(Received for publication, March 31, 1995; and in revised form, June 6, 1995)
From the
The homodimeric SecA protein is the peripheral subunit of the translocase, and couples the hydrolysis of ATP to the
translocation of precursor proteins across the bacterial cytoplasmic
membrane. The high affinity ATP binding activity of SecA resides in the
amino-terminal domain of SecA. This domain contains a tandem repeat of
the ``so-called'' Walker B-motif, hXhhD (Walker, J. E.,
Saraste, M., Runswick, M. J., and Gay, N. J.(1982) EMBO J. 1,
945-951), that in combination with motif A is responsible for the
Mg-phosphate protein interaction. Two aspartate
residues at positions 207 and 215 of the Bacillus subtilis SecA, and Asp-217 in the Escherichia coli SecA, that
could be Mg
ion ligands, were individually mutated to
an asparagine. Mutant SecA proteins were unable to growth-complement an E. coli secA amber mutant strain, and the E. coli SecA mutant interfered with the translocation of precursor
proteins in vivo. B. subtilis mutant SecA proteins were
expressed to a high level and purified to homogeneity. The high
affinity ATP and Mg
-ion binding activity was reduced
in the Asp-207 mutant, and completely lost in the Asp-215 mutant. Both
SecA proteins were defective in lipid-stimulated ATPase activity.
Proteolytic studies suggest that the two subunits of the mutated
dimeric SecA proteins are present in different conformational states.
These data suggest that Asp-207 and Asp-215 are involved in the binding
of the Mg
-ion when Mg
-ATP is bound
to SecA, while Asp-207 fulfills an additional catalytic role, possibly
in accepting a proton during catalysis.
SecA is an essential component of the general protein translocation pathway in Escherichia coli (Schmidt et al., 1988). The homodimeric SecA (Akita et al., 1991; Driessen, 1993) is involved in multiple catalytic and regulatory interactions (Wickner et al., 1991; Driessen, 1994). SecA associates with cytosolic precursor proteins bound or unbound to SecB, and guides them to the translocation sites at the cytoplasmic membrane (Hartl et al., 1990). At the membrane, SecA interacts with the heterotrimeric SecY/E/G integral membrane protein complex (Wickner et al., 1991). SecA is an ATPase, and is stimulated for ATP hydrolysis by its interactions with translocation-competent precursor proteins, the SecY/E/G complex and acidic phospholipids (Cunningham and Wickner, 1989; Brundage et al., 1990; Lill et al., 1989, 1990).
In the early stages of translocation, SecA uses the energy of ATP binding to insert into the membrane and to release the signal sequence domain of the bound precursor protein (Schiebel et al., 1991). SecA may undergo nucleotide-modulated cycles of membrane insertion and exclusion (Breukink et al., 1992; Van der Wolk et al., 1993). Precursor proteins may be stepwise translocated across the membrane (Schiebel et al., 1991) through co-insertion with SecA (Economou and Wickner, 1994), during which SecA may completely transverse the membrane (Kim et al., 1994). Further translocation may be driven by the protonmotive force and/or repeated nucleotide-modulated SecA cycles (Schiebel et al., 1991). A detailed understanding of the molecular mechanism of the ATPase activity of SecA will clarify the coupling between the cycles of ATP binding and hydrolysis, and the binding and release of the precursor proteins.
SecA homologues have been identified in
higher plants, primitive algae, and in Gram-negative and -positive
bacteria. The Bacillus subtilis SecA protein shares an overall
65% homology with the E. coli SecA (Overhoff et al.,
1991; Sadaie et al., 1991) and appears to be functionally
similar (Takamatsu et al., 1992; Klose et al., 1993;
Van der Wolk et al., 1993). SecA contains two
nucleotide-binding sites, i.e. a high (K
150 nm) (Matsuyama et al., 1990; Klose et
al., 1993; Van der Wolk et al., 1993; Mitchell and
Oliver, 1993) and a low (K
300
µm) (Mitchell and Oliver, 1993) affinity site. The amino terminus
of SecA contains the amino acid sequence motif, GXXXXGKT (the
Walker motif A) that is characteristic of a major class of
nucleotide-binding sites (Walker et al., 1982). Substitutions
at Lys-108 (Lys-106 of the B. subtilis SecA) block the
translocation ATPase activity of SecA (Van der Wolk et al.,
1993; Mitchell and Oliver, 1993), and interfere with release of SecA
from the membrane (Van der Wolk et al., 1993). This suggests a
coupling between translocation and the temporal insertion/de-insertion
of SecA into the membrane (Economou and Wickner, 1994; Driessen, 1994).
The Walker B-motif, hXhhD, that in combination with motif A completes
the Mg
-phosphate protein interaction, is found as a
tandem repeat at residues 205-217 (residues 203-215 in the B. subtilis SecA) (Fig.1). The conserved aspartate is
thought to be needed for the coordination of the Mg
ion. The first occurrence of the B-motif shows some homology to
the DEAD box found in helicases (Koonin and Gorbalenya, 1993), while
second occurrence is positioned in a highly conserved region, and
aligns with the corresponding domain in members of the ATP-Binding Cassette (ABC) family (Mitchell
and Oliver, 1993). Substitution of Asp-209 of the E. coli SecA is known to block the translocation ATPase activity,
confirming its role in catalysis (Mitchell and Oliver, 1993). However,
the tandem duplication of this region suggests that the
Mg
-binding domain has a more complicated spatial
organization. To determine which of the aspartate residues is needed
for Mg
binding, we have constructed site-directed
mutants at Asp-207 and Asp-215 of the B. subtilis SecA, and at
Asp-217 of the E. coli SecA (Fig.1A). Our
data indicate that both aspartate residues are indispensable for SecA
function, both in vivo and in vitro.
Figure 1: Aligned sequences of SecA proteins showing the B-region of the high-affinity ATP-binding site (A) and the internal duplication of the B-motif (B). Mutations in the B. subtilis and E. coli SecA are indicated with an arrow. Genebank accession codes: B. subtilis (D10279; D90218); Staphylococcus carnosus (X79725); Streptomyces lividans (U21192); Listeria monocytogenes (L32090); E. coli (M20791); Caulobacter crescentus (U06928), Antithamnion sp. (X64705); Pavlova lutherii (X65961); Synechoccus sp. (X74592); and Olisthodiscus luteus (Z35718). Positions of the introduced mutations in the B. subtilis and E. coli SecA are indicated by arrows.
B. subtilis SecA proteins were purified
as described (Van der Wolk et al., 1993). For the purification
of the D207N and D215N SecA proteins, the phenyl-Sepharose
chromatographic step was replaced for a weaker hydrophobic interaction
medium, i.e. butyl-Sepharose. It was not possible to elute the
mutant SecA proteins to an appreciable yield from the phenyl-Sepharose
resin, suggesting that they differ from the wild-type SecA in that they
expose a greater hydrophobic surface. Pooled SecA-containing peak
fractions from the MonoQ chromatographic step (Van der Wolk et
al., 1993) were brought to 1.3 M ammonium sulfate and
applied to a butyl-Sepharose column, equilibrated with 1.3 M
ammonium sulfate in Buffer A (50 mM Tris-HCl, pH 7.6, 10%
(v/v) glycerol, 1 mM DTT) at 4 °C. The SecA protein was
eluted with a linear gradient of 1.3-0 M ammonium
sulfate in Buffer A. Mutant SecA proteins eluted at approximately 80
mM ammonium sulfate shortly after the endogenous E. coli SecA. To remove remaining minor impurities, the material was
further purified using again MonoQ and a HiLoad Superdex® 200
column (Van der Wolk et al., 1993). SecA proteins eluted from
the gel filtration column at the position of the dimeric wild-type SecA
protein (Driessen, 1993). Pooled fractions were concentrated in
Centriprep concentrators, aliquoted, and stored frozen at -80
°C at 3-5 mg ml. Purified proteins were
analyzed by Coomassie-stained SDS-PAGE and by Western blotting using
polyclonal antisera directed against the B. subtilis and E. coli SecA protein. Western blots confirmed that the protein
samples were free of residual E. coli SecA.
For measurements of the intrinsic tryptophan fluorescence, MgOAc was added at various concentrations ranging from 500 µM to 50 mM to a solution of 600 µl containing: 0.8-1 µM SecA, 50 mM Tris-OAc, pH 7.5, and 100 mM KOAc. Tryptophan fluorescence emission measurements were referenced against the excitation intensity. Excitation and emission was at 290 and 330 nm, respectively. Bandwidths were set at 2 nm. Quenching data was analyzed according to the modified Stern-Volmer equation (Lakowicz, 1986):
were F and F are the
tryptophan fluorescence in the absence and presence of
Mg
, respectively. f
is
the fraction of the fluorescence signal accessible to the quencher
Mg
, K is the Stern-Volmer constant in
mM
, and [Q], is the
Mg
concentration in mM. All fluorescence
measurements were performed at 20 °C using an SLM 4800C
time-resolved fluorometer (Aminco, Urbana, IL).
pMKL400 (encoding B. subtilis SecA wild-type protein),
pMKL440 (B. subtilis SecA(D215N)), pMKL441 (B. subtilis SecA(D207N)), pMKL180 (E. coli SecA wild-type), and
pMKL183 (E. coli SecA(D217N)) were introduced into the
conditional-lethal E. coli secA mutants MM52 (secA51) (Oliver and Beckwith, 1981), BA13 (secA13
, supF
) (Cabelli et al., 1988), and MM66 (geneX
, supF
) (Oliver and Beckwith, 1982). Compared to
pMKL400 and pMKL180, the transformation efficiency of pMKL440, pMKL441,
and pMKL183 in these strains was significantly lower but could be
improved by including, in addition to 0.5% glucose, the anti-inducer ortho-nitrophenylgalactofucoside (1 mM) throughout
the whole transformation procedure. Whereas the transformants harboring
pMKL400 or pMKL180 grew normally at the permissive temperature in the
presence of 0.5% glucose, we noticed that the pMKL440, pMKL441, or
pMKL183-containing cells grew somewhat more slowly at 30 °C under
repressed conditions, a finding which was especially pronounced in
liquid medium (data not shown). Full induction of B. subtilis
secA(D207N) or secA(D215N) expression with IPTG at the
permissive temperature was lethal in all E. coli secA mutant
strains tested, and also in their MC4100 parental strain. In addition,
we have observed that full induction of the B. subtilis secA wild-type gene frequently resulted in lethality in MC4100-derived E. coli strains. In non MC4100-derived strains (e.g. JM109; see above), the B. subtilis secA genes (wild-type
and mutant) could be fully induced without noticeable effects on cell
growth. The reason(s) for these strain-specific differences are
unknown. Induction of the E. coli secA(D217N) gene was lethal
in all conditional-lethal E. coli secA strains tested.
Substantial steady-state accumulation of precursor proteins was
observed in pMKL183-containing cells (Fig.2, lanes
5-8) when cell extracts were analyzed by Western blotting
experiments using antisera directed against maltose binding protein
(MBP) (Fig.2B) and outer membrane protein A (OmpA)
(data not shown), although D217N SecA level was comparable to wild-type
levels (Fig.2A). This suggests that the corresponding
gene product strongly interferes with protein translocation.
Figure 2:
Accumulation of precursor proteins in E. coli JM109 expressing the wild-type and D217N E. coli SecA proteins. E. coli JM109 cells harboring plasmids
pMKL180 (lanes 1-4) or pMKL183 (lanes
5-8) were grown on LB supplemented with glucose (lanes 1 and 5), IPTG (lanes 2 and 6), maltose (lanes 3 and 7), or maltose and IPTG (lanes 4 and 8). The cellular levels of SecA (A) and MBP (B; , precursor of MBP;
, mature MBP) were
determined by Western blotting as described under ``Experimental
Procedures.''
Previously, we have found that the B. subtilis SecA
wild-type protein (encoded by pMKL400) could partially complement the
growth and secretion defects of E. coli secA mutants MM52 (secA51), BA13 (secA
, supF
), and MM66 (geneX
, supF
) at the nonpermissive temperature (Klose et al., 1993). Successful complementation required that the B. subtilis SecA protein was not overproduced. Therefore,
complementation of the E. coli secA mutant strains was
performed in the presence of glucose. In contrast to our previous
results, we noticed that the B. subtilis secA wild-type gene
was no longer able to complement the growth defect of the MM52 (secA51
) mutant at the nonpermissive temperature.
This result is in agreement with Takamatsu et al.(1993), and
suggests an erroneous MM52 strain had been used in our earlier study.
However, as also reported earlier (Klose et al., 1993), the B. subtilis secA gene (pMKL400) reproducibly allowed growth of
the BA13 (secA
) (data not shown) and MM66 (geneX
) (Fig.3) mutant strains at 42
°C. In contrast to MM52, no full-length SecA protein is synthesized
in BA13 and MM66 at the nonpermissive temperature (Oliver and Beckwith,
1982; Schmidt et al., 1988; data not shown), suggesting that
the presence of the SecA51 mutant protein precludes successful growth
complementation by the B. subtilis SecA. Contrary to MM66
cells containing pMKL400 or pMKL180, cells of MM66 harboring pMKL440,
pMKL441, or pMKL183 were unable to grow at the nonpermissive
temperature (Fig.3). These results suggest that both aspartate
residues positioned in the putative B-domain are critical for SecA
function.
Figure 3: Growth complementation of E. coli strain MM66 at the non-permissive temperature by wild-type and mutant E. coli and B. subtilis SecA proteins. Overnight cultures of E. coli MM66 harboring the indicated plasmids were cross-streaked on selective medium and incubated at 30 and 42 °C respectively. Plasmids contained the following inserts: pTRC99A, none; pMKL400, B. subtilis wild-type secA; pMKL440, B. subtilis secA(D215N); pMKL441, B. subtilis secA(D207N); pMKL180, E. coli wild-type secA; and pMKL183, E. coli secA(D217N).
Figure 4:
SecA ATPase mutants interfere with in
vitro translocation of proOmpA. In vitro translocation
reactions with urea-treated inverted inner membranes of E. coli strain D10 supplemented with purified E. coli SecA (5
µg ml) (lane 1) were performed in the
presence of a 10-fold excess of purified wild-type (lane 2),
K106N (lane 3), D207N (lane 4), and D215N (lane
5) B. subtilis SecA protein.
Figure 5:
Phospholipid-stimulated ATPase activities
of wild-type and mutant B. subtilis SecA proteins. B.
subtilis SecA proteins (100 µg ml) were
incubated at 37 °C with the indicated amounts of E. coli lipid in buffer B supplemented with 2 mM ATP. After 30
min, reactions were terminated and analyzed for the released inorganic
phosphate as described under ``Experimental Procedures.''
Wild-type
; K106N
; D207N
; D215N
.
The high-affinity
nucleotide binding site at the amino terminus of the E. coli SecA can be photoaffinity cross-linked with
[-
P]ATP (Matsuyama et al., 1990).
At 50-200 nM [
-
P]ATP, hardly
any cross-linking of the low-affinity ATP-binding site occurs (Mitchell
and Oliver, 1993). Wild-type, K106N, and D207N SecA were readily
cross-linked with [
-
P]ATP upon irradiation
with UV (Fig.6A). The efficiency of cross-linking of
D207N SecA was low as compared to the wild-type. No cross-linking was
observed with the D215N mutant (Fig.6A). Addition of
ATP to the reaction mixture prior to UV irradiation efficiently
prevented cross-linking (Fig.6B), and inhibition was
half-maximal at about 100-150 nM and 2 µM for the wild-type and D207N SecA, respectively. When SecA was
dialyzed against a buffer containing EDTA, photocross-linking was
strictly dependent on the presence of Mg
(data not
shown). These results indicate that the D207N and D215N SecA mutants
differ in their ability to bind Mg
-ATP. Although the
D207N SecA is unable to hydrolyze ATP, it retains ATP binding activity
albeit with a lowered affinity.
Figure 6:
[-
P]ATP
photoaffinity cross-linking of wild-type and mutant B. subtilis SecA proteins. A, SecA proteins were incubated in Buffer
B in the presence or absence of 0.1 mM ATP and subjected to UV
cross-linking as described under ``Experimental Procedures.'' B, [
-
P]ATP photoaffinity
cross-linking of wild-type (
) and D207N (
) SecA protein in
the presence of increasing amounts of ATP. The
[
-
P]ATP concentration was 25 nM.
Autoradiograms were densitometrically scanned, and the relative amounts
of [
-
P]ATP cross-linking of the SecA
proteins were plotted as a function of the total ATP
concentration.
Figure 7:
Binding of Mg ions to
wild-type and mutant B. subtilis SecA proteins. Mg
quenching of the intrinsic tryptophan fluorescence of wild-type
(
), D207N (
), and D215N (
) SecA. Inset,
relative decrease of the fluorescence of Magnesium Green® in the
presence of 200 nM Mg
upon the addition of
wild-type and mutant SecA proteins at a final concentration of 300
nM. Further details are as described under ``Experimental
Procedures.''
Measurements
with Magnesium Green® require that a significant fraction of the
Mg present in solution is bound to SecA. This method
is therefore less suited for the determination of the kinetic constants
of Mg
binding. The B. subtilis SecA contains
a tryptophanyl residue at positions 651 and 723. The Trp fluorescence
emission spectra of wild-type and K106N SecA peaks at 325 nm, typical
for Trp residues shielded from the aqueous phase. The emission spectra
of the D205N and D215N was red-shifted to approximately 332 and 335 nm,
respectively (data not shown). We noted that Mg
addition to wild-type SecA caused a partial quenching of the Trp
fluorescence without a shift of the emission maximum. Quenching
saturated at millimolar Mg
concentration (Fig.7,
). Analysis of the quenching data according to the
modified Stern-Volmer equation yields an apparent K
of 0.58 mM. About 13.6% of the Trp fluorescence was
accessible to the quencher Mg
. D207N SecA appears to
bind Mg
with lower affinity, i.e. 2.8 mM (
), while the fraction of accessible Trp fluorescence
remained the same, i.e. 13.9%. The Trp fluorescence of D215N
SecA was not quenched by Mg
(
), even when the
concentration was raised up to 50 mM. These results suggest
that Asp-207 and Asp-215 are both involved in coordinating the
Mg
in the catalytic high-affinity ATP-binding site of
SecA. Asp-215 seems to be critical for Mg
binding.
Figure 8:
Protease digestion of wild-type and mutant B. subtilis SecA proteins. V8 protease (9 µg
ml) digestion of wild-type and mutant SecA proteins
in the absence and presence of 1 µM or 2 mM of
the non-hydrolyzable ATP analogue, ATP
S. The untreated D207N SecA
stains as a doublet due to partial proteolysis during the purification
procedure. Proteolysis of the D207N and D215N SecA proteins resulted in
the accumulation of a 88-kDa fragment.
A complete understanding of how SecA functions requires
knowledge of the molecular mechanism of the action of ATP. The region
in the B. subtilis (Van der Wolk et al., 1993) and E. coli (Mitchell and Oliver, 1993) SecA responsible for
high-affinity ATP-binding harbor the GXXXXGK(T/S) motif found
in many nucleotide triphosphatases (Walker et al., 1982). The
residues of the Walker A-type motif typically form a loop (P-loop) that
provides a binding pocket for the phosphoryl groups of the
Mg-nucleotide complex (Müller and
Schultz, 1992; Story and Steitz, 1992; Berchtold et al., 1992;
Pai et al., 1990; Howard and Rees, 1994; Abrahams et
al., 1994). The Mg
ion is stabilized by at least
two residues, i.e. the serine or threonine of the A-motif, and
an aspartate that is located at the end of an hydrophobic
-strand
contained in the B-motif (Walker et al., 1982). Tentative
assignment of the region in the primary amino acid sequence of SecA
suggests that the high-affinity ATP-binding site harbors a tandem
repeat of the B-motif (Fig.1B). Rigorous conservation
of aspartate residues at positions 207 and 215 in the B. subtilis SecA (i.e. Asp-209 and Asp-217 in the E. coli SecA) leaves only these two candidates as possible important
residues in the coordination of the Mg
ion.
Mutagenesis of Asp-209 of the E. coli SecA inactivates the
ATPase activity of SecA (Mitchell and Oliver, 1993). We now show that
this region has a more complex structure. Both Asp-207 and Asp-215 of
the B. subtilis SecA, and in conjunction to Asp-209 (Mitchell
and Oliver, 1993), Asp-217 of the E. coli SecA, render the
protein inactive for in vivo complementation of the E.
coli secA mutant strain MM66 (Fig.3).
Mg is needed for nucleotide binding to SecA. Asp-215 appears to be
most intimately involved in Mg
ion binding. Due to
the inability to bind Mg
, this mutant is also
defective in nucleotide binding and hydrolysis. The pronounced effect
of the substitution of the acidic aspartate by the cognate amine
asparagine at position 215 implies that the precise position or
electrostatic environment of the Mg
is crucial for
binding. Mutation of Asp-207 has a much smaller effect on
Mg
binding, and the D207N mutant is still able to
bind nucleotides. The apparent perturbation of the Mg
environment, however, also results in a substantial decrease in
the lipid-stimulated ATP hydrolytic activity. It, therefore, seems
likely that Asp-207 is not only involved in ligating the
Mg
, but that it is also needed for a catalytic
function. In the bovine Hsc70 ATPase at least two aspartate residues
are involved in Mg
binding (Wilbanks et al., 1994; Flaherty et al., 1994), while several acidic groups
in the ATP-binding region participate in accepting a proton during
release of the
-phosphate. The function of Asp-207 of SecA may lie
primarily in accepting a proton during ATP hydrolysis, possibly in
conjunction with the invariable Lys of the A-motif.
SecA is functional as a dimer (Driessen, 1993). The coupling mechanism that connects the two subunits of the dimer to the translocation of precursor protein across the membrane is one of the unanswered problems in the SecA mechanism. Size-exclusion chromatography demonstrates that the D215N and D207N SecA proteins retain their dimeric structure. Proteinase digestion and tryptophan fluorescence studies, however, indicate that their conformation has changed as compared to the wild-type. Intriguingly, the V8 protease digestion pattern of the D215N SecA (and to a lesser extent indicated by the intrinsic instability of the purified D207N SecA) indicates that only half of the molecules are proteolytically attacked. It is tempting to speculate that this phenomenon reflects proteolysis of only one of the two monomers of the SecA dimer, possibly as they differ in their respective conformations. Alternatively, one could propose that the mutant proteins have folded in an aberrant fashion, yielding two populations that differ in conformation. Both mutants strongly interfere with translocation in vitro, suggesting that their conformation (or certain aspects of it) has retained at least some aspects of an intermediate state in the ATP binding and hydrolysis cycle of SecA. This interference may be due to a stable association of the SecA mutants with the translocation site as shown before for the K106N mutant (Van der Wolk et al., 1993). The B-site mutants may interfere by a similar mechanism as they still bind to inner membranes, while effective interference in vitro requires the prior inactivation of membrane-bound SecA.
Wild-type SecA adopts a protease-resistant conformation only when both the high- and low-affinity nucleotide-binding sites are occupied. The D207N and D215N SecA proteins are cleaved at a different position as compared to the wild-type, and only half of the molecules are proteolyzed at the amino terminus. If this proteolysis concerns only one of the monomers of the functional dimer (Driessen, 1993), it is suggested that the two subunits of the dimer may be conformationally coupled. Conformational coupling may be of relevance for the process in which the precursor protein is threaded through the membrane in a stepwise fashion. It is not known if the complete dimeric SecA inserts into the membrane when it binds ATP, or whether the subunits insert in an alternate order. In the latter case, zig-zag movements between the two subunits of the SecA dimer would thread the precursor protein across the membrane, much like a two-tact engine where each stroke results in alternate movements of the cylinders. We are currently investigating if this fascinating, but still speculative, combination of protein-protein interactions and conformational changes coupled to ATP hydrolysis may have significance to the action of the SecA dimer in threading the precursor proteins across the membrane.
The alteration of the conformation of SecA by the introduced mutations at Asp-207 and Asp-215 is also evident from the major red-shift in the Trp fluorescence emission maximum. The relative exposure of Trp residues in proteins to solvent generally correlates well with the fluorescence emission maxima (Lakowicz, 1986). It thus appears that the exposure of the two Trp residues, i.e. at positions 651 and 723, to the aqueous solution has increased in these mutants, and probably reflect a long-range conformational change in SecA. Both mutants also bind more tightly to hydrophobic interaction media, suggesting that they expose a surface with greater hydrophobicity.
Mg quenches
the tryptophan fluorescence of SecA in a saturable manner. This may
imply that the Mg
ion is bound in close proximity to
one of the two Trp residues. Alternatively, Mg
binding results in a conformational change sensed by these
residues. An intriguing point, however, is that the two
carboxyl-terminal Trp residues of SecA and the site-directed mutations
are far apart in the primary sequence. One possibility is that the
carboxyl-terminal portion of the molecule folds back onto the
amino-terminal domain. Intimate contact between both regions would
explain why the mutations in the Mg
-binding domain
result in a long range conformational changes in the SecA protein.
Alternatively, binding of Mg
effects a long range
conformational change in the SecA molecule.
In conclusion, the
mutagenesis data identifies two conserved aspartate residues in SecA
involved in coordinating the Mg ion and catalytic ATP
hydrolytic function. These residues perform an important role in the
function of SecA as an ATP-driven force generator and are needed to
couple ATP binding to a conformational change in the SecA dimer that
drives translocation.