©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Magnesium-binding Domain of the High-affinity ATP-binding Site of the Bacillus subtilis and Escherichia coli SecA Protein (*)

(Received for publication, March 31, 1995; and in revised form, June 6, 1995)

Jeroen P. W. van der Wolk Michael Klose (1) Janny G. de Wit Tanneke den Blaauwen Roland Freudl (1) Arnold J. M. Driessen (§)

From theDepartment of Microbiology and Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 approx 150 nm) (Matsuyama et al., 1990; Klose et al., 1993; Van der Wolk et al., 1993; Mitchell and Oliver, 1993) and a low (K approx 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.




EXPERIMENTAL PROCEDURES

Bacterial Strains and Growth Media

B. subtilis NIG1121 (Takamatsu et al., 1992) and the E. coli strains JM109 (recA1, endA1, gyrA96, thi, hsdR17, relA1, supE44, , Delta(lac-proAB), [F`, traD36, proAB, lacIDeltaM15]) (Yanish-Perron et al., 1985), BW313 (dut, ung, thi rel, spoT [F`, lysA]) (Warner and Duncan, 1978), MC4100 (F, araD139, Delta(argF-lac)U169, rpsL150, relA1, flbB5301, deoC1, ptsF25) (Casadaban, 1976), MM52 (MC4100, secA51) (Oliver and Beckwith, 1981), BA13 (MC4100 secA13, supF) (Cabelli et al., 1988), and MM66 (MC4100 geneX, supF) (Oliver and Beckwith, 1982) were grown in L-broth or on L-agar (Miller, 1972) supplemented with 50 µg ml of ampicillin, 0.5% (w/v) glucose, 0.4% (w/v) maltose, 1 mM isopropyl-1-thio-beta-D-galactopyranoside (IPTG), (^1)or 1 mMortho-nitrophenylgalactofucoside as required.

Materials

E. coli SecA (Cunningham et al., 1989), SecB (Weiss et al., 1988), and proOmpA (Crooke et al., 1988) proteins were purified from overproducing strains as described. S-proOmpA was synthesized from plasmid pRD87, carrying the E. coli OmpA gene (Freudl et al., 1985), using an in vitro transcription/translation reaction (De Vrije et al., 1987). Radiolabeled precursor protein was affinity-purified as described (Crooke and Wickner, 1987). Inverted inner membrane vesicles were prepared from E. coli strain D10 (unc, rna10, relA1, spoT1, metB1) by the procedure of Chang et al.(1978) and treated with 6 M urea (Cunningham et al., 1989). Endoproteinase Asp-N was obtained from Boehringer. Staphylococcal V8 protease was from Sigma. [alpha-P]ATP (3000 Ci mmol) and [S]methionine (>1000 Ci mmol) were obtained from Amersham, U.K.

DNA Techniques and Plasmid Constructions

Isolation of chromosomal DNA, preparation of plasmid DNA, and other DNA techniques followed standard procedures (Sambrook et al., 1989). The B. subtilis wild-type secA gene was amplified by PCR using oligonucleotides OMKL23 (5`-GGCTCTAGAGATGATAGAAGGAGCG) and OMKL24 (5`-TGTGGTACCCATTTCATTCACCTC) as primers and chromosomal DNA of B. subtilis NIG1121 as template. The resulting DNA fragment was digested with XbaI and PstI and cloned into the expression vector pTRC99A (Amann et al., 1988) which has been digested with the same restriction enzymes. The corresponding plasmid pMKL400 contained the B. subtilis wild-type secA gene under the control of the lac regulatory elements and its nucleotide sequence (Sadaie et al., 1991) was confirmed by DNA sequencing. Plasmid pMKL180, containing the E. coli wild-type secA gene, was described previously (Klose et al., 1993).

Mutagenesis of secA Genes

Oligonucleotide-directed mutagenesis of the B. subtilis secA gene was performed essentially according to the method of Kunkel(1985) using the Mutagene phagemid in vitro mutagenesis kit (Bio-Rad Laboratories). A 2.6-kilobase BamHI/SphI fragment from pMKL400, containing the B. subtilis secA gene, was cloned into the BamHI/SphI-digested phagemid pTZ18U (Mead et al., 1986). The resulting plasmid pTZ181 was transformed into E. coli BW313 and uracil-containing single-stranded phagemid DNA was isolated. The oligonucleotides OMKL40 (5`-TGTTCTTGCTTCATTAATTAAAATAG) or OMKL41 (5`-GAGTCAACTTCATTTATTACCGCAAA) were used as mismatch primers for the replacement of Asp-215 or Asp-207 by Asn in the B. subtilis SecA. Using OMKL40 or OMKL41 as primers, the complementary DNA strand was synthesized by T4 polymerase and the resulting double-stranded phagemid DNAs were transformed into JM109. The presence of the desired mutations was screened by DNA sequencing and the resulting plasmids were named pTZ182 (containing the secA D215N allele) and pTZ183 (containing the secA D207N allele). From these plasmids, a 2.6-kilobase XbaI/PstI fragment was subcloned into the XbaI/PstI-digested expression vector pTRC99A, resulting in plasmids pMKL440 (secA D215N) and pMKL441 (secA D207N). Replacement of Asp-217 in the E. coli SecA by Asn was done by two-step PCR as described by Landt et al.(1990). A 0.63-kilobase DNA fragment corresponding to the 5`-end of the E. coli secA gene was amplified by PCR using plasmid pMF8 (Schmidt and Oliver, 1989) as template and oligonucleotides OMKL25.1 (5`-ATGAATCTAGAGGCGTTTGAGATTTTAT) as 5`-primer and OMKL70 (5`-GAGTCCACTTCGTTCACCAGCGCA) as mismatch primer. The resulting DNA fragment was isolated and, together with an equimolar amount of OMKL71 (5`-AACGATGTAGTCGACGTCACGGGT), was used in a second PCR as primer, in which pMF8 again was used as template. The DNA fragment, which resulted from this second PCR, was digested with XbaI and SalI and ligated into pMKL180, from which the corresponding XbaI/SalI fragment had been removed. The presence of the desired mutation (E. coli secA D217N) was screened by DNA sequencing and the corresponding plasmid was designated pMKL183.

In Vivo Interference

To test for steady-state accumulation of MalE and OmpA precursor proteins, the corresponding cells were grown in the presence of glucose to A = 0.6, washed once with L-broth and resuspended in the original volume of L-broth containing 0.4% maltose and 1 mM IPTG. After 1-2 h incubation at 37 °C, 1-ml samples were precipitated with 200 µl of 20% trichloroacetic acid and kept on ice for 2 h. The samples were pelleted by centrifugation and washed once with 30 mM Tris-HCl, pH 8.0, and acetone. Subsequently, samples were subjected to SDS-PAGE (Laemmli, 1970) and Western blotting using MBP, OmpA, or SecA antibodies as described (Overhoff et al., 1991).

Expression and Purification of SecA

E. coli strain JM109 harboring the plasmids pMKL400, pMKL21, pMKL441, or pMKL440, was used for the isolation of the B. subtilis wild-type, K106N, D207N, and D215N SecA protein, respectively. Plasmid-bearing cells were grown overnight in L-broth containing glucose and ampicillin at 37 °C. Cells were diluted 50-fold in fresh L-broth containing ampicillin, and induced for protein expression by the addition of 200 µM IPTG. Cells were harvested by centrifugation after 4 h, and resuspended in an equal weight of 10% (w/v) sucrose, 50 mM Tris-HCl, pH 7.5. The suspension was frozen as nuggets by rapid pipetting in liquid nitrogen and stored at -80 °C.

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.

ATPase Assay

ATPase activity of SecA was assayed as described before (Van der Wolk et al., 1993). Released orthophosphate was determined by a colorimetric assay of Lanzetta et al.(1979) as described by Lill et al.(1990).

ATP Binding

Photoaffinity [alpha-P]ATP cross-linking experiments were conducted as described by Matsuyama et al.(1990). SecA (30 pmol) was incubated in Buffer B (50 mM Tris-OAc, pH 7.5, 100 mM KOAc, 2 mM MgOAc, 1 m DTT) with 25 or 100 nM [alpha-P]ATP for 15 min at 0 °C, and then subjected to photoaffinity cross-linking for 40 min at 0 °C using a 254-nm lamp (Model UVG-54, UVP Live Sciences Inc., Cambridge, United Kingdom) at a distance of 2 cm. Cross-linking reactions were carried out with or without UV radiation in the presence or absence of ATP at the indicated concentrations. Samples were analyzed by SDS-PAGE on 10% acrylamide gels. The gels were dried and exposed to Kodak X-Omat AR Film. Autoradiograms were densitometrically scanned using a Dextra DF-2400T scanner and analyzed using SigmaScan/Image (Jandel Corp., San Rafael, CA).

Proteolytic Digestion

SecA conformation was probed by the sensitivity to Staphylococcus aureus V8 protease according to Shinkai et al.(1991). Reaction mixtures (50 µl) contained: 60 µg of SecA, 50 mM Tris-HCl (pH 8.0), 50 mM KCl, 5 mM MgCl(2), and 1 mM DTT. ATPS (1 µM or 2 mM) was added when indicated. After 10 min preincubation at 37 °C, 100-450 ng of V8 protease was added, and the incubation was continued for 10-30 min. Reactions were terminated by adding 5 µl of blocking buffer (10 mM phenylmethylsulfonyl fluoride, 6% (w/v) SDS, 300 mM Tris-HCl, pH 6.8, 30% (v/v) glycerol, 6% (w/v) DTT, and 0.03% (w/v) bromphenol blue) to 10 µl of the reaction and heating for 5 min at 95 °C. Samples were analyzed by SDS-PAGE on 10% acrylamide gels.

Binding of MgIons

Binding of Mg ions to SecA was measured with the fluorescent indicator Magnesium Green® (Molecular Probes Inc., Eugene, OR) and by quenching of the intrinsic tryptophan fluorescence. Magnesium Green® measures the free concentration of Mg. Solutions (150 µl) contained: 200 µM MgOAc, 50 mM Tris-OAc, pH 7.5, 100 mM KOAc, 1 mM DTT, and 30 µM Magnesium Green®. SecA (300 nM) was added to the solution, and the decrease in fluorescence was followed at 532 nm, using an excitation wavelength of 476 nm and a bandwidth of 4 nm. Binding experiments were carried out in the absence of ATP, as the high Mg ion binding capacity of ATP precluded the detection of specific binding of Mg ions to 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).

Translocation Reactions

Assays of translocation of S-proOmpA into inverted inner membrane vesicles of E. coli strain D10 were performed as described (Cunningham et al., 1989). Reactions mixtures (50 µl) contained Buffer C (50 mM Hepes-KOH, pH 7.5, 50 mM KCl, 5 mM MgCl(2), 0.5 mg ml bovine serum albumin, 2 mM DTT), 5 µg mlE. coli SecA, 10 µg ml SecB, 2 mM ATP, 10 mM phosphocreatine, 50 µg ml creatine kinase, and inverted inner membrane vesicles (44 µg of protein ml). When indicated, reaction mixtures were supplemented with 50 µg mlB. subtilis wild-type or mutant SecA. Translocation was initiated by the addition of 1 µl of S-proOmpA in 6 M urea, 50 mM Tris-HCl, pH 7.6. After 30 min incubation at 37 °C, samples were treated with proteinase K (0.2 mg ml, 30 min, 0 °C), and precipitated with trichloroacetic acid. Reactions were analyzed by SDS-PAGE and autoradiography.

Other Analytical Techniques

Protein concentrations were determined as described (Bradford, 1976). Amino-terminal sequencing of proteolytic fragments of SecA was performed by Eurosequence, Groningen, The Netherlands.


RESULTS

Construction of Mutant secA Genes and in Vivo Analysis

The high affinity nucleotide binding site of SecA is located in the amino-terminal region (Matsuyama et al., 1990; Klose et al., 1993; Van der Wolk et al., 1993; Mitchell and Oliver, 1993). Residues 203-207 in the B. subtilis SecA (and residues 205-209 in the E. coli SecA) (Fig.1A) show sequence similarity to the Walker B-motif, hXhhD (Walker et al., 1982) of a typical nucleotide binding fold. The motif seems to occur as a repeat (Fig.1B), with Asp-207 and Asp-215 (Asp-209 and Asp-217 in the E. coli SecA) as possible candidates for the conserved aspartate that coordinates the Mg ion of the bound Mg-ATP complex. To reveal their function in catalysis and Mg-binding, Asp-207 and Asp-215 of the B. subtilis SecA were individually mutated to the cognate amide residue, asparagine (pMKL441, D207N; and pMKL440, D215N; respectively). A complementary mutation was introduced in the E. coli SecA, i.e. D217N (corresponding to Asp-215 in the B. subtilis SecA; pMKL183). A previous study on E. coli SecA (Mitchell and Oliver, 1993) has shown that the D209N (i.e. Asp-207 in the B. subtilis SecA) mutation blocks the translocation ATPase activity. Mutated secA genes were placed under the control of the lac regulatory elements in the expression vector pTRC99A, and plasmids were transformed into E. coli JM109. Expression was monitored under repressed (0.5% glucose) or fully induced (1 mM IPTG) conditions. The B. subtilis or E. coli SecA mutant proteins were expressed to levels found for the respective wild-type proteins (data not shown).

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; bullet, 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).



B. subtilis D207N and D215N SecA Interfere with in Vitro Protein Translocation

In vivo data demonstrate that the E. coli D217N SecA interferes with protein export when expressed in a host containing wild-type SecA (Fig.2). For in vitro analysis, purification of E. coli SecA mutant proteins is complicated by the difficulty to separate them from the wild-type (Mitchell and Oliver, 1993). In contrast, B. subtilis wild-type and mutant SecA proteins can be conveniently separated from E. coli SecA (see ``Experimental Procedures''; Van der Wolk et al.(1993)). Therefore, the E. coli D217N SecA was not further characterized for in vitro activities. Wild-type and mutant B. subtilis SecA proteins were purified to homogeneity from E. coli strains expressing these proteins to a high level, and tested for their ability to interfere with E. coli SecA dependent-translocation of S-proOmpA into everted inner membrane vesicles (Fig.4, lane 1). As shown previously (Van der Wolk et al., 1993), the presence of an excess wild-type B. subtilis SecA (Fig.4, lane 2) does not interfere with proOmpA translocation. As a control, the K106N SecA was used (lane 3). In this protein the invariable lysine residue of the phosphate-binding loop of the high-affinity ATP-binding site is mutated (Klose et al., 1993), and it has been shown to interfere with translocation by occupying the high-affinity SecA membrane binding sites (Van der Wolk et al., 1993). As in the case of K106N, both D207N and D215N SecA proteins retained the capacity to bind to the high-affinity membrane binding sites as assessed by co-sedimentation analysis (data not shown). D207N SecA (lane 4), and even more pronounced the D215N SecA, showed a stronger inhibitory effect (lane 5) as the K106N SecA (lane 3). Only weak interference was observed when E. coli inner membrane vesicles were used that were not stripped with urea to remove endogenous SecA from the translocation sites (data not shown).


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.



D207N and D215N SecA Are Impaired in ATP Binding and Hydrolysis

The ATPase activity of SecA is obligatory for in vitro protein translocation (Lill et al., 1990). Since both mutants are incapable of complementing the E. coli secA strain, we investigated their ATP binding and hydrolysis characteristics. Wild-type SecA harbors a low endogenous ATPase activity that is stimulated by liposomes containing acidic phospholipids (Lill et al., 1990; Van der Wolk et al., 1993). As shown previously, K106N SecA retained lipid-stimulated ATPase activity, albeit at a lower level as compared to the wild-type. Both the D207N and D215N SecA proteins showed a dramatically reduced endogenous ATPase activity (Fig.5), which was not stimulated by E. coli phospholipid. These results demonstrate that the mutations introduced at Asp-207 and Asp-215 interfere with the SecA ATPase activity.


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 bullet; D207N black square; D215N .



The high-affinity nucleotide binding site at the amino terminus of the E. coli SecA can be photoaffinity cross-linked with [alpha-P]ATP (Matsuyama et al., 1990). At 50-200 nM [alpha-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 [alpha-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: [alpha-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, [alpha-P]ATP photoaffinity cross-linking of wild-type (bullet) and D207N () SecA protein in the presence of increasing amounts of ATP. The [alpha-P]ATP concentration was 25 nM. Autoradiograms were densitometrically scanned, and the relative amounts of [alpha-P]ATP cross-linking of the SecA proteins were plotted as a function of the total ATP concentration.



Asp-207 and Asp-215 Are Needed for High-affinity MgBinding

To test if Asp-207 and Asp-215 are involved in Mg binding, experiments were performed with the fluorescent probe Magnesium Green® that measures the free concentration of Mg with a Kof 0.9 mM. Addition of wild-type or K106N SecA to a solution containing 200 nM Mg resulted in a substantial decrease in Magnesium Green® fluorescence (Fig.7, inset), indicative for a lowering of the free Mg concentration. D207N and D215N SecA had little effect on the fluorescence (Fig.7, inset), suggesting that they are impaired in high-affinity Mg binding.


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 (black square) 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 (black square), 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.

Conformational State of Mutant SecA Proteins

Nucleotides protect E. coli SecA against proteolytic cleavage by Staphylococcal V8 protease (Shinkai et al., 1991) that cleaves peptide bonds at the carboxyl terminus of glutamic acid residues. In analogy, B. subtilis SecA was readily digested by V8 protease in the absence of nucleotides (Fig.8). By following the digestion pattern in time, two major fragments could be identified with molecular masses of 49 and 38 kDa, respectively. The latter fragment was unstable and further digested when the incubation time was prolonged. Both fragments were separated on SDS-PAGE, blotted onto polyvinylidene difluoride membranes, and amino-terminal sequenced. The 49-kDa fragment showed the sequence ``LGILN. . . . ..'' corresponding to the amino-terminal domain of SecA, while the 38-kDa fragment had the amino-terminal sequence ``LISKL. . . . ..''. The latter sequence corresponds to a region adjacent to Glu-443 of the B. subtilis SecA. SecA was not digested by endoproteinase Asp-N, that cleaves at the peptide bond preceding an aspartate residue (data not shown). Incubation of SecA with ATPS (Fig.8), ATP, or ADP in the presence of Mg conferred protection to V8 protease digestion, while Mg alone was not sufficient (Fig.8, controls). Protection required a high ATPS concentration, i.e. in the millimolar range, indicating that both the high- and low-affinity ATP-binding sites need to be occupied. As compared to wild-type SecA, ATPS protected the K106N SecA to a lesser extent against V8 protease digestion. The D207N or D215N mutant SecA proteins, however, showed an intrinsic resistance to V8 protease digestion, resulting in the accumulation of a 88-kDa fragment. D215N SecA appeared to be proteolyzed at the amino terminus, as both the intact and the truncated forms were readily labeled with biotin-maleimide (data not shown) that reacts with the carboxyl-terminal cysteines (Driessen, 1993). The digestion pattern of the D215N SecA stabilized in about equal quantities of native and proteolyzed SecA. The D207N SecA behaved in a similar fashion, although it appeared to be less stable than the D215N SecA at prolonged incubation times. Addition of ATP had little effect on the digestion pattern. These data demonstrate that the D207N and D215N SecA proteins are intrinsically resistant to V8 protease, unlike the wild-type SecA protein that acquires this resistance only when both the high- and low-affinity ATP-binding sites are occupied by nucleotide.


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, ATPS. 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.




DISCUSSION

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 beta-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.


FOOTNOTES

*
This work was supported by a PIONIER grant of the Netherlands Organization for Scientific Research (N.W.O.), the Netherlands Foundation for Chemical Research (S.O.N.), the Bundesministerium für Forschung und Technologie, and the E.C. as part of the BIOTECH program BIO2-CT-930254. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 31-50-632164; Fax: 31-50-632154; (Internet):A.J.M.DRIESSEN{at}BIOL.RUG.NL.

^1
The abbreviations used are: IPTG, isopropyl-1-thio-beta-D-galactopyranoside; ATPS, adenosine 5`-O-(3-thiotriphosphate); DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; MBP, maltose-binding protein; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank the members of the BIOTECH program BIO2-CT-930254 for valuable discussions, E. Kuiper and S. Crombach for expert technical assistance, D. B. Oliver for providing the SecA mutant strains, and Y. Sadaie for the B. subtilis NIG1121 strain.


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