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• W2001905916 abstract "SecYEG translocase mediates the transport of preproteins across the inner membrane of Escherichia coli. SecA binds the membrane-embedded SecYEG protein-conducting channel with high affinity and then drives the stepwise translocation of preproteins across the membrane through multiple cycles of ATP binding and hydrolysis. We have investigated the kinetics of nucleotide binding to SecA while associated with the SecYEG complex. Lipid-bound SecA was separated from Se-cYEG-bound SecA by sedimentation of the proteoliposomes through a glycerol cushion, which maintains the SecA native state and effectively removes the lipid-bound SecA fraction. Nucleotide binding was assessed by means of fluorescence resonance energy transfer using fluorescent ATP analogues as acceptors of the intrinsic SecA tryptophan fluorescence in the presence of a tryptophanless variant of the SecYEG complex. Binding of SecA to the SecYEG complex elevated the rate of nucleotide exchange at SecA independently of the presence of preprotein. This defines a novel pretranslocation activated state of SecA that is primed for ATP hydrolysis upon preprotein interaction. SecYEG translocase mediates the transport of preproteins across the inner membrane of Escherichia coli. SecA binds the membrane-embedded SecYEG protein-conducting channel with high affinity and then drives the stepwise translocation of preproteins across the membrane through multiple cycles of ATP binding and hydrolysis. We have investigated the kinetics of nucleotide binding to SecA while associated with the SecYEG complex. Lipid-bound SecA was separated from Se-cYEG-bound SecA by sedimentation of the proteoliposomes through a glycerol cushion, which maintains the SecA native state and effectively removes the lipid-bound SecA fraction. Nucleotide binding was assessed by means of fluorescence resonance energy transfer using fluorescent ATP analogues as acceptors of the intrinsic SecA tryptophan fluorescence in the presence of a tryptophanless variant of the SecYEG complex. Binding of SecA to the SecYEG complex elevated the rate of nucleotide exchange at SecA independently of the presence of preprotein. This defines a novel pretranslocation activated state of SecA that is primed for ATP hydrolysis upon preprotein interaction. The SecA protein functions as a molecular motor to drive precursor proteins (preproteins) across the bacterial cytoplasmic membrane (1Hartl F.U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Google Scholar, 2Ulbrandt N.D. London E. Oliver D.B. J. Biol. Chem. 1992; 267: 15184-15192Google Scholar, 3Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Google Scholar). It is a dimeric peripheral ATPase subunit of the preprotein translocase and binds with high affinity to the translocation channel formed by an oligomeric assembly of the SecYEG complex. The translocation channel allows membrane passage of preproteins in their unfolded state (4Manting E.H. Driessen A.J.M. Mol. Microbiol. 2000; 37: 226-238Google Scholar, 5Driessen A.J.M. Manting E.H. van der Does C. Nat. Struct. Biol. 2001; 8: 492-498Google Scholar). SecA couples the energy of ATP binding and hydrolysis to the preprotein translocation. ATP hydrolysis also drives the cycling of SecA between the membrane-bound and free cytosolic state (6Economou A. Wickner W. Cell. 1994; 78: 835-843Google Scholar, 7van der Does C. den Blaauwen T. de Wit J.G. Manting E.H. Groot N.A. Fekkes P. Driessen A.J.M. Mol. Microbiol. 1996; 22: 619-629Google Scholar). This cycling reaction occurs on the same time scale as the overall preprotein translocation reaction (6Economou A. Wickner W. Cell. 1994; 78: 835-843Google Scholar), but it is unclear if cycling is a necessary step in catalysis. Evidence for the high affinity binding of SecA to the SecYEG complex is demonstrated by an increased number of SecA membrane binding sites upon overproduction of the SecYEG complex (7van der Does C. den Blaauwen T. de Wit J.G. Manting E.H. Groot N.A. Fekkes P. Driessen A.J.M. Mol. Microbiol. 1996; 22: 619-629Google Scholar), the reduction of specific binding of SecA to SecYEG by an antibody directed against SecY (1Hartl F.U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Google Scholar), and the presence of a significant SecYEG-dependent fraction of SecA that cannot be membrane-extracted by urea, alkali, or high salt (1Hartl F.U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Google Scholar, 8Cabelli R.J. Dolan K.M. Qian L.P. Oliver D.B. J. Biol. Chem. 1991; 266: 24420-24427Google Scholar). SecA can also bind to the lipid surface, where it associates with acidic phospholipids (1Hartl F.U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Google Scholar, 3Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Google Scholar) and seems to insert into the fatty acid acyl phase of the membrane (1Hartl F.U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Google Scholar, 2Ulbrandt N.D. London E. Oliver D.B. J. Biol. Chem. 1992; 267: 15184-15192Google Scholar). This process of lipid association occurs with low affinity and is inhibited by ATP (9Breukink E. Demel R.A. de Korte-Kool G. de Kruijff B. Biochemistry. 1992; 31: 1119-1124Google Scholar). The translocation of preproteins across the membrane involves several rounds of nucleotide binding and hydrolysis by SecA (1Hartl F.U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Google Scholar, 10Schiebel E. Driessen A.J.M. Hartl F.U. Wickner W. Cell. 1991; 64: 927-939Google Scholar, 11Economou A. Pogliano J.A. Beckwith J. Oliver D.B. Wickner W. Cell. 1995; 83: 1171-1181Google Scholar). On the basis of mutational and functional studies and the use of photoactivatable nucleotide analogues (11Economou A. Pogliano J.A. Beckwith J. Oliver D.B. Wickner W. Cell. 1995; 83: 1171-1181Google Scholar, 12Lill R. Cunningham K. Brundage L.A. Ito K. Oliver D. Wickner W. EMBO J. 1989; 8: 961-966Google Scholar, 13Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Google Scholar, 14den Blaauwen T. van der Wolk J.P. van der Does C. van Wely K.H. Driessen A.J.M. FEBS Lett. 1999; 458: 145-150Google Scholar), it was suggested that SecA contains a high and a low affinity nucleotide binding domain (NBD). 1The abbreviations used are: NBD, nucleotide binding domain; FRET, fluorescence resonance energy transfer; MANT, 2′-(or-3′)-O-(N-methylanthraniloyl); PMF, proton motive force; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOPG, 1,2-dioleoyl-sn-glycero-3-phosphoglycerol; IMV, inner membrane vesicle. The high affinity NBD corresponds to the catalytic site, whereas the low affinity NBD may act as a regulatory site. The structures of the Bacillus subtilis (15Hunt J.F. Weinkauf S. Henry L. Fak J.J. McNicholas P. Oliver D.B. Deisenhofer J. Science. 2002; 297: 2018-2026Google Scholar) and Mycobacterium tuberculosis SecA (16Sharma V. Arockiasamy A. Ronning D.R. Savva C.G. Holzenburg A. Braunstein M. Jacobs Jr., W.R. Sacchettini J.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2243-2248Google Scholar), however, show no evidence of a second catalytic NBD (17van der Wolk J.P. de Wit J.G. Driessen A.J.M. EMBO J. 1997; 16: 7297-7304Google Scholar). The low affinity NBD may correspond to a nonfunctional nucleotide binding domain (11Economou A. Pogliano J.A. Beckwith J. Oliver D.B. Wickner W. Cell. 1995; 83: 1171-1181Google Scholar, 18Sianidis G. Karamanou S. Vrontou E. Boulias K. Repanas K. Kyrpides N. Politou A.S. Economou A. EMBO J. 2001; 20: 961-970Google Scholar). The nucleotide binding characteristics of the soluble SecA protein have been studied extensively (13Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Google Scholar, 14den Blaauwen T. van der Wolk J.P. van der Does C. van Wely K.H. Driessen A.J.M. FEBS Lett. 1999; 458: 145-150Google Scholar, 19Kourtz L. Oliver D. Mol. Microbiol. 2000; 37: 1342-1356Google Scholar) and used as a model to describe the conformational states of SecA when actively involved in preprotein translocation. The translocation active state of SecA may, however, not be reflected by those studies, since this requires binding of SecA to the SecYEG complex (3Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Google Scholar, 12Lill R. Cunningham K. Brundage L.A. Ito K. Oliver D. Wickner W. EMBO J. 1989; 8: 961-966Google Scholar). On the other hand, nucleotide binding experiments with the SecYEG-bound SecA using natural membranes or membranes containing overexpressed SecYEG complex are complicated by the presence of many unrelated ATPases. Whereas purification and reconstitution provide a suitable functional alternative, high affinity binding of SecA to the reconstituted SecYEG complex has never been demonstrated for the purified proteins. This has been attributed to the very high nonspecific binding of SecA to the lipid surface (20Hendrick J.P. Wickner W. J. Biol. Chem. 1991; 266: 24596-24600Google Scholar). We have investigated the binding of nucleotides to SecA when bound to the SecYEG complex. For that purpose, we have devised a method that efficiently separates the high and low affinity-bound SecA fractions to SecYEG proteoliposomes. This method was combined with fluorescence resonance energy transfer (FRET) experiments that monitor the interaction between fluorescent ATP analogues as acceptors of the intrinsic SecA tryptophan fluorescence in the presence of a tryptophanless variant of the SecYEG complex. The data demonstrate that binding of SecA to the SecYEG complex stimulates nucleotide exchange at SecA. This defines a novel intermediate step in the activation of SecA for preprotein translocation. Materials—SecA, SecB, pro-OmpA, and SecYEG and the tryptophanless SecYEG mutant were purified as described (4Manting E.H. Driessen A.J.M. Mol. Microbiol. 2000; 37: 226-238Google Scholar). SecYEG was reconstituted into liposomes composed of Escherichia coli phospholipids or synthetic liposomes as described (21van der Does C. Manting E.H. Kaufmann A. Lutz M. Driessen A.J.M. Biochemistry. 1998; 37: 201-210Google Scholar). Synthetic phospholipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) (Avanti Polar Lipids, Inc., Birmingham, AL) were mixed in a 60:40 (w/w) ratio, and liposomes were prepared as described (22van der Does C. Swaving J. van Klompenburg W. Driessen A.J.M. J. Biol. Chem. 2000; 275: 2472-2478Google Scholar). 2′-(or-3′)-O-(N-methylanthraniloyl) (MANT)-labeled nucleotides were obtained from Molecular Probes, Inc. (Leiden, The Netherlands). Binding of Nucleotides to SecA-SecYEG Proteoliposomes—Liposomes or SecYEG proteoliposomes (0.05 mg of SecYEG protein/ml) were incubated with 50 nm [α-32P]ATP (Amersham Biosciences) in buffer A (50 mm HEPES-KOH, pH 7.6, 100 mm KCl, 5 mm MgAc2, 10 mm dithiothreitol, 1 mg/ml bovine serum albumin) with or without 250 nm SecA dimer. After a 20-min incubation at room temperature, proteoliposomes were spun (Beckman Airfuge, 30 p.s.i., 5 min) through a 15% (w/v) glycerol cushion in buffer A. Bound nucleotides were extracted from the pellet fraction and visualized as described (23Scheffers D.J. den Blaauwen T. Driessen A.J.M. Mol. Microbiol. 2000; 35: 1211-1219Google Scholar). For a kinetic analysis of nucleotide binding to SecA, proteoliposomes (0.67 μm SecYEG) were first preincubated (15 min, room temperature) in buffer A with SecA (400 nm dimer) and spun through a 15% (w/v) glycerol cushion in buffer A (Airfuge, 30 p.s.i., 5 min). The SecA-SecYEG proteoliposomes were recovered from the pellet fraction, resuspended in buffer A (0.13 μm SecYEG), and incubated with [32P]ADP (0–2 μm) for 15 min at room temperature. When indicated, SecB and pro-OmpA were added (0.5 μm). To separate bound from free nucleotide, the proteoliposomes were once again spun through a 15% (w/v) glycerol cushion, and the pellet and supernatant fractions were analyzed by scintillation counting. The binding data were analyzed by curve fitting (SigmaPlot 2000; SPSS Inc.) using nonlinear regression. Binding of SecA to SecYEG Proteoliposomes—[14C]SecA was prepared by reductive methylation (24van der Wolk J.P. Klose M. Breukink E. Demel R.A. de Kruijff B. Freudl R. Driessen A.J.M. Mol. Microbiol. 1993; 8: 31-42Google Scholar) and found to retain full in vitro translocation and translocation ATPase activity. Binding of [14C]SecA to SecYEG proteoliposomes was performed essentially as described for nucleotide binding. SecYEG proteoliposomes were incubated with 2.5–650 nm [14C]SecA in buffer A with or without 1 mm ADP. After a 20-min incubation at room temperature, proteoliposomes were spun (Beckman Airfuge, 30 p.s.i., 5 min) through a 15% (w/v) glycerol cushion in buffer A. Bound and free SecA were quantitated by liquid scintillation counting, and the data were analyzed by Sigma Plot 2000 (SPSS Inc.) using nonlinear regression assuming biphasic binding kinetics. Construction of Tryptophanless SecYEG Complex—To construct a tryptophanless SecYEG (Trp-less SecYEG) complex, the tryptophan residues of the individual proteins (SecY, 4 residues; SecE, 3 residues; SecG, 1 residue) were mutated to phenylalanine by site-directed mutagenesis of the respective genes using mismatch oligonucleotides as described in Table I. The PCR products were then exchanged with the corresponding fragments in pET610 (25Kaufmann A. Manting E.H. Veenendaal A.K. Driessen A.J.M. van der Does C. Biochemistry. 1999; 38: 9115-9125Google Scholar) to obtain Trp-less His-tagged secYEG under control of the isopropyl-β-d-thiogalactopyranoside-inducible trc promoter (7van der Does C. den Blaauwen T. de Wit J.G. Manting E.H. Groot N.A. Fekkes P. Driessen A.J.M. Mol. Microbiol. 1996; 22: 619-629Google Scholar, 21van der Does C. Manting E.H. Kaufmann A. Lutz M. Driessen A.J.M. Biochemistry. 1998; 37: 201-210Google Scholar). All constructs were verified by DNA sequence analysis.Table IMismatch primers used for site-directed mutagenesis of the secYEG genesGeneAmino acid substitutionMismatch oligonucleotidesaThe resulting amino acids (underlined) are shownsecYW173F5′-TTCCTGATGTTCTTGGGCGAACW300F/W302F5′-GCGGTACTGGTTTCAACTTCCTGACAACAATTTCGCTGW293F5′-GGCGACCATCGCGTCATTCTTCGGGGGCGGTACCGGTTTCsecEW19F5′-GCGATGAAGTTCGTCGTTGTGGTGW84F5′-GTAAGGTCATTTTCCCGACTCGCCAGGW109F5′-CACTGATCCTGTTCGGACTGGATGGsecGW84F5′-CACTCAGATTTTCAAATTCGCTACCa The resulting amino acids (underlined) are shown Open table in a new tab Steady-state Fluorescence Measurements—Tryptophan emission spectra were measured on a Aminco-Bowman Series 2 luminescence spectrometer (SLM-Aminco, Urbana, IL) with excitation and emission wavelengths of 295 and 350 nm and a slit width of 4 nm. Binding of Fluorescent Nucleotides by SecA—Binding of MANT nucleotides was followed by FRET measurements using a temperature-controlled SLM-Aminco 4800C spectrofluorometer (SLM-Aminco, Urbana, IL) with Glan-Thompson polarizers. The slit widths for the excitation and emission beam were set open, the excitation beam polarizer was set to 90°, and the emission polarizer was set to 0° to reduce light scatter. Tryptophan fluorescence was measured in time at excitation and emission wavelengths of 295 and 350 nm, respectively. FRET to MANT-ATP or MANT-ADP was measured in time at excitation and emission wavelengths of 295 and 450 nm, respectively. To measure binding of MANT-nucleotide to free SecA or Trp-less SecYEG proteoliposome-bound SecA, 0.147 μm MANT-nucleotide was added to a solution containing 0.26 μm SecA dimer in 50 mm Tris-HCl, pH 8.0, 50 mm KCl, and 5 mm MgCl2 (or 0.2 mg/ml 60% DOPG, 40% DOPG liposomes). Trp-less SecYEG proteoliposome-bound SecA was obtained by incubating the proteoliposomes (2.6 μm Trp-less SecYEG and 0.2 mg/ml of phospholipid) and 5.2 μm SecA dimer for 20 min at room temperature and spun (Beckman Airfuge, 30 p.s.i., 5 min) through a 15% (w/v) glycerol cushion in 50 mm Tris-HCl, pH 8.0, 50 mm KCl, and 5 mm MgCl2. The pellet was resuspended in 50 mm Tris-HCl, pH 8.0, 50 mm KCl, and 5 mm MgCl2, and the final proteoliposome-bound SecA concentration was equalized to 0.26 μm SecA dimer by comparative SDS-PAGE. Dissociation of the MANT-nucleotide from SecA was measured after 30 min of binding and initiated by the addition of nucleotides (0.6 mm). Data were corrected for background and fitted using nonlinear regression on single exponential curves (SigmaPlot 2000, SPSS Inc.). Kinetics of Fluorescent Nucleotide Binding of SecA—The kinetics of MANT-nucleotide binding and release was monitored over a wide range of nucleotide concentrations (0.01–2 μm). MANT-nucleotide was added to a solution containing either 0.165 μm SecA dimer or 0.07 μm SecA dimer bound to Trp-less SecYEG proteoliposomes in 50 mm Tris-HCl, pH 8.0, 50 mm KCl, and 5 mm MgCl2, and the observed association rate (kobs) was calculated from the progress curves. The dissociation rate (koff) was determined after the addition of 0.6 mm ATP or ADP. The kobs values obtained at various MANT-nucleotide concentrations were analyzed according to the following relationship (26Fersht A. Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding. W. H. Freeman & Co., New York1999: 143Google Scholar): kobs=koff+kon×[MANT-nucleotide](Eq. 1) Data were fitted by linear regression, whereby the line was forced through the y intercept, which equaled the experimentally determined koff of the reaction. The slope of the line was used to calculate the kon for MANT-nucleotide binding to SecA. Other Methods—In vitro translocation of pro-OmpA into Trp-less SecYEG proteoliposomes (27de Keyzer J. van der Does C. Driessen A.J.M. J. Biol. Chem. 2002; 277: 46059-46065Google Scholar) and pro-OmpA-stimulated translocation ATPase activity of SecA assays (12Lill R. Cunningham K. Brundage L.A. Ito K. Oliver D. Wickner W. EMBO J. 1989; 8: 961-966Google Scholar) were performed as described. SecA concentration was determined spectroscopically at 280 nm using a molar extinction coefficient of 83,000 m-1 cm-1 for monomeric SecA. Protein concentration was determined with the DC protein assay (Bio-Rad). Nucleotide Binding to SecA-SecYEG Proteoliposomes—Binding of nucleotides to SecA free in solution has been studied extensively (13Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Google Scholar, 14den Blaauwen T. van der Wolk J.P. van der Does C. van Wely K.H. Driessen A.J.M. FEBS Lett. 1999; 458: 145-150Google Scholar), but no information is available on nucleotides binding to SecA associated with SecYEG complex (28Schmidt M. Ding H. Ramamurthy V. Mukerji I. Oliver D. J. Biol. Chem. 2000; 275: 15440-15448Google Scholar). Inner membrane vesicles (IMVs) of E. coli bind SecA with high affinity at the SecYEG complex (1Hartl F.U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Google Scholar), but since this system contains many other ATPases that interfere with an accurate analysis of nucleotide binding, it is unsuitable for the analysis of nucleotide binding to SecYEG-bound SecA. Therefore, we have performed nucleotide binding experiments with purified SecA and reconstituted SecYEG proteoliposomes. Although the functional reconstitution of SecYEG into proteoliposomes is well established (29Brundage L. Hendrick J.P. Schiebel E. Driessen A.J.M. Wickner W. Cell. 1990; 62: 649-657Google Scholar, 30Hanada M. Nishiyama K.I. Mizushima S. Tokuda H. J. Biol. Chem. 1994; 269: 23625-23631Google Scholar), the high affinity SecA binding has never been demonstrated for this system. Such experiments are complicated by the high nonspecific binding of SecA to the lipid surface, which masks the specifically bound fraction (20Hendrick J.P. Wickner W. J. Biol. Chem. 1991; 266: 24596-24600Google Scholar). We have therefore devised a method that allows the detection of nucleotide binding to the high affinity SecYEG-bound SecA in proteoliposomes. For this purpose, liposomes and SecYEG proteoliposomes were incubated with [32P]ATP with and without SecA (Fig. 1). The specific binding of [32P]ATP to the (proteo)liposomes was determined after centrifugation through a glycerol instead of a sucrose cushion to separate the lipid-bound and free nucleotide fraction. In previous studies, sedimentation through a sucrose cushion was shown to be ineffective with SecYEG proteoliposomes due to the high nonspecific SecA association with the lipid surface (20Hendrick J.P. Wickner W. J. Biol. Chem. 1991; 266: 24596-24600Google Scholar). Glycerol has been reported to preserve the native state of SecA (31Weinkauf S. Hunt J.F. Scheuring J. Henry L. Fak J. Oliver D.B. Deisenhofer J. Acta Crystallogr. D Biol. Crystallogr. 2001; 57: 559-565Google Scholar), and therefore, sedimentation to a glycerol cushion more likely minimizes denaturation of SecA at the lipid surface. When [32P]ATP was incubated with liposomes (Fig. 1, lane 3) or SecYEG proteoliposomes (lane 5) in the absence of SecA, no radiolabel was recovered in the pellet fraction after glycerol cushion centrifugation. In contrast, a high level of nucleotide binding was observed in the pellet fraction when SecYEG proteoliposomes were incubated with SecA (lane 6), whereas only a small amount of radiolabel was recovered when liposomes were incubated with SecA (lane 4; less than 10% of the control SecA-SecYEG proteoliposomes). During the incubation, even at 4 °C, all bound [32P]ATP was hydrolyzed to ADP. These data suggest that the glycerol cushion effectively reduces the nonspecific binding of SecA to the lipid surface. To determine the kinetic parameters of nucleotide binding to the SecYEG-bound SecA, SecYEG proteoliposomes were prepared with specifically bound SecA by the method described above. Subsequently, the SecA-SecYEG proteoliposomes were incubated with [32P]ADP, and the bound and free nucleotides were separated by a second glycerol cushion centrifugation step. Fig. 2A shows a plot of the free ADP versus the ADP bound to the SecA-SecYEG proteoliposomes. The Kd for ADP binding to SecYEG-bound SecA was 63 ± 9 nm. The presence of the preprotein pro-OmpA (and SecB) had a minor effect on the ADP binding affinity (Kd 107 ± 14 nm, whereas the Bmax remained unchanged (56 ± 3 and 53 ± 3 nm, respectively). The number of ADP binding sites determined after the second glycerol cushion centrifugation was independent of the SecA concentration, provided that SecA was present in sufficient amounts to saturate all SecYEG binding sites (300 nm SecA dimer). The binding reaction was linearly dependent on the concentration of SecYEG in the range of 5–20 μg/ml. To determine the number of nucleotide binding sites per SecA dimer bound to SecYEG, SecA binding experiments were performed with the SecYEG proteoliposomes. Since this assay involves only a single glycerol centrifugation step, biphasic SecA binding kinetics were observed (Fig. 2B) that can be attributed to a small fraction of remaining nonspecifically bound SecA. SecA binds SecYEG with a Kd of 31 ± 5 nm. This value is similar to the one reported for IMVs (32de Keyzer J. van der Does C. Swaving J. Driessen A.J.M. FEBS Lett. 2002; 510: 17-21Google Scholar). The background SecA binding appeared nonsaturable in the range tested. The Bmax of 24 ± 2 nm for specific SecA dimer binding corresponds to about half (0.43 ± 0.07) the number of high affinity ADP binding sites that were determined with SecYEG proteoliposomes. This is equivalent to 1.1 ± 0.1 mol of ADP/mol of monomeric SecA. Therefore, we conclude that each SecYEG-bound SecA dimer contains two high affinity nucleotide binding sites. Binding of Fluorescent Nucleotides by SecA—To analyze the kinetics of nucleotide binding to SecA, we employed fluorescence (MANT)-labeled nucleotides (33Jameson D.M. Eccleston J.F. Methods Enzymol. 1997; 278: 363-390Google Scholar) in a nucleotide exchange assay. MANT-ATP was found to support the in vitro pro-OmpA translocation into SecYEG proteoliposomes (Fig. 3A), demonstrating that it functionally interacts with SecA and that it can drive protein translocation (27de Keyzer J. van der Does C. Driessen A.J.M. J. Biol. Chem. 2002; 277: 46059-46065Google Scholar), albeit with an almost 3-fold lower efficiency as compared with ATP. Nucleotide binding and release of SecA can be monitored by changes in MANT-nucleotide anisotropy (19Kourtz L. Oliver D. Mol. Microbiol. 2000; 37: 1342-1356Google Scholar). Although anisotropy is a fast and easy way to monitor the association and dissociation of MANT-nucleotide to SecA, the presence of liposomes or SecYEG proteoliposomes severely interfered with the measurements (data not shown). A fraction of the MANT-ATP or ADP appears to intercalate with the lipid bilayer, which results in a huge rise in anisotropy. To circumvent this problem, we shifted to FRET measurements (34Lakowicz J.R. Principles of Fluorescence Spectroscopy. Kluwer Academic/Plenum Publishers, New York1999: 368Google Scholar). Herein, the intrinsic protein fluorescence of SecA is used as a donor for the excitation of the bound MANT-nucleotide. The Förster distance (R0) of tryptophan residues varies up to 40 Å (35Wu P. Brand L. Anal. Biochem. 1994; 218: 1-13Google Scholar), which lies within the dimensions of SecA (36Ding H. Hunt J.F. Mukerji I. Oliver D. Biochemistry. 2003; 42: 8729-8738Google Scholar). This makes FRET measurements insensitive to nonspecific background binding of MANT-nucleotides to lipids. Upon excitation of the Trp residues of SecA at 295 nm in the presence of MANT-ATP, the Trp fluorescence at 350 nm is decreased (Fig. 3B) with a concomitant increase in MANT fluorescence at 450 nm (Fig. 3C). The FRET could be reversed by the addition of an excess of nonlabeled ATP that chases the MANT-ATP from the SecA bound state (Fig. 3C). This chase reaction was accompanied by an increase in the Trp fluorescence (Fig. 3B). This effect was not observed when SecA was absent from the reaction or when Mg2+ was excluded from the system. MANT-ATP binding is strictly dependent on the presence of Mg2+ with an apparent dissociation constant (Kd,Mg2+) of 40 μm (data not shown). Construction and Functional Characterization of a Tryptophanless SecYEG Complex—To use FRET for measurements of nucleotide binding to SecA in the presence of the SecYEG complex, a tryptophanless (Trp-less) variant was constructed. The SecYEG complex contains eight tryptophan residues: four in SecY (Trp173, Trp293, Trp300, and Trp302), three in SecE (Trp19, Trp84, and Trp109), and a single Trp in SecG (Trp84). All tryptophan residues were mutated to phenylalanine by site-directed mutagenesis (Table I), and the Trp-less variant of SecYEG was cloned in an expression vector under the control of the trc promoter with an N-terminal hexahistidine tag on SecY. To check the overexpression and functionality of the Trp-less SecYEG complex, IMVs were isolated from the SecYEG-over-producing strain. The Trp-less SecYEG complex could be overexpressed to the same level as the wild-type SecYEG complex (Fig. 4A). This result was verified for the individual subunits by immunoblot analysis (data not shown). Wild type and Trp-less SecYEG complex were purified by ion exchange and Ni2+-nitrilotriacetic acid affinity chromatography and reconstituted into liposomes composed of synthetic lipids (DOPE/DOPG, 60: 40, w/w) (Fig. 4A, lanes 3 and 4). The Trp-less and wild type SecYEG complexes were equally active in supporting pro-OmpA translocation (Fig. 4B), SecA translocation ATPase activity (Fig. 4C), and SecA binding (27de Keyzer J. van der Does C. Driessen A.J.M. J. Biol. Chem. 2002; 277: 46059-46065Google Scholar) (data not shown). Taken together, these data demonstrate that none of the Trp residues of the SecYEG complex are essential for activity. Fig. 4D shows the fluorescence emission spectra at an excitation at 295 nm of the purified and reconstituted wild type and Trp-less SecYEG complexes. As expected, the Trp-less SecYEG variant showed no significant fluorescence above that of liposomes, which makes the Trp-less SecYEG complex suitable for our FRET measurement. Binding of Fluorescent Nucleotides to the SecYEG-bound SecA—To measure the nucleotide binding characteristics of SecA bound to SecYEG, SecA was incubated with Trp-less SecYEG proteoliposomes, and the complex was recovered by sedimentation through a glycerol cushion (Fig. 4E). FRET measurements on nucleotide binding to SecYEG-bound SecA protein showed a very fast rate of MANT-ATP binding and release upon the addition of an excess of ATP (Fig. 5, dark line). No FRET was observed when the Trp-less SecYEG proteoliposomes were prepared in the absence of SecA (dark gray line). Moreover, the rate of MANT-nucleotide release was nearly identical when, instead of ATP (dark line), ADP was used for the chase (light gray line). Next, the kinetic parameters of MANT-ATP binding were determined for SecA associated with the Trp-less SecYEG complex and compared with soluble SecA and SecA bound to DOPE/DOPG liposomes. In the presence of DOPE/DOPG liposomes, SecA readily associates with the lipid surface, and the majority of the SecA co-sediments with the liposomes after ultracentrifugation (20Hendrick J.P. Wickner W. J. Biol. Chem. 1991; 266: 24596-24600Google Scholar) (data not shown). The kinetics of MANT-ATP binding and release with SecA in solution (Fig. 6, A and D) or in the presence of liposomes (Fig. 6, B and E) were indistinguishable within the experimental error. Remarkably, substantial higher rates of MANT-ATP binding and release were observed with SecA bound to SecYEG proteoliposomes (Fig. 6, C and F). The enhanced rate is in particular pronounced with the nucleotide release. Whereas free or lipid-bound SecA only slowly releases the nucleotide, a 6–7-fold increase in the nucleotide dissociation rate, koff, is observed with the SecYEG-bound" @default.
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