PhoA(CCSS)) were
constructed individually or in sequence. pMS016 (for PhoA(SSSS)) was
constructed by replacing the EcoT14I-KpnI segment
of pMS004 with that of pMS015. All the mutations were confirmed by
sequencing.
Cells were grown at
37 °C to an exponential phase in M9 medium (29) supplemented with
0.4% glycerol and 20 µg/ml each of amino acids, except methionine
and cysteine. The phoA gene under the lac
promoter control was induced with 1 mM
isopropyl--D-thiogalactoside. After 15 min, cells were
pulse-labeled with 50 µCi/ml [35S]methionine (1100 Ci/mmol, obtained from American Radiolabeled Chemicals) followed by
chase with unlabeled L-methionine (200 µg/ml) for
indicated periods. A portion of the culture was treated with
trichloroacetic acid, and protein precipitates were dissolved in SDS
for subsequent immunoprecipitation as described previously (11).
Anti-PhoA serum was purchased from 5 Prime
3 Prime, Inc., Boulder,
CO. Radioactive proteins were visualized and quantitated using a
Bioimaging Analyzer BAS2000 (Fuji Film) in combination with a Discovery
Series Densitometer (PDI).
Cells of MS5 that
carried a phoA plasmid were grown at 37 °C in L-broth
containing 1 mM isopropyl--D-thiogalactoside
and 50 µg/ml ampicillin to an exponential phase (turbidity of 40 as measured by a Klett colorimeter with filter no. 54), mixed with a final
concentration of 1 mM iodoacetamide, pelleted by
centrifugation (placed in a microcentrifuge for 10 min), and
resuspended in 1 M Tris-HCl (pH 8.1), 1 mM
iodoacetamide (30). The procedures for the enzyme assay and unit
definition were as described by Manoil and Beckwith (7). For assessing
relative accumulation of PhoA proteins by immunoblotting, a portion of
each culture was mixed directly with trichloroacetic acid (final
concentration, 5%) to precipitate whole cell proteins that were
solubilized in SDS-containing solution. Two fixed culture equivalents
of samples (10 and 30 µl) were subjected to
SDS-PAGE3 and immunoblotting using
anti-PhoA serum, as described previously (31). PhoA activities of
different mutant constructions were compared after normalizing the
enzyme unit by relative accumulation levels of the PhoA proteins.
To examine dimerization states of PhoA, cells of MS5 that carried a phoA plasmid were grown as described above, and the periplasmic fraction was prepared from 5 ml of culture as described previously (32). This fraction (480 µl) was layered on top of a 4.5-ml gradient of 5-20% sucrose in 10 mM Tris-HCl. After centrifugation at 45,000 rpm for 19 h in a Beckman SW65 rotor, the gradient was fractionated into 16 fractions that were then analyzed by 10% SDS-PAGE, followed by immunoblotting for distribution of PhoA proteins (3). Periplasmic fractions were also separated by gel electrophoresis without denaturation (33), and PhoA species were detected by immunoblotting.
Analysis of Intramolecular Disulfide Bonds by SDS-PAGE under Non-reducing ConditionsA 200-µl portion of a culture as described above was mixed with an equal volume of 10% trichloroacetic acid. Protein precipitates were washed with acetone and then dissolved in SDS/Tris-HCl/iodoacetamide solution (34). Samples were subjected to SDS-PAGE in the absence of any reducing reagent, followed by immunoblotting with anti-PhoA serum. In some experiments, the SDS solution used was slightly different (35), but the results were essentially not affected by this variation.
Trypsin Sensitivity AssayTrypsin resistance of various PhoA constructions in crude cell lysates was assessed essentially as described previously (5, 31), except that iodoacetamide was included (30).
Purification of PhoA and PhoA(SSCC)To overproduce these proteins, the phoA genes were cloned under the control of the ara promoter. pMS002 and pMS004 were linearized with KpnI and then partially digested with EcoRI. An approximately 1.5-kilobase fragment containing the phoA open reading frame was recovered and cloned into pBAD22 (36) that had been digested with EcoRI and KpnI. The resulting plasmids (named pMS019 and pMS021, respectively, for PhoA and PhoA(SSCC)) were introduced into strain MS5. Cells were cultivated in 4 liters of M9 amino acid medium described above (except containing 0.4% arabinose), and periplasmic fractions were prepared as described previously (32). PhoA proteins were purified according to the procedure of Chaidaroglou et al. (37). Approximately 10 and 0.5 mg of proteins of at least 90% purity (as judged from the SDS-PAGE profiles) were obtained for PhoA and PhoA(SSCC), respectively.
The phoA gene, cloned under the control of the lac operator/promoter on a plasmid vector, was subjected to site-directed mutagenesis to construct a series of 8 mutants in which a Cys codon was converted into a Ser codon singly or in combination. For the sake of convenience, these mutant forms of PhoA are desginated by four-letter notations in which residues 168, 178, 286, and 336, are indicated by C (Cys) or S (Ser) in this order, respectively. Thus, for instance, PhoA(SSCC) indicates the PhoA mutant in which Cys residues at positions 168 and 178 have been replaced by Ser.
The mutant forms of PhoA were expressed in a phoA strain.
All of them were found to be unstable in vivo. Pulse-chase
experiments showed that, in contrast to the wild-type PhoA protein
(Fig. 1A), mutant forms were degraded after
the synthesis (Fig. 1, B-D, closed symbols). The mutants that are not shown in Fig. 1 were similarly unstable. Their approximate half-lives were 5-10 min at 37 °C. They
were significantly stabilized in the
degP::Tn5 mutant strain (Fig. 1,
B-D, open symbols). Although some
proteolysis persisted even in the degP strain (Fig. 1), it
was possible to accumulate mutant forms of PhoA using this periplasmic
protease-deficient strain as a host.
The Cys-286-Cys-336 Disulfide Bond Is Essential, whereas the Cys-168-Cys-178 Disulfide Bond Is Dispensable for the PhoA Activity
The wild-type and mutant forms of PhoA were allowed to accumulate in the degP mutant cells, and their relative amounts were determined by subjecting whole cell extracts to SDS-PAGE and visualizing PhoA by immunoblotting. PhoA activities of the same cultures were assayed, and relative specific activities were determined by dividing the enzymatic activities by relative intensities of PhoA in the immunoblotting experiments (Table I). Only PhoA(SSCC) and PhoA(SCCC) were found to have significant enzyme activity (Table I). Since PhoA(CCSS), PhoA(CCCS), PhoA(CCSC), PhoA(SSSC), and PhoA(SSCS) were all inactive (see Table I for (CCSS); data not shown for the other mutants), the presence of both Cys-286 and Cys-336 was required for the activity. In contrast, Cys-168 and Cys-178 were dispensable for the enzymatic activity.
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PhoA(SSCC) was as active as the wild-type enzyme (Table I), suggesting that Cys-286-Cys-336 is the sole activity-supporting disulfide bond in PhoA. To confirm this notion, PhoA and PhoA(SSCC) were overproduced and purified. Specific activities of these preparations were about the same (Table I). Interestingly, PhoA(SCCC), which contains an additional cysteine, was less active than PhoA(SSCC). We have evidence indicating that PhoA(SCCC) tends to form an aberrant disulfide bond.4
The Fully Active PhoA(SSCC) Forms a Dimer but Does Not Form the Protease-resistant ConformationNormal PhoA sediments at about 6 S in sucrose gradient centrifugation, whereas an unfolded monomer
subunit sediments at around 2 S (3, 19). Periplasmic fractions were
fractionated by sucrose gradient centrifugation and examined for
sedimentation behaviors of PhoA proteins (Fig. 2). It
was found that enzymatically active PhoA(SSCC) sedimented to similar
fractions as the wild-type enzyme (Fig. 2, compare A and
B). In contrast, PhoA(CCSS) (which was inactive) sedimented
much more slowly (Fig. 2C) to the position similar to that
of PhoA(SSSS) (data not shown) or that of reduced and denatured PhoA
(3). These results indicate that PhoA(SSCC), but not PhoA(CCSS), is
competent for the dimer formation.
The PhoA variants were also examined by PAGE at pH 8.8 under
non-denaturing conditions. PhoA(SSCC) migrated to a similar position as
the wild-type PhoA (Fig. 3, compare lanes 1 and 2). PhoA(CCSS) migrated much slower (Fig. 3, lane
3). PhoA(SSSS) also migrated slowly under the conditions used
(data not shown). The basis for the slow migration of PhoA(CCSS) and
PhoA(SSSS) is not known. It may be due to the lack of a compactly
folded structure and/or different exposure of charged residues. In any
case, the results of native PAGE are consistent with the notion that
the overall structure of PhoA(SSCC) is similar to that of the wild-type
enzyme.
To know whether the remaining cysteines in the mutant proteins are
disulfide-bonded, we examined profiles of PhoA proteins in SDS-PAGE
under non-reducing conditions. Normal PhoA with two disulfide bonds
migrated faster than its reduced form (Fig. 4, compare
lanes 1 and 2). Electrophoretic mobility of
PhoA(SSCC) was indistinguishable from that of the oxidized wild-type
enzyme (Fig. 4, compare lanes 3 and 4). After
reduction with -mercaptoethanol, all the PhoA molecules shown in
Fig. 4 migrated identically at the position of the reduced wild-type
PhoA protein (data not shown). Thus, Cys-286 and Cys-336 that remain in
PhoA(SSCC) form an intramolecular disulfide bond.
Electrophoretic mobility of PhoA(CCSS) (Fig. 4, lane 5) was indistinguishable from that of PhoA(SSSS) (Fig. 4, lane 6) or of reduced PhoA. Either Cys-168 and Cys-178 in this mutant remain reduced or the Cys-168-Cys-178 disulfide bond affects the electrophoretic mobility only negligibly. The close proximity of these residues in the primary sequence may suggest the latter possibility.
It was already shown in Fig. 1 that PhoA(SSCC) was unstable in
vivo. Protease susceptibility of the mutant forms of PhoA was studied in vitro by trypsin digestion of cell extracts.
Wild-type PhoA was resistant to up to 50 µg/ml trypsin examined (Fig.
5A, showing data for up to 10 µg/ml
trypsin). PhoA(SSCC) was not resistant to even 2.5 µg/ml trypsin
(Fig. 5B, lane 2). PhoA(CCSS) was not resistant
to 5 µg/ml trypsin (Fig. 5C, lanes
2-5). These results show that the fully active and
dimeric PhoA(SSCC) is still in loosely folded conformation.
Roles of Disulfide Bonds in Activity and Stability of Alkaline Phosphatase
The results presented above indicate that of the two disulfide bonds in PhoA, the one between Cys-286 and Cys-336 is sufficient for the enzymatic activity. However, it is not sufficient to confer the protease-resistant conformation normally observed for this enzyme. The other disulfide bond (between Cys-168 and Cys-178) is important for stabilization of the protein.
PhoA is one of the enzymes that exhibit a high degree of thermal stability and protease resistance (38). Intramolecular disulfide bond formation is a prerequisite for its folding into an active dimer (3). PhoA has been widely used as a reporter of protein export from the cytosol (7, 8). The reducing environment of the cytosol prevents folding and activation of PhoA, and thioredoxin reductase plays a key role in this prevention (6, 9). In contrast, the periplasmic space contains DsbA, which facilitates oxidative folding of newly synthesized proteins (10, 11). PhoA molecules that are internalized because of a defect in signal sequence as well as those exported to the periplasm of a dsbA-defective mutant strain lack enzymatic activity and are degraded by cellular or exogenous proteases (4-6, 10, 11). Our results now show directly that only one of the disulfide bonds (Cys-286-Cys-336) is essential for enzymatically active conformation of PhoA.
The active site of this enzyme contains two atoms of zinc and one of
magnesium and involves at least the following amino acid residues:
Asp-51, Ser-102, Asp-153, Thr-155, Arg-166, Glu-322, Asp-327, Lys-328,
His-331, His-372, and His-412 (23, 24, 39). The Cys-286-Cys-336
disulfide bond, which connects two different loops that originate from
different -strands, will constrain the central
-sheet (with 10 strands) so that the active site residues are arranged in proper
geometric configuration (24). In contrast, the conformational
constraint imposed by the Cys-168-Cys-178 disulfide bond is limited
within a single loop. Such a restriction may be trivial with respect to
the formation of an active site and dimerization, but should be
important for establishing a tight overall or local folding state that
is crucial for the resistance of this enzyme to the action of
proteases.
The finding that the Cys-168-Cys-178 disulfide bond is required for the tight folding without a major contribution to the active site formation (Table I) may corroborate the proposal (40) that the acquisition of a catalytic ability of a protein is often made possible at the expense of stability of the protein. By making a disulfide bridge, the Cys-168 and Cys-178 residues may have evolved to overcome such an intrinsic incompatibility between activity and stability of the enzyme. Subramaniam et al. (22) reported that during renaturation of guanidine hydrochloride-denatured PhoA, full enzymatic activity is achieved well before the establishment of the final conformation of the native enzyme. The PhoA(SSCC) molecule deserves structural studies.
We thank Yoshinori Akiyama for useful discussion, Noriko Kusukawa for the polymerase chain reaction product of the phoA gene, and Kiyoko Mochizuki and Kuniko Ueda for technical and secretarial support.