From the Departments of Biochemistry & Molecular
Biology and Chemistry, University of Massachusetts, Amherst,
Massachusetts 01003-4510, ¶ Department of Pharmacology and Program
in Molecular Biophyics, University of Texas Southwestern Medical
Center, Dallas, Texas 75235, and ** Department of Biology, Georgia State
University, Atlanta, Georgia 30303
Received for publication, January 5, 2001, and in revised form, February 28, 2001
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ABSTRACT |
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The selective recognition of pre-secretory
proteins by SecA is essential to the process of protein export from
Escherichia coli, yet very little is known about the
requirements for recognition and the mode of binding of precursors to
SecA. The major reason for this is the lack of a soluble system
suitable for biophysical study of the SecA-precursor complex.
Complicating the development of such a system is the likelihood that
SecA interacts with the precursor in a high affinity, productive manner
only when it is activated by binding to membrane and SecYEG. A critical
aspect of the precursor/SecA interaction is that it is regulated by
various SecA ligands (nucleotide, lipid, SecYEG) to facilitate the
release of the precursor, most likely in a stepwise fashion, for
translocation. Several recent reports show that functions of SecA can
be studied using separated domains. Using this approach, we have
isolated a proteolytically generated N-terminal fragment of SecA, which is stably folded, has high ATPase activity, and represents an activated
version of SecA. We report here that this fragment, termed SecA64,
binds signal peptides with significantly higher affinity than does
SecA. Moreover, the ATPase activity of SecA64 is inhibited by signal
peptides to an extent that correlates with the ability of these signal
peptides to inhibit either SecA translocation ATPase or in
vitro protein translocation, arguing that the interaction with
SecA64 is functionally significant. Thus, SecA64 offers a soluble, well
defined system to study the mode of recognition of signal peptides by
SecA and the regulation of signal peptide release.
SecA plays a central role in bacterial protein export: recognition
of the protein precursor and facilitation of its translocation across
the membrane (1-4). Critical to this role is the ability of SecA to
bind specifically to precursors with functional signal sequences. The
structural origin of this binding is a major question, since many
different sequences can act as signal sequences, but specific patterns
of residues are required and define the targeting function (5). To
date, no systems have been described that enable direct
characterization of SecA/signal peptide interactions without including
other species that participate in the translocation reaction
(membranes, SecY). Some success in dissecting the requirements for
signal sequence recognition by SecA has been achieved by the use of
idealized signal sequences in in vitro and in
vivo protein translocation assays (6-8) and including, in some
cases, cross-linking experiments as a measure of interaction with SecA
(9, 10). For example, a recent study by Miller et al. (11)
demonstrates interaction of synthetic signal peptides with SecA in a
vesicle system. Still, the complexity of these systems has made it
difficult to explore the intriguing structural question of how a
variety of diverse signal sequences can be specifically bound by one
protein. We have been attempting to simplify the interacting species,
SecA and the precursor protein, to develop a system for detailed
biophysical studies.
SecA is a large (901 amino acids), homodimeric, multifunctional protein
with highly interdependent ligand binding activities (1-4). For
example, pre-protein binding is stimulated by binding to phospholipids
(12). Moreover, binding to pre-protein and lipid stimulates the ATPase
activity of SecA, yet higher activity is seen (so-called
"translocation ATPase") in the presence of SecYEG, the putative
membrane translocation pore (13). Several years ago, Wickner and
co-workers (13, 14) reported that signal peptides inhibit the
translocation ATPase of SecA in the presence of membranes and SecY. In
contrast, the recent study by Miller et al. (11) finds that
signal peptides stimulated SecA ATPase activity in the presence of
lipids. In earlier work, we observed that signal peptides inhibit
in vitro protein translocation (15), although the site of
inhibition in the in vitro system has not been definitively
established. In these latter effects, mutant signal peptides that have
low in vivo activity (monitored by the proportion of the
precursor protein that attains an extracytoplasmic location and is thus
cleaved by signal peptidase) are less effective in modulating ATPase or
translocation activities.
Clearly, it would be of great advantage to dissect the functions of
SecA in order to carry out detailed studies of its interactions with
ligands. Recent work from several laboratories suggests that the study
of separated domains of SecA may provide an opportunity to analyze its
functions in greater detail (16-19). Our particular interest is to
elucidate the means by which SecA recognizes many different secreted
proteins and to clarify how precursor binding may be regulated. In the
present study, we report the isolation and characterization of SecA64,
a stable proteolytic fragment of SecA lacking about one-third of the
sequence from the C terminus and 10 residues from the N terminus. Our
studies with SecA64 suggest that it mimics the state of SecA that
recognizes and binds precursors and is then active in translocation, as
described above. This fragment corresponds closely with proteolytic
fragments described previously (18, 19); comparable fragments have
recently been expressed and characterized for some
biochemical functions (16, 17). Like the N-terminal fragments described
by these researchers, SecA64 has high ATPase activity, presumably
because it lacks the proposed intramolecular regulatory region
(16).
Since SecA64 has properties suggesting that it resembles the activated
state of SecA critical for pre-protein delivery to the membrane, we
were particularly interested in determining how signal sequence binding
modulates its functions. We have used peptides corresponding to
wild-type and mutant signal sequences from the Escherichia
coli outer membrane proteins, LamB and OmpA, to explore signal
sequence binding to SecA64. Additionally, we have compared the effects
of the same signal peptides on intact SecA membrane and translocation
ATPase activities as well as on in vitro protein
translocation in a well defined assay system. We find that the signal
peptides bind to SecA64 and inhibit its ATPase activity to an extent
that parallels their ability to facilitate export in
vivo.
Reagents--
Unless specifically mentioned, standard
laboratory reagents were purchased from Sigma, Fisher, or Aldrich. The
source of the lipids used in the ATPase and circular dichroism
(CD)1 experiments is Avanti
Polar Lipids, Inc. Typically the lipid mixture used consisted of a 3:1
molar ratio of L- SecA Preparation--
SecA was purified from an overexpressing
E. coli strain BL21( SecA64 Preparation--
SecA freezer stock was thawed and mixed
with an equal volume of 2× digestion buffer (50 mM
Tris-HCl, pH 8.0, 30 mM KCl, 15 mM ATP, 16 mM MgCl2, 1 mM DTT) at 5 °C.
Pre-chilled
To obtain a SecA64 sample relatively free of impurities, three
chromatographic steps were used. Undigested SecA was removed from the
sample by passing the reaction mixture over a Cibacron blue affinity
column in the original digestion buffer. The flow-through material from
the previous step was concentrated using a Centricon 30 unit and
exchanged into the column equilibration buffer (10 mM
Tris-HCl, pH 8.5, 30 mM KCl, 300 mM guanidine
(aminomethanamidine) hydrochloride, 1 mM PMSF, 1 mM DTT). Chromatography on Sephadex S200 at 4 °C
with a flow rate of 0.325 ml/min yielded SecA64. Pooled SecA64
fractions were concentrated in a Filtron 30 concentrator, exchanged
into 50 mM Tris, pH 8.0, 30 mM KCl, 1 mM DTT, 25% glycerol, and stored at Gel Electrophoresis--
Samples were analyzed using the
Tricine-based method of Schägger and von Jagow (23). Unless
otherwise noted, 10% acrylamide gels were used. Nondenaturing gels
were prepared using the same reagents as denaturing gels but omitting
SDS from all the buffers.
Determination of N-terminal Protein and Peptide
Sequences--
N-terminal sequences were determined by separating
proteolytic fragments using Tricine-SDS-polyacrylamide gel
electrophoresis, followed by electroblotting to polyvinylidene fluoride
(Immobilon P, Millipore Corp., Bedford MA) at 250 mA for 2.5 h in
10 mM CAPS, pH 11, 10% methanol. Protein bands were
visualized by staining for 1 min with Amido Black (0.1% in 45%
methanol, 10% ethanol) and then destaining with H2O until
a clear background was obtained. Protein bands selected for sequencing
were cut from the membrane and sequenced using an ABI 470A automated
peptide sequencer (Applied Biosystems, Inc., Foster City, CA).
ATPase Measurements--
ATPase activity was estimated by either
of two methods (24, 25), which were found to give consistent results.
To test the effect of lipid-binding on activity, purified protein was incubated in assay buffer at 37 °C at a concentration of 100 nM or the hydrolysis of ATP was monitored
spectrophotometrically using the pyruvate kinase/lactate
dehydrogenase-coupled enzyme assay in the presence or absence of small
unilamellar vesicles composed of a 3:1 mixture of the synthetic
phospholipids L- Densitometry--
Densitometry was carried out using a Molecular
Dynamics scanning laser densitometer. The density of the
largest/darkest band was defined as the upper end of the density scale,
and the values of the remaining bands were normalized to and expressed
as a percentage of this value.
Circular Dichroism--
CD spectra of proteins were measured in
1 mM citrate-phosphate-borate buffer, pH 7.6, at a
concentration of 1.6 or 1.1 µM on an AVIV 62DS CD
spectrophotometer (Aviv Associates, Inc., Lakewood, NJ). Secondary
structure content was estimated from averaged, smoothed spectra using
the method of Provencher and Glöckner (26) as implemented in the
computer program CONTIN. The CD spectra reported here consist of the
experimental data points overlaid with the curve of calculated values
as reported by CONTIN.
Signal Peptides--
Signal peptides were synthesized using
standard solid phase methods using either Applied Biosystems 430A or
Milligen 9050 automated peptide synthesizer.
t-Butyloxycarbonyl- or 9-fluorenylmethoxycarbonyl-protected amino acids were purchased from Peptides International (Louisville, Kentucky). Each crude peptide was purified using C4,
C18, or phenyl reversed phase columns from Vydac (25 × 2 cm); the peptide was eluted using an
H2O/CH3CN gradient in 0.1% trifluoroacetate in all cases. After lyophilization, the resulting peptide preparation was
treated to remove of residual trifluoroacetic acid by re-lyophilization from a solution of 50 mM ammonium acetate. The composition
and purity of the peptides were verified using automated sequencing on
an ABI 477A sequencer (Applied Biosystems, Inc.) and quantitative amino
acid analysis on a Beckman 6300 amino acid analyzer (Beckman Instrument
Co.). Peptide concentrations were calculated from amino acid analysis
and were estimated to be accurate to within ~5%.
Biotin-labeled Peptide Binding Assay Using Nondenaturing Gel
Electrophoresis and Transfer to Nitrocellulose--
To monitor
interaction between a biotin-labeled signal sequence and SecA64, a gel
binding assay was developed based on a reported procedure (27). The
biotin label was added during synthesis using standard solid phase
methods, and the labeled peptide was purified by high pressure liquid
chromatography using the procedures described above. Samples were
prepared in 1× sample buffer (112 mM Tris-HCl, pH 8.4, 5%
glycerol, 30 mM KCl, 1 mM Translocation ATPase Assays--
SecA-depleted membrane vesicles
were prepared from CK1801.4 cells as previously described (28).
Translocation ATPase reactions were carried out in 50-µl volumes,
with 5-µg membranes, 0.2 µg of SecA, 0.2 µg of proOmpA, 50 µg
of bovine serum albumin, 2 mM ATP, 1× buffer (50 mM Tris/HCl, pH 7.6, 20 mM NH4Cl, 2 mM magnesium acetate, 20 mM KCl). The proOmpA
used for this assay was purified according to published procedures
(13). We found that 100 µg/ml CK1801.4 membranes support a linear
translocation ATPase activity at 40 °C up to 10 min.
Translocation Assay--
In vitro protein synthesis
of proOmpA was carried out at 40 °C for 15 min using mRNA
enriched for OmpA (prepared from strain HJM114 containing plasmids
pOmpA and pC1857 upon induction of the
ompA gene) (29), and the precursors were purified by an immuno-affinity column as described (30, 31). The proOmpA molecules
labeled with [35S]methionine were eluted from the OmpA
IgG-agarose column with 0.2 M glycine-HCl, pH 2.2, 1 mM DTT. The eluants were neutralized with Tris base to pH
7.6 and used immediately for translocation.
The translocation mixture (0.1 ml) contained 50 mM
Tris-HCl, pH 7.6, 1 mM spermidine HCl, 8 mM
putrescine HCl, 1 mM DTT, 2 mM magnesium
acetate, 60 µl of immuno-purified proOmpA (final concentration of 120 mM glycine), 0.05 A280 units of
membrane vesicles, 2 µg of purified SecA unless otherwise indicated,
and an ATP-regenerating system (1 mM ATP-Tris, 0.02 mM GTP-Tris, 5 mM phosphoenol pyruvate-Tris, 3 µg of pyruvate kinase). Synthetic signal peptides were added at the
start of the translocation assay. After translocation had proceeded for
15 min at 40 °C, samples were chilled and exposed to proteinase K
(100 µg/ml) for 15 min at 0 °C. Membrane vesicles were isolated,
and the translocated products were analyzed in SDS-gel electrophoresis
as described (30, 31). To visualize the radioactive polypeptides, gels were treated with Autofluor (National Diagnostic), dried, and exposed
to Kodak XR-5 film at
For quantitation, fluorograms were scanned by densitometry as described
(31). To calculate the efficiency of translocation, the amount of
translocated OmpA (resistant to proteinase K treatment and recovered
with the membrane vesicles) was compared with the total input of
purified proOmpA (corrected for difference in methionine content)
present in translocation assays.
Limited proteolysis of SecA with
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dioleoyl phosphatidylcholine to
L-
-dioleoyl phosphatidylglycerol.
DE3)pT7-SecA (20) kindly provided by
J. Deisenhofer and D. Oliver. The protein was purified using a
published procedure based on affinity chromatography on the resin
Cibacron blue-agarose 3GA (21) or by a second widely used protocol
(22), with some minor modifications. Specifically, the pelleted cells
were resuspended in
-chymotrypsin was added to a final concentration of 0.15 mg/ml, and the sample was incubated for a time between 70 and 90 min in
a 5 °C bath with mixing. When the amount of SecA64 produced had
reached a maximum and most of the original SecA had been digested, the
reaction was quenched by adding PMSF to 1 mM final concentration.
80 °C. Samples
that had been purified to ~95% purity, as assessed by densitometry
of SDS gels, were used in subsequent experiments.
-dioleoyl phosphatidylcholine and
L-
-dioleoyl phosphatidylglycerol at a concentration of
150 µg/ml. SecA64 ATPase activity was measured using a
malachite green-ammonium molybdate colorimetric assay (24) at a
concentration of 400 nM SecA64 and 0.25 mM ATP
(all experiments shown except that in Fig. 7A) or 560 nM SecA64 and 5 mM ATP (Fig. 7A
only) in a volume of 25 µl at 37 °C.
-mercaptoethanol) and incubated for 30 min to 1 h at 4 °C to allow the system to reach equilibrium. After electrophoresis, the contents of the gels were
transferred to nitrocellulose using standard electroblotting methods.
The blot was probed with a streptavidin-peroxidase conjugate (Roche
Molecular Biochemicals) at 0.1 unit/ml after the ECL (enhanced chemiluminescence) Western blotting protocol from Amersham Pharmacia Biotech). The location of the bound streptavidin-peroxidase conjugate was then visualized by exposure to photographic film (HyperFilm-ECL, Amersham Pharmacia Biotech), which was quantitated by densitometry. To
standardize exposure times and facilitate comparison between samples,
nitrocellulose strips containing known amounts of the labeled peptides
were prepared from a stock labeled peptide solution of known
concentration using a Schleicher and Schuell Minifold II slot-blotting device.
76 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chymotrypsin in the presence
of ATP gives rise to a 64-kDa polypeptide that is remarkably stable
(Fig. 1A). During the course
of chymotryptic digestion, the ATPase activity of SecA and its
proteolytic fragments increases severalfold, whereas lipid stimulation
of the ATPase activity decreased during the same time period (Fig.
1B). We have isolated and purified the 64-kDa fragment,
which we term SecA64, and found that the elevated ATPase activity
co-purifies with it (Fig. 1C). The specific activity of
purified SecA64, 2850 ± 236 pmol ATP min
1 µg of
protein
1, is in the same range as reported
for SecA translocation ATPase activity (11-13). N-terminal sequencing
of SecA64 showed that it is derived from the N-terminal portion of SecA
through the loss of the C-terminal one-third of the protein and the
loss of the first 10 amino acids from the N terminus. Examination of
the sequence leads to the conclusion that SecA64 most likely spans a
region from amino acids 11 to 586.
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Fig. 1.
Limited
-chymotrypsin digestion of SecA in the presence of
ATP results in stabilization of a 64-kDa band that has elevated ATPase
activity. A, SDS-polyacrylamide gel electrophoresis gel
of the digestion. Lane 1 shows molecular weight markers,
lane 2 contains 1.8 µg of SecA incubated in buffer for 120 min in the absence of protease. Lane 3 contains the same
amount of protein incubated for 120 min in the presence of 0.14 mg/ml
-chymotrypsin. Lane 4 contains SecA treated under the
same conditions but in the presence of 15 mM ATP. The
addition of ATP results in the stabilization of a 64-kDa band, which is
indicated by the lower arrow. B, ATPase activity
alone or in the presence of 150 µg/ml 3:1 L-
-dioleoyl
phosphatidylcholine:L-
-dioleoyl phosphatidylglycerol
small unilamellar vesicles. The assay of ATPase activity was carried
out as described in the text. As digestion proceeds, the ATPase
activity of the sample increases (shaded bars). In the
presence of lipids, the ATPase activity is stimulated relative to the
activity in the absence of lipids (early time points), but the
difference decreases as SecA becomes depleted and SecA64 accumulates
(black bars). C, Top,
Tricine-SDS-polyacrylamide gel electrophoresis of fractions from
Sephacryl S-200 column run in 300 mM guanidine
(aminomethanamidine) hydrochloride, buffer (10 mM Tris-HCl,
pH 8.5, 30 mM KCl, 300 mM guanidine
(aminomethanamidine) hydrochloride, 1 mM PMSF, 1 mM DTT) at 4 °C. Each fraction was precipitated in 15%
trichloroacetic acid and resuspended in sample buffer. Lane
1, SecA; lane 2,
-chymotrypsin digested material;
lanes 3-14, column fractions 1-12. Bands due to SecA and
SecA64 are indicated by arrows. Bottom, ATPase
activities of column fraction show that the highest activity
co-purifies with SecA64.
As shown in Fig. 2, the CD spectrum of
SecA64 is consistent with a stably folded domain or combination of
domains. Curve-fitting analysis of the SecA64 CD spectrum leads to an
estimate of ~49% helix, 26% sheet, and 24% other, which compares
quite closely with the fit for the SecA CD, 66% helix, 18% sheet, and
16% remainder.
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To explore the interaction of SecA64 with signal sequences, we utilized
a series of synthetic signal peptides (Fig.
3) based on signal sequence mutations in
LamB, the phage receptor of E. coli (32-35), and a
second series from the signal sequence of the outer membrane protein
OmpA (36-39). The interaction between protein and signal sequence was
probed using a gel binding assay in which complex formation was
detected by co-migration of a biotin-labeled signal peptide with SecA
in a non-denaturing polyacrylamide gel (27). Due to its net positive
charge, the free biotinylated peptide did not enter the gel to any
appreciable extent, whereas SecA, a predominantly negative protein,
migrated into the gel, carrying bound peptide with it.
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Binding of the signal peptide to SecA is relatively weak (Fig.
4), as expected from the low
affinity interaction of SecA with pre-proteins in solution (12). In
contrast, the SecA64 sample showed a clearly visible signal (Fig. 4),
indicating that the peptide bound substantially more strongly to this
fragment of SecA.
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Functional, unlabeled signal sequences competed effectively for the
binding of the biotin-labeled peptide to SecA64, whereas non-functional
signal peptides did not (Fig. 5). A
peptide corresponding to the wild-type LamB signal sequence effectively
competed with the biotin-labeled peptide for binding, but the peptide
containing the four-residue deletion in the hydrophobic core,
LamB78, competed poorly. A peptide that harbors a single proline to
leucine change, which restores function to the deletion mutant in
vivo, LamB
78r2 (32), competed for binding at near wild-type
levels, indicating that effective competition requires a functional
signal sequence.
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The addition of LamBWT signal peptide inhibits the ATPase activity of
SecA64 (Fig. 6). Under the same
conditions, the ATPase activity of native SecA was affected only
slightly by the signal peptide. For the family of LamB signal
peptides, the capacity to inhibit SecA64 ATPase paralleled binding
affinity as monitored by competition (Fig.
7A). At concentrations where
the wild-type peptide clearly inhibits activity, the signal peptide
corresponding to the nonfunctional deletion mutant, LamB78, had no
observable effect on the activity of SecA64. The signal peptide from
the functional revertant strain, LamB
78r2, was able to inhibit
activity. These results argue that ATPase inhibition arises from
functionally relevant interactions of signal peptides with SecA64.
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To test the generality of the above observations, we examined the
ability of a set of OmpA signal peptides (Fig. 3) (36-39) to inhibit
SecA64 ATPase activity. Inhibition of the SecA64 ATPase by the OmpA
peptides correlated with their in vivo function (Fig. 7B). The wild-type peptide is an effective inhibitor of
activity at concentrations similar to those required for inhibition by the LamBWT peptide. The mutants OmpA8, OmpA
6-9, and OmpAI8N are
essentially without effect on the ATPase activity, consistent with
their export defects. Puzzlingly, the OmpA
9 peptide, which functions
at near wild-type levels in vivo, is able to inhibit SecA
ATPase activity only slightly, decreasing the ATPase by 20% at the
highest concentration tested.
Variants of the LamB signal peptide were tested for their ability to
inhibit the SecA64 ATPase. A peptide with three additional basic
residues in the N region of the LamB signal peptide (KRR-LamB, Fig. 3)
potently inhibits SecA64 ATPase activity, with a 50% maximal inhibition near 5 µM (Fig.
8). In contrast, half-maximal inhibition by the LamBWT peptide required ~25 µM under similar
conditions. In addition, the inhibition curve for the more highly
water-soluble KRR-LamB signal peptide is quite clearly hyperbolic.
Comparison of the ability of the L- and
D-isomers of the LamBWT signal sequence to inhibit SecA64
ATPase activity showed that the L-peptide inhibits activity
more strongly than the D-peptide (Fig. 8). Since the physical and chemical properties of these peptides are otherwise identical, we can conclude that the recognition of signal sequence by
SecA is sensitive to the chirality of the peptide backbone.
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To ensure the physiological relevance of the interactions between
SecA64 and synthetic signal peptides, we have examined the effects of
several of the same signal peptides on translocation ATPase and
in vitro protein translocation, both of which were previously shown to be inhibited by the addition of
signal peptides (13-15). Strikingly,
translocation ATPase (Fig. 9A) and in vitro translocation activities (Fig.
10A) are inhibited by the
family of OmpA signal peptides to an extent that closely parallels
their interaction with SecA64. Moreover, the order of effectiveness of
the peptides is as expected from their in vivo export
activities with the exception of an unexpectedly strong inhibition of
translocation ATPase by the 8 mutant. It is notable that
significantly more signal peptide is required to inhibit the SecA
translocation ATPase than in vitro translocation of proOmpA.
This difference can be attributed to the substantially lower quantity
of precursor protein present in the latter assay and, hence, the much
higher ratio of signal peptide to precursor. Also, signal peptide may
inhibit the in vitro translocation assay at more than one
site, for example the SecYEG complex, as well as SecA.
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A similar result was obtained when we compared the effect of the two
enantiomers of the LamBWT signal peptide. The natural, L-enantiomer inhibited the SecA translocation ATPase (Fig.
9B) and in vitro protein translocation (Fig.
10B) to a greater extent than did the
D-enantiomer, as had been seen for SecA64 ATPase inhibition. In all cases, the D-enantiomer does inhibit but
requires a higher concentration. Again, these results point to a
fundamental parallel between the behavior of the "stripped down"
proteolytic domain, SecA64, and SecA when engaged with the export
machinery in the process of protein translocation.
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DISCUSSION |
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Removal of approximately one-third of the SecA molecule by limited chymotrypsinolysis provided us with SecA64, a soluble, activated 64-kDa form of this large molecule with which to examine its function as a signal sequence receptor. SecA64 is predicted to contain the signal sequence/pre-protein binding region of SecA based on the location of PrlD mutations, which rescue maltose-binding protein signal sequence mutants (40), and on biochemical evidence, which places the pre-protein-binding site between residues 267 and 340 of SecA (41). The pre-protein-binding site has a low affinity for pre-proteins in free SecA, but the binding affinity increases when SecA becomes bound to a lipid bilayer (12). The increase in binding affinity is triggered by a conformational change in SecA, which also activates its ATPase activity (13, 42, 43).
Our data argue that removal of the C-terminal domain from SecA functionally mimics its binding to the cytoplasmic membrane. Proteolytic removal of the C-terminal domain leads to increased signal sequence binding as well as activation of ATPase to near translocation ATPase levels. The limited chymotryptic digestion conditions we employed results in removal of ~10 residues from the N terminus as well, but we have also shown that such an N-terminally trimmed SecA is fully func-tional.2 In an extension of earlier work showing that a comparable C-terminal truncation of SecA to that observed here activated its ATPase activity (18), Economou and co-workers (16) recently identified a segment of the C-terminal domain that they postulate acts as an intramolecular switch. They dubbed this region the intramolecular inhibitor of ATP hydrolysis or IRA. These researchers further hypothesized that this region mediates binding between the N- and C-terminal portions of SecA and that its removal causes a conformational change in the N-terminal region of SecA. Our data are entirely consistent with this model and add the important functional dimension that the conformational change accompanying activation of ATP hydrolysis also remodels the binding site for signal peptide in a manner that enhances affinity. Thus the C terminus of SecA must regulate the accessibility or structure of the signal sequence-binding site either directly by obstructing the binding site or indirectly by allosteric effects on the tertiary structure of the N-terminal region.
Several previous reports have shown signal peptide modulation of SecA lipid and translocation ATPase activities. Synthetic signal peptides inhibited SecA translocation ATPase in in vitro systems (13, 14). Signal peptides also inhibit translocation of proOmpA into E. coli membrane vesicles (15). In all of these cases, the signal peptide apparently acts by competing with the signal sequence of the protein to be translocated from the signal sequence-binding site on SecA. By preventing access of the pre-protein to the signal sequence recognition site, export is blocked. There is also evidence that the binding of the signal sequence is coupled to the ATPase activity of SecA during translocation. Activation of SecA-dependent translocation ATPase requires the presence of a functional signal sequence at the N terminus of the secretory protein (13). This suggests that productive binding of the signal sequence directly triggers changes in the conformation of SecA, since the translocation ATPase is not observed in the absence of a functional signal sequence even when all the other required molecules are present. The signal sequence need not be covalently linked to the secretory protein to stimulate ATPase activity; a synthetic signal sequence and the mature portion of proOmpA together can stimulate the translocation ATPase of SecA nearly as well as the addition of the intact pre-protein, but either one alone has no effect (13). This observation suggests that there are probably binding sites on SecA for both the signal sequence and the mature part of the secreted protein and, further, that both sites must be occupied to result in a productive change in SecA conformation. It is also clear that the interaction of SecA with lipid and with the SecYEG complex modulates the conformational change to its translocation-active state and the ATPase activities (44).
From past work, we had expected that signal sequence binding would also have an effect on the interaction of SecA64 with ATP. Moreover, we had anticipated that signal peptides would inhibit the ATPase activity of SecA64 as we observed them to do by analogy to their effect on the translocation ATPase activity of SecA (11-13), which SecA64 appears to mimic. The exact mechanism of this inhibition is not clear. In studies where synthetic signal peptides were shown to act as competitive inhibitors of translocation ATPase, the inhibition was postulated to arise from competition at the pre-sequence-binding site, preventing functional binding of proOmpA (13). In the case of SecA64, this cannot be the mechanism of inhibition, since this is a purified system, and no functional coupling between SecA64 and another polypeptide is taking place that the signal sequence can block. Binding of the signal sequence to its binding site must therefore cause a conformational change in SecA64, which alters either ATP binding, ADP release, or the rate of hydrolysis. It was noted previously that increasing the ATP concentration could reverse the inhibitory effect of signal peptides (13). Although we did not explore this effect systematically, we also observe a weaker inhibition at higher ATP concentration (compare Fig. 6 and 7A); both these results suggest that the functional coupling between these two sites is bi-directional. The basis for this inhibition is likely to reflect the relationship between ATP binding/hydrolysis and pre-secretory protein binding/release by SecA during export. It is intriguing that Kendall and co-workers observe a stimulation of SecA ATPase by signal peptides in the presence of lipid vesicles (11) and that this stimulatory effect also correlates with the functionalities of the signal peptides (45). Their observations most likely reflect an earlier step in precursor recognition before the conformational change that accompanies activation of SecA translocation ATPase. In our simplified system where SecA64 behaves like SecA after activation, the inhibition could be due to the inability of the protein to reach the next step of the cycle due to the absence of the other components of the translocase. A similar observation is seen in the case of the signal recognition particle (SRP); signal peptides inhibit the GTPase activity seen when SRP is combined in a simple in vitro system with its detergent-solubilized receptor (46, 47). We also show that the ATPase inhibition is reduced by signal sequence mutations that block export in vivo, suggesting that at least one of the effects of these mutations is to disrupt the interaction between SecA and the pre-secretory protein. Since evidence suggests that the pre-protein encounters SecA early in the translocation process (1-4), these mutations probably block export at the SecA recognition step rather than a later step in secretion.
All of our inhibition plots show a complex curve shape that is not consistent with simple inhibition as a result of peptide binding. If binding of the signal peptides is taking place at a single binding site and if the ATPase activity is diminished as a result of peptide binding, then the inhibition plot would be hyperbolic. Instead the curves are sigmoidal; at low peptide concentrations the effect on ATPase is negligible, but as the peptide concentration increases, the onset of inhibition occurs rapidly. The reason for this apparently cooperative behavior is not known. If one assumes a two-binding-site model, then the apparent cooperativity could arise from an interaction between these two sites. Binding of a peptide to the "mature protein" site, for example, might be weak if the signal sequence-binding site is unoccupied; binding could be strengthened by placing a functional signal sequence in the signal sequence-binding site. If both sites need to be occupied to inhibit the ATPase activity, then the apparent cooperativity could arise as the binding of signal sequences to the signal sequence site affects the binding affinity for binding to the mature site (or vice versa). Additionally, these peptides are highly amphiphilic and of low water solubility. The apparent cooperativity may arise from signal peptide self-association. Supporting this interpretation is the hyperbolic inhibition curve of the more soluble KRR-LamB signal peptide.
The basis for the recognition of signal sequences by SecA is not well
understood but is thought to involve the overall physical and chemical
properties of the signal sequence rather than sequence-specific interactions. SecA64 provides a system to explore directly the requirements for signal sequence interaction with SecA by following either binding (competition for binding to biotinylated LamBWT) or
ATPase inhibition or both. Previous work has established that the
essential features of E. coli signal sequences include both positive charges in the N-terminal region and a hydrophobic core of
minimum length approximately seven residues and average residue hydrophobicity between Ala and Leu (5, 48, 49). To distinguish the
relative importance of hydrophobicity of the central h-region from possible requirements for specific secondary structure, we have
compared enantiomers of LamBWT. These peptides have identical amino
acid sequences and, thus, identical hydrophobicities, but they differ
in the handedness of any secondary structure they adopt. In interfacial
environments, under conditions where the LamBWT adopts a right-handed
-helical conformation (33-35), the D-enantiomer forms a
left-handed
-helix, as indicated by a circular dichroism spectrum of
opposite sign (data not shown). If the binding of the signal sequence
to SecA64 depends on stereochemically specific contacts and not merely
on the overall hydrophobicity of the peptide, then the
D-peptide should bind and thus inhibit less well than the
natural ligand, the L-peptide, as was observed.
The N-terminal positive charge has been proposed to play an important role in SecA/signal peptide interaction (9), although recent work suggests that the hydrophobic core may compensate for lack of positive charges (8, 10). The increased charge of the KRR-LamB signal peptide indeed led to enhanced binding to SecA64. This result is consistent with the published reports that a greater positive charge in the N region of the signal sequence enhanced cross-linking of the pre-protein to SecA (9).
We found that the signal peptides had largely the same relative
effectiveness in inhibition of SecA64 ATPase and in inhibition of
translocation ATPAse or in vitro translocation, with one
exception (OmpA8). This result argues strongly that their actions on
SecA64 have the same origin as their effect on intact
translocation-active SecA. Moreover, their inhibitory effect on SecA
provides the most likely mechanism for their inhibition of
translocation in the more complex system. However, the potency of the
signal peptides as inhibitors is lower in the SecA64 system than in
either the translocation ATPase or in vitro translocation
assays. Biophysical data have previously shown that there is a tight
correlation between the hydrophobicity of the hydrophobic cores of
these sequences and their in vivo function (38, 39). It is
likely that the signal peptides that are most effective in
vivo partition into the membrane in either the translocation
ATPase or translocation assays and, thus, may be acting at a higher
effective concentration in the region of membrane-bound SecA. We
conclude that SecA64 is providing a simplified system devoid of
membranes to analyze the nature of the interaction of the signal
sequence with the export apparatus and, furthermore, that a key
interaction in the complex translocation apparatus occurs between SecA
and the signal sequence as expected.
In the present study, we have taken advantage of our identification of
a proteolytically stable domain of SecA, SecA64, to develop a system
for characterization of functionally relevant binding of signal
peptides to SecA. We have shown that the effects of signal peptides on
SecA64 closely parallel their effects on SecA translocation ATPase and
SecA-mediated in vitro translocation. We are now in a
position to develop biophysical approaches such as NMR and fluorescence
to explore in greater detail the structural origins of signal peptide
recognition by activated SecA. These studies will complement the
emerging structural picture of the Bacillus subtilis SecA
from x-ray crystallography (50).
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ACKNOWLEDGEMENTS |
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We thank Don Oliver and John Hunt for stimulating discussions and sharing unpublished work, Guenther Wittrock, Robert Chou, and Joanna Swain for critical reading of the manuscript, and Sarah Stradley for peptide synthesis.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM34962 (to L. M. G.) and GM34766 (to P. C. T.).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.
§ This author carried out the bulk of the experimental work while a student in the Molecular Biophysics Program at the University of Texas Southwestern Medical Center and with the training support of National Institutes of Heath Grant GM08297. Present address: Pfizer Global Research & Development, 2800 Plymouth Rd., Ann Arbor, MI 48105.
Present address: Kent and Queen Anne's Hospital, Dept. of
Pathology, 100 Brown St., Chestertown, MD 21620.
Present address: Dept. of Physiology, J-426, University of
California, San Francisco, CA 94143.
§§ Present address: Dept. of Microbiology and Immunology, School of Medicine, University of California, Los Angeles, CA 90095.
¶¶ To whom correspondence should be addressed. Tel.: 413-545-6094; Fax: 413-545-1289; E-mail: gierasch@biochem.umass.edu.
Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M100098200
2 J. P. You and P. C. Tai, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
CD, circular
dichroism;
CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid;
DTT, dithiothreitol;
ECL, enhanced chemiluminescence;
LamB, phage
receptor protein;
OmpA, outer membrane protein A;
PMSF, phenylmethylsulfonyl fluoride;
WT, wild type;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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