(Received for publication, September 20, 1995; and in revised form, January 16, 1996)
From the
The maltose-binding protein (MalE) of Escherichia coli is the periplasmic component of the transport system for
malto-oligosaccharides. We have examined the characteristics of a
Mal mutant of malE corresponding to the
double substitution Gly
Asp/Ile
Pro, MalE31, previously obtained by random mutagenesis. In
vivo, the MalE31 precursor is efficiently processed, but the
mature protein forms inclusion bodies in the periplasm. Furthermore,
the accumulation of insoluble MalE31 is independent of its cellular
localization; MalE31 lacking its signal sequence forms inclusion bodies
in the cytoplasm. The native MalE31 protein can be purified by affinity
chromatography from inclusion bodies after denaturation by 8 M urea. The renatured protein exhibits full maltose binding affinity (K
= 9
10
M), suggesting that its folded structure is similar to
that of the wild-type protein. Unfolding/refolding experiments show
that MalE31 is less stable (-5.5 kcal/mol) than the wild-type
protein (-9.5 kcal/mol) and that folding intermediates have a
high tendency to form aggregates. In conclusion, the observed phenotype
of cells expressing malE31 can be explained by a defective
folding pathway of the protein.
In Escherichia coli, the export of proteins to the periplasm follows the general secretion pathway ((1) ). Considerable genetic and biochemical information about the export machinery has accumulated(2) , but little is known about the conformational state of the polypeptide chain, either during its translocation through the membrane or upon its release into the periplasm.
Maltose-binding protein (MalE or MBP), the malE gene product, serves as the periplasmic receptor for the high affinity transport of maltose and maltodextrins (reviewed in (3) ). Because of its key role in maltose transport, correct export of MalE into the periplasm is essential for cells to utilize maltose as a carbon source. This feature facilitated the use of genetic selections for analyzing MalE export. Furthermore, the isolation of strains synthesizing MalE-LacZ hybrid proteins led to a novel genetic approach that identified several genes encoding the secretory machinery, the sec genes(4) . As a protein model of translocation across the cytoplasmic membrane, MalE has also been extensively studied in the laboratories of the late P. Bassford and of L. Randall(5, 6) . The MalE protein is synthesized in the cytoplasm as a precursor protein, pre-MalE, with an amino-terminal extension, the signal sequence. As the precursor enters the periplasm, the signal sequence is cleaved by signal peptidase. Translocation requires that the precursor exist in an export-competent conformation representing a partially unfolded state (7) . Both the signal peptide (8) and the binding of SecB, the molecular chaperone involved in protein export(1) , participate in the maintenance of this initial conformation. Although Randall and Hardy (7) showed that MalE folding and export are kinetically competing processes, we do not know the extent of secondary and tertiary structures of precursor proteins. Another important unsolved problem concerns the releasing and folding steps of the polypeptide chain in the periplasm. Do specific proteins or molecular chaperones catalyze or facilitate this step in the periplasm? Two integral membrane proteins, SecD and SecF, are required for efficient protein translocation(9) . Interestingly, treatment of spheroplasts with anti-SecD antibodies decreased the processing of pre-MalE and increased the protease susceptibility of the mature species(10) . These results would be consistent with a molecular chaperone function of SecD on the periplasmic face of the inner membrane(11) .
Protein aggregates are formed in
vivo as a result of: (i) mutations that affect the folding
pathway; (ii) expression of heterologous proteins; (iii) exposure of
the cell to certain environmental stresses. It is generally accepted
that aggregation and folding are competing processes and that
aggregation is favored at elevated protein concentrations. A model
suggesting that a folding intermediate is responsible for inclusion
body formation has been proposed by Mitraki and King(12) . Such
a kinetic competition between the folding pathway and an aggregation
reaction has previously been recognized as a major determinant for
apparent irreversibility in refolding studies in
vitro(13) . Since aggregation is a second (or higher)
order process, it can be much faster than first-order folding and
therefore, at high protein concentrations, aggregation dominates over
folding and leads to the formation of insoluble protein(14) .
The observation that temperature-sensitive folding (tsf)
mutations in the tailspike protein of phage P22 increased the fraction
of newly synthesized tailspike chains forming aggregates demonstrated
the importance of folding intermediates in the mechanism of inclusion
body formation(15) . The tsf mutations prevent
formation of the native protein at the restrictive temperature, but
once folded at a lower permissive temperature, the mutant tailspike
proteins have stabilities similar to that of the native protein.
Intragenic suppressors of these mutations inhibit aggregation of
folding intermediates and increase the folding efficiency of tsf mutants(16) . Mutations influencing inclusion body
formation were also identified for human interleukin-1, expressed
in the cytoplasm of E. coli(17) . The mutated
residues were mainly located either in a
strand or in a flexible
loop. However, there was no strong correlation between the
thermodynamic stability of the mutant interleukin-1
proteins and
their tendency to form inclusion bodies. Another example of studies of
inclusion body formation in E. coli is provided by the enzyme
-lactamase(18) . In the case of such an exported protein,
the formation of inclusion bodies depends on the protein synthesis rate
and the amino acid sequence of the signal peptide(19) . High
level of expression of
-lactamase resulted in the accumulation of
precursor chains: aggregation occurred both in the cytoplasm from the
precursor and in the periplasm from the mature
-lactamase. Despite
the widespread reports of inclusion body formation resulting from very
high level production of recombinant proteins in E. coli,
there is little information on the molecular basis governing this
process.
In this study, we characterized a Mal mutant of the maltose binding protein (MalE31), previously
isolated by random linker insertion into the malE gene(20) . Our in vivo analysis revealed that the
mutant MalE31 polypeptide chain forms inclusion bodies after
translocation and signal peptide processing. We further report the in vitro analysis of the stability and aggregation of this
mutant protein compared with the wild-type protein. These data
confirmed that the observed phenotype of cells expressing malE31 can be explained by a defective folding pathway of the protein.
Plasmids pPD1 and pPD364 carrying
the wild-type malE gene (21) and the mutant malE31 gene(20) , both under the control of the mal promoter, were designated here as pHCME and pHCME31,
respectively. Plasmid p709, which carries a deletion of the
ribosome binding site and the first 256 codons of the malE gene(22) , was used as a negative control.
The mutations were confirmed by dideoxynucleotide
sequencing. The EcoRI-BglII fragments were isolated
from mutant M13mp18 RF DNAs and subcloned into plasmid pHCME to
construct pHCME-G32D, pHCME-I33P, and pHCME312-26.
Preceding all
biochemical characterizations, both purified proteins were extensively
dialyzed three times against 3 liters of buffer A at 4 °C. The
first five amino acid residues of purified MalE31 protein were
identified by using an Applied Biosystems 473 protein sequencer. The
protein concentration of MalE-wt and MalE31 solutions was determined
from the absorbance at 280 nm with an extinction coefficient of
= 68,750 M
cm
. (
)
In order to take into account the effect of the solvent on the fluorescence signal, the following equation was used,
where Y is the fluorescence signal in the
presence of
molar GdnHCl, Y
and Y
are the signal of the native and denatured
forms, respectively, at the same denaturant concentration; M
and M
are the solvent
effects on the native and denatured protein signal, respectively.
Experimental data were fitted by using a simplex procedure based on the
Nelder and Mead algorithm(30) . To study the reversibility of
the unfolding process, MalE-wt and MalE31 proteins at two
concentrations (1 and 10 µM) were incubated with varying
concentrations of GdnHCl for 6 h. They were then diluted in the
Tris-HCl buffer, incubated for 15 h at 25 °C, and centrifuged at
10,000 rpm for 10 min in an Eppendorf microcentrifuge. Finally, the
amount of native protein in the supernatant was determined by using the
two-antibody sandwich assay described by Martineau et
al.(31) . The monoclonal antibody 56.5, which was used in
this assay, bound to a conformational epitope of MalE or MalE31 protein
with the same affinity (K
= 5
10
M). (
)
We previously described a set of MalE mutant proteins
generated by random insertion of an oligonucleotide linker into the malE gene(20) . Among this collection, six mutations
prevented both growth on maltose as a carbon source and the periplasmic
release of the corresponding proteins by cold osmotic
shock(32) . In all cases but one, a deletion of several amino
acid residues from the mature region of the protein could explain their
defective phenotypes. However, in one case, malE31, the linker
insertion did not modify the length of the protein, but changed six
nucleotides, resulting in Gly Asp and Ile
Pro
substitutions at position 32 and 33 of the mature sequence,
respectively(20) .
Figure 1: Subcellular fractionation of MalE31 and derivatives. Periplasmic (A), cytoplasmic (B), and membrane fractions (C) were separated by SDS-PAGE and Coomassie-stained bands scanned by densitometry. The bands corresponding to GroEL and DnaK are indicated by arrows (GroEL is the faster migrating of the two bands). D, the proportions were calculated by summing the area of MalE band in the three subcellular fractions and are shown under the corresponding lane.
First, the kinetics of signal
sequence processing was determined using a pulse-chase analysis.
Processing of the precursor, a late step in export, is a reliable
indicator that the protein has, at least partially, crossed the inner
membrane(1) . Cells were pulse-labeled with
[S]methionine and then chased with
nonradioactive methionine for different times. The labeled MalE
proteins were analyzed by immunoprecipitation. Fig. 2shows that
the MalE31 precursor is processed very rapidly and that an export
defect cannot be observed at the earliest time point (10 s). In
conclusion, there were no differences in the processing kinetics
observed for pre-MalE31 species compared with those of wild-type
pre-MalE. Furthermore, amino-terminal microsequence analysis of
purified MalE31 protein (see below) confirmed that pre-MalE31 was
processed at the normal cleavage site.
Figure 2:
In
vivo processing of MalE31 precursor. Cells were radiolabeled with
60 mCi of [S]methionine for 15 s at 30 °C
and then chased with cold methionine for 10, 30, and 60 s. In lanes
B, cells were treated with 5 mM sodium azide for 5 min
prior to radiolabeling and chasing as described under ``Materials
and Methods.'' The positions of precursor (p) and mature (m) polypeptides are indicated by arrows.
Second, transmission electron
microscopic studies of cells harboring pHCME31 revealed the presence of
electron dense material between the inner and outer membranes of the
bacteria (Fig. 3A). Periplasmic inclusion bodies of
MalE31 were small and frequently included more than one per cell. In
contrast, cells producing the signal sequence deletion MalE31 protein
(pHCME312-26) had rare inclusion bodies that were clearly
localized within the cytoplasm and that had blurred boundaries (Fig. 3B). By analogy with other proteins which form
inclusion bodies in E. coli(33) , the amount of
insoluble MalE31 was temperature-dependent, with higher growth
temperatures promoting inclusion body formation (data not shown). These
results indicated: (i) that the MalE31 precursor was correctly
processed and thus that the mature protein is localized in the
periplasm, (ii) that the malE31 mutation causes MalE
aggregation whatever its cellular localization, and (iii) that
inclusion body formation in the periplasm arose from the association of
processed MalE31 protein
Figure 3:
Inclusion bodies of MalE31. Transmission
electron micrographs of ultrathin sections of E. coli pop6499
strain containing either plasmid pHCME31 (A) or plasmid
pHCME312-26 (B). Black and white
arrows indicate the position of the periplasmic and cytoplasmic
inclusion bodies, respectively.
Figure 4:
Heat-shock induction by MalE31. The
induction of the lon promoter fused to the lacZ gene
was tested in E. coli SR1364 strain harboring the various
pHCME31 plasmids (see Fig. 1). Miller activities of the lacZ encoded -galactosidase are calculated using the average of
five independent determinations with their standard error of the
mean.
Figure 5: Purification of MalE31. Fractions from the steps of purification were analyzed on SDS-PAGE stained with Coomassie blue. Lanes: 1, whole cell lysate; 2, soluble cell extract; 3, crude insoluble extract solubilized in 8 M urea; 4, pellet suspended in Tris-HCl buffer after renaturation; 5, supernatant after renaturation; 6, maltose eluate from amylose column.
To investigate the folding properties of MalE31,
the GdnHCl-induced unfolding transitions of the wild-type and MalE31
proteins were determined using tryptophanyl fluorescence as a probe of
the tertiary structure (Fig. 6). At the final protein
concentration of 0.2 mM, both transitions were found to be
reversible (see below) and symmetrical. The concentration of GdnHCl at
the midpoint of the transition (C) for MalE-wt was
0.92 ± 0.02 M, whereas the midpoint occurred at 0.62
± 0.02 M GdnHCl for MalE31 (Table 2). This
reflects the lower stability of the mutant MalE31; the
G
Figure 6:
Equilibrium denaturation curves. The
GdnHCl-induced transition was assessed by the fluorescence emission at
345 nm (excitation wavelength: 290 nm). , MalE-wt;
,
MalE31. The normalized data were obtained by using the relation f
= (Y
- Y
)/(Y
- Y
), where Y
refers to the observed fluorescence intensity, Y
and Y
are the fluorescence
intensity of the native and unfolded protein, respectively, and f
is the fraction of folded protein at
molar GdnHCl concentration.
Light scattering and apparent irreversibility were detected when the MalE31 concentration was increased in the unfolding experiments described above. To test the effects of protein concentration on renaturation, we performed a two-step dilution experiment(37) . Starting from the native state, MalE31 was incubated in various GdnHCl concentrations for 6 h at 25 °C and then diluted into the renaturation buffer (Fig. 7). Two different protein concentrations in the first step were explored, and we used a quantitative immunoassay to monitor the recovery of native MalE31 because of its high sensitivity(31) . The renaturation became partially irreversible when the protein had been exposed to concentrations of GdnHCl corresponding to the end of the unfolding transition zone. However, when refolding was initiated from 3 M GdnHCl, this phenomenon was less pronounced. The extent of irreversible denaturation of MalE31 was strongly dependent on the protein concentration, while no such effect of the MalE-wt protein concentration could be detected up to 10 mM. This phenomenon was detected only upon monitoring the native protein with the immunoassay. Indeed, using the same procedure as described for Fig. 7, but with a MalE31 protein concentration of 1 µM, complete reversibility was found by fluorescence measurements (data not shown). These results revealed the occurrence of an aggregation reaction arising mainly from MalE31 equilibrium folding intermediates. It is tempting to speculate that the aggregation of MalE31 folding intermediates is correlated with the formation of inclusion bodies.
Figure 7:
In vitro aggregation of MalE31.
The reversibility of the unfolding process was determined by measuring
the soluble protein after renaturation of MalE31 previously incubated
in various GdnHCl concentrations. Recovery of native protein was
assessed by an immunoassay. The concentrations of protein in the
denaturant were: , MalE-wt, 10 µM;
, MalE-wt, 1
µM;
, MalE31, 10 µM;
, MalE31,
1 µM.
In vivo and in vitro analyses of MalE31
folding revealed that the variant polypeptide chain had a high tendency
to aggregate. The three-dimensional structure of MalE consists of two
globular domains separated by a cleft in which maltose and
maltodextrins bind(38) . Each of the two domains is constructed
from secondary structural elements belonging to both the amino- and
carboxyl-terminal halves of the protein, thereby forming a central
-sheet framed by
-helices. The structural location of amino
acid substitution corresponding to the malE31 mutation is
shown in Fig. 8. Positions 32 and 33 are located in a turn
connecting helix I to strand B forming the first
supersecondary structure of the amino-terminal domain. In other
proteins, similar structural locations were also found for amino acid
substitutions which promote aggregation(17, 40) . This
observation is in agreement with the significant role of turns and
loops in the folding pathway of
/
proteins(41) . The
double substitution, Gly
Asp/Ile
Pro, destabilizes the protein by about 4 kcal/mol,
probably as a result of changes to geometrical constraints at the turn
region. Furthermore, despite a synergistic effect, the single
Ile
Pro substitution was the major factor
responsible for the aggregation. It is possible that a turn which
includes Pro
could introduce a conformational strain in
the folded state because of the limited value of the
angle(42) , even though the turn is in a solvent-accessible
region. Moreover, if the introduction of a proline residue in this turn
destabilizes the native structure, it does not affect the affinity of
maltose binding of MalE (Table 2).
Figure 8:
Ribbon diagram showing the altered
turn. A, diagram of the x-ray structure of MalE was
generated from coordinates kindly provided by F. Quiocho using the
program Molscript(39) . The location of the altered
turn is arrayed. B, the two substituted residues, Gly
and Ile
, connecting helix 1 and strand B are shown
by thick lines.
By studying the
unfolding-refolding transition of MalE variants corresponding to
intragenic suppressors of export-defective signal peptides, Randall and
co-workers (43) have suggested that the rate-limiting step in
the MalE folding pathway could be the formation of a supersecondary
structure localized in the amino-terminal domain. Strikingly, the
altered turn which leads to in vivo and in vitro aggregation of MalE31 connects an -helix and a
-strand
belonging to the same substructure of the amino-terminal domain.
Intragenic suppression of the export-defective signal peptide is
accomplished by slowing the folding of the precursor, thereby
increasing the time during which the protein is competent to enter the
export pathway(44) . cis-trans isomerization of prolyl
imide bonds might be expected to introduce slow steps in protein
folding, thus increasing the lifetime of intermediates and promoting
inclusion body formation. Preliminary results on the kinetics of
refolding have shown that, like intragenic suppressors of defective
peptide signal, MalE31 refolds more slowly than MalE-wt in
vitro. However, this slowest refolding step could not be explained
by a slow isomerization step in the unfolded state of MalE31. (
)
Inclusion body formation and in vitro aggregation are similar phenomena, originating from the failure of folding intermediates to achieve final folding. The putative cellular pathway for export and folding of precursors is shown in Fig. 9. As the precursor protein elongates, it begins to acquire secondary structure. This newly synthesized precursor may face many competing pathways, including binding molecular chaperones, folding, degradation, aggregation, and export. All of these possible outcomes depend on the intrinsic folding properties of the precursor being synthesized (or its folding rate relative to the rate of other processes). In this schematic drawing, molecular chaperones play an essential role in determining the choice between folding and export, but they may also participate in proteolysis by maintaining proteins in an unfolded state accessible to proteases. MalE belongs to a subset of precursors that interact preferentially with SecB, which seems to be entirely dedicated to export(45) . However, heat-shock proteins can substitute for SecB during protein export. Indeed, overproduction of DnaK and DnaJ improved the export of wild-type pre-MalE in a secB::Tn5 mutant(46) . Although periplasmic molecular chaperones involved in general protein folding have not yet been identified, we assume that such molecules could exist in this compartment. Another class of periplasmic proteins is enzymes that can either directly catalyze disulfide bond formation or maintain an appropriate oxidized environment in the periplasm. The lack of Cys residues in MalE eliminates the possible complication of intra- and/or extramolecular disulfide bond formation.
Figure 9: Model for protein export and folding in E. coli. This schematic representation illustrates the present discussion and tries to emphasize the different kinetic partitioning between folding, binding to chaperone, aggregation, degradation, and export. The outcome of the pathway depends on the folding parameters of the protein being exported.
Our results suggest that the step at which
productive folding of MalE31 is blocked is likely to be a folding
intermediate localized in the periplasm. These data do not fit the
model of alternative pathways open to protein precursors within the
cell, as defined by Randall and Hardy (48) . Indeed, they
proposed that the kinetic partitioning between folding and aggregation
depends strongly on binding to SecB in the cytoplasm. Although direct
experiments on the interactions between SecB and MalE31 have not been
performed, we have independently confirmed by several approaches that
the MalE31 precursor did not accumulate in the cytoplasm. This behavior
would indicate that the altered turn of the MalE31 precursor affects
neither its export-competent conformation nor its ability to bind SecB.
Structurally, a nascent precursor in an export-competent conformation
may be similar to misfolded proteins. Indeed, Wild et al.(49) showed that accumulation of secretory protein
precursors in strains lacking the SecB chaperone generates a signal for
induction of heat-shock proteins. Obviously, the absence of increased
activity in bacteria producing pre-MalE31
eliminates this possibility. However, the formation of periplasmic
inclusion bodies of MalE31 specifically triggers the
-dependent stress. (
)This alternative
heat-shock sigma factor is involved in response to stress occurring in
extracytoplasmic compartments(50) . It is worth noting that the
soluble fraction of MalE31 that is periplasmic (2%) is less than the
soluble fraction of MalE31 that is cytoplasmic (27%). This observation
suggests that in the latter case, the increased level of GroEL and
DnaK, that results from induction of the heat-shock response by MalE31,
can partially suppress the cytoplasmic aggregation of MalE31. It
appears that under these experimental conditions, there are
insufficient molecular chaperones in the periplasm to prevent or reduce
aggregation of MalE31
Finally, this work on the aggregation of
MalE31 opens the way to the selection of mutants affected in protein
folding and/or aggregation in the E. coli periplasm. Indeed, malE31 carried by plasmid pHCME31 was not able to complement
the deletion malE444 of strain pop6499. If general
molecular chaperones exist in the periplasm of E. coli, they
might modulate the aggregation reaction from the defective folding
intermediate of MalE31 as they do in the cytoplasm. Thus, mutations
that improve protein folding in the periplasm could be selected by
their ability to promote growth on maltose.