Biochemistry 2004, 43, 12009-12019
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Two-Site Autoinhibition of the ADP-Ribosylating Mosquitocidal Toxin (MTX) from Bacillus sphaericus by Its 70-kDa Ricin-like Binding Domain† Irina Carpusca, Jo¨rg Schirmer, and Klaus Aktories* Institut fu¨r Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-UniVersita¨t Freiburg, Albertstrasse 25, D-79104 Freiburg, Germany ReceiVed June 18, 2004; ReVised Manuscript ReceiVed July 26, 2004
ABSTRACT: The mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1 is an ∼97-kDa argininespecific ADP-ribosyltransferase that is activated by proteolytic cleavage, thereby releasing the active 27kDa enzyme (MTX30-264) and a 70-kDa C-terminal fragment (MTX265-870). In solution, the cleaved 70kDa fragment is still a potent inhibitor of the ADP-ribosyltransferase activity of MTX. Here we studied the interaction of the 70-kDa fragment with the enzyme domain of MTX. Several C-terminal deletions of the 70-kDa fragment inhibited the enzymatic activity of MTX30-264. However, the IC50 values were about 2 orders of magnitude higher for the deletions than for the 70-kDa fragment. A peptide covering amino acid residues 265-285 of the holotoxin exhibited the same inhibitory potency as the C-terminal deletions of the 70-kDa fragment. MTX265-285 contains several acidic residues, of which D273 and D275 were found to be essential for the inhibitory effect. Exchange of these residues in the 70-kDa fragment (MTX265-870) reduced its inhibitory potency. Kinetic analysis showed that the peptide MTX265-285 had no effect on the Vmax of MTX30-264 but increased the Km for NAD. By contrast, the 70-kDa fragment deleted of residues Ile265 through Asn285 inhibited the enzyme activity of MTX30-264 mainly by decreasing the Vmax of the enzyme. A second binding site for interaction of MTX265-870 with MTX30-264 was localized to the C-terminus within the region of residues 750-870. The data support a two-site binding model for inhibition of the ADP-ribosyltransferase activity of MTX30-264 by the 70-kDa fragment MTX265-870 with an interaction of amino acid residues 265-285 at the active site and an allosteric inhibition by the C-terminal part of the 70-kDa fragment.
Bacillus sphaericus is a mosquito pathogen and produces several mosquitocidal toxins, including the binary toxin (BTX or Bin toxin) consisting of 42- and 51-kDa proteins, which assemble in crystals (therefore also called crystal toxins) and become visible during sporulation (1, 2). In addition, they produce several mosquitocidal toxins called MTX,1 MTX2, and MTX3 (1, 3, 4). The mosquitocidal toxin MTX is an arginine-specific ADP-ribosylating toxin, which was first identified in the low-toxicity strain SSII-1 of B. sphaericus; however, it is also produced by numerous other B. sphaericus strains (1, 5-8). The toxin is lethal to Culex quinquefasciatus and Aedes aegypti mosquito larvae. Without the putative signal sequence (29 residues), the mature MTX is a 97-kDa protein (MTX30-870). MTX30-870 is cleaved into an enzymatically active 27-kDa N-terminal fragment and a 70-kDa C-terminal fragment (putative binding component) by crude mosquito larval gut extracts. Both unprocessed and proteolytically cleaved MTX30-870 are lethal for C. quinquefasciatus larvae; however, NH2-terminal or COOH-terminal † This work was financially supported by the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie. * Corresponding author. Tel: +49-761-2035301. Fax: +49-7612035311. E-mail:
[email protected]. 1 Abbreviations: MTX, mosquitocidal toxin from Bacillus sphaericus strain SSII-1; C2 toxin, Clostridium botulinum C2 toxin; C3 toxin, Clostridium botulinum exoenzyme C3; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; SBTI, soybean trypsin inhibitor.
truncations of the toxin alone have no lethal activity toward mosquito larvae (1, 6, 9). In vitro the 27-kDa enzyme fragment of MTX (MTX30-264) ADP-ribosylates numerous proteins in insect, mammalian, and even E. coli cell lysates (6, 10, 11). So far, the in vivo substrate of MTX is not known. Production of recombinant MTX30-264 in Escherichia coli is hampered by the fact that active MTX appears to be toxic toward the bacterial cell. This toxicity depends on the transferase activity of the enzyme, as catalytically inactive MTX30-264 constructs can be produced in E. coli. Recently, we identified a major in vitro substrate of MTX in E. coli lysates, which was identified as the E. coli elongation factor Tu (EF-Tu) (11). ADP-ribosylation of purified EF-Tu prevented formation of the stable ternary EF-Tu‚aa-tRNA‚GTP complex. The inactivation of EF-Tu by MTX-catalyzed ADP-ribosylation and the resulting inhibition of bacterial protein synthesis suggest that the active enzyme may be toxic toward toxin-producing bacteria and its production requires an effective and elaborate autoinhibitory regulation (11). Recently, we and others reported on the proteolytic activation of MTX (9, 10). Following proteolytic cleavage of the holotoxin MTX30-870 (without signal sequence), the 70-kDa putative MTX binding component, which has a ricinlike structure (12), remains noncovalently bound to the enzyme domain, thereby blocking the enzyme activity. Here, we report on the molecular mechanism underlying the
10.1021/bi048729y CCC: $27.50 © 2004 American Chemical Society Published on Web 08/28/2004
12010 Biochemistry, Vol. 43, No. 38, 2004 inhibition of the ADP-ribosyltransferase activity of MTX30-264 by the 70-kDa fragment (MTX265-870). EXPERIMENTAL PROCEDURES Materials. Chymotrypsin and soybean trypsin inhibitor were from Roche Diagnostics (Mannheim, Germany). [adenylate-32P]NAD (30 Ci/mmol) was purchased from NEN (Vilvoorde, Belgium). All other reagents were from Sigma (Deisenhofen, Germany) unless otherwise indicated. Cloning of mtx Truncations and Mutagenesis. For cloning, genes encoding MTX265-308, MTX265-456, MTX265-605, MTX265-750, and MTX265-840 were amplified from the plasmid pTH21 (encoding for MTX30-870) by polymerase chain reaction (PCR) with the following primers: mtx(265)sen (5′GGATCCATACTAGATTTAGATTATAATCAAG-3′) for all five constructs and mtx(308)antisen (5′-GTCGACCTTTTATTTTTGATTTGATATTCTG-3′) for MTX265-308; mtx(456)antisen (5′-GTCGACTAATTTTGAAGATACTTGATAAG-3′) for MTX265-456; mtx(605)antisen (5′-GTCGACCAAGTCAGCAATTAAATCCTG-3′) for MTX265-605; mtx(750)antisen (5′-GTCGACTTTATTTGAGTTCATAGTAATAATTTTA-3′) for MTX265-750 and mtx(840)antisen (5′-GTCGACAAAATTTCCTTGAAATACATCAAGTAC3′) for MTX265-840. For cloning the gene encoding MTX30-285, the following primers were used: mtx(30)sen (5′-AGATCTGCTTCACCTAATTCTCCAAAAG-3′) and mtx(285)antisen (5′-GTCGACATTAGGTATTTCTCCATTGGG-3′). For cloning the genes encoding MTX276-870 and MTX285-870, the following primers were used for polymerase chain reaction amplification from the pTH21 plasmid: mtx(276)sen (5′-GGATCCATGTTTGCCCCCCAATGGA-3′) for MTX276-870; mtx(285)sen (5′-GGATCCAATAATAATTTATTAAATAATAATAG-3′) for MTX285-870 and mtx(870) antisen (5′-GTCGACTCTAGGTTCTACACCTAATG-3′) for both constructs. After cloning into the pCRII vector (Invitrogen, Kroningen, Netherlands) the mtx constructs were cut with BamHI (except the gene encoding MTX30-285, which was cut with BglII) and SalI, purified, and ligated into the digested pGEX vector (except the gene encoding MTX30-285 and MTX265-308, which were ligated into the digested pET-21a vector) (Amersham Pharmacia Biotech, Freiburg, Germany). The proper constructs were checked by DNA sequencing (ABI PRISM; Perkin-Elmer, Weiterstadt, Germany). The primers were from MWG (Ebersberg, Germany). The commercially synthesized peptide MTX265-285, consisting of amino acids 265-285, was purchased from Peptide Specialty Laboratories GmbH (Heidelberg, Germany). Dilution was performed in thrombin cleavage buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 5 mM MgCl2), and the pH was subsequently adjusted to 7.4. Mutagenesis of the gene encoding for MTX265-870 was performed by round circle PCR-based site-directed mutagenesis (Quick Change, Stratagene, Amsterdam, The Netherlands) using the following sense primers and corresponding antisense primers: mtx_D267A,D269Asen (5′-CCATACTAGCTTTAGCTTATAATCAAG-3′) and mtx_D273A,D275Asen (5′-TATAATCAAGCTTTTGCCATGTTTGCC-3′). These primers were from Qiagen Operon (Hilden, Germany), and mutations were verified by DNA sequencing. Purification of MTX Proteins. For expression and purification of the GST fusion proteins, vectors were transformed
Carpusca et al. into E. coli TG1 strains. The plasmids encoding for the 6-Histagged proteins (MTX30-285 and MTX265-308) were transformed in E. coli BL21(λDE3) strains. Cells were grown in LB medium and induced with 0.2 mM isopropyl 1-thio-βD-galactopyranoside at an OD of 0.6 and overnight incubated at 29 °C. Induction of MTX30-308 and MTX30-285 protein synthesis was carried out as described recently (10). Briefly, the E. coli cells were grown to stationary phase at 37 °C, harvested, resuspended in fresh medium, containing 1 mM isopropyl 1-thio-β-D-galactopyranoside, and incubated for 1 h at 30 °C. TG1 strain cells were harvested, lysed by sonication in lysis buffer (20 mM Tris-HCl, pH 7.4, 10 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride, and 5 mM dithiothreitol), and purified by affinity chromatography with glutathione-Sepharose beads (Amersham Pharmacia Biotech, Freiburg, Germany). Loaded beads were washed twice with lysis buffer and twice with thrombin cleavage buffer. MTX constructs were cleaved with thrombin directly from the beads in thrombin cleavage buffer. Thrombin was removed with benzamidine-Sepharose beads (Amersham Pharmacia Biotech, Freiburg, Germany). BL21(λDE3) strain cells were harvested, lysed by sonication in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.2 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4) containing 1% Tween 20, and applied to a Ni-NTAagarose column (Qiagen, Hilden, Germany) preequilibrated with PBS, pH 8. The column was washed twice with PBS (pH 6), containing 10% glycerol, and three times with 20 mM imidazole. The recombinant protein was then eluted from the column with 250 mM imidazole. Thereafter, the elution fractions were further purified by size exclusion chromatography on a Superdex peptide PC3.2/30 column (Amersham Pharmacia Biotech, Freiburg, Germany) in PBS, pH 7.4. CleaVage of MTX Constructs with Chymotrypsin. MTX30-308 or MTX30-285 constructs were incubated with chymotrypsin in a protease/toxin ratio of 1/200 for 60 min at room temperature. For cleavage of GST-MTX30-308 loaded on glutathione-Sepharose beads, chymotrypsin was added in a 1/100 ratio to the toxin for 2 h at room temperature. Chymotrypsin was inactivated with 2 µg of aprotinin/µg of protease. To check whether full cleavage was achieved, the proteins were subjected to SDS-PAGE. ADP-Ribosyltransferase Assay. ADP-ribosylation was performed as follows: 10 µM soybean trypsin inhibitor was incubated with 100 µM [32P]NAD and 100 nM MTX enzymatic fragment for 10 min at room temperature in the presence of 1 µM BSA, 1 mM dithiothreitol, 2.5 mM MgCl2 and 50 mM Tris-HCl, pH 7.4, in a total volume of 20 µL. The reaction was stopped by addition of Laemmli buffer and heating for 5 min at 95 °C, and the samples were subsequently subjected to SDS-PAGE according to the methods of Laemmli (13). [32P]ADP-ribosylated proteins were detected with a phosphorimager from Molecular Dynamics (Amersham Pharmacia Biotech, Freiburg, Germany). To study the inhibition of MTX ADP-ribosyltransferase activity by its putative binding component, truncations, or mutants, increasing concentrations of inhibitory fragments, ranging from 20 to 105 nM, were preincubated for 5 min with MTX30-264 (100 nM). Thereafter, ADP-ribosyltransferase assays were performed as described above.
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FIGURE 1: MTX constructs. (A) Schematic presentation of MTX constructs. MTX30-870 is the 97-kDa holotoxin lacking the putative signal sequence. The chymotryptic cleavage site resulting in the MTX30-264 (27 kDa) and the MTX265-870 (70 kDa) fragments is indicated (arrow). For production of MTX30-264, a 32-kDa N-terminal toxin fragment (MTX30-308) was cleaved by chymotrypsin. The MTX265-870 (70 kDa), MTX265-840, MTX265-750, MTX265-605, MTX265-456, MTX265-308, MTX276-870, and MTX285-870 fragments were generated to study interaction with MTX30-264. (B) Analysis of MTX proteins by SDS-PAGE. MTX proteins (except MTX265-308) were expressed as GST fusion proteins in E. coli and cleaved with thrombin from glutathione-Sepharose beads. Further cleavage of MTX30-264 was accomplished by addition of chymotrypsin in a 1:200 ratio to the toxin (60 min at room temperature). MTX265-308 was cloned into a pET vector and expressed in E. coli. The protein was further purified by size exclusion chromatography on a Superdex peptide column (PC 3.2/30). Each protein (2-3 µg) was subjected to SDS-PAGE and stained with Coomassie blue (shown).
Kinetic Experiments. For kinetic studies, initial rate data for the ADP-ribosyltransferase reactions were determined with activated MTX30-308 in the presence of increasing NAD
concentrations (7.5-250 µM). All experiments were performed at a fixed concentration (10 µM) of soybean trypsin inhibitor (SBTI) as substrate. The amount of SBTI utilized
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FIGURE 2: Inhibition of ADP-ribosyltransferase activity by C-terminal truncations of the putative binding component. (A) ADPribosyltransferase assays were performed with activated MTX30-308 (100 nM) plus SBTI in the presence of [32P]NAD and the indicated concentrations of MTX265-870 ([), MTX265-605 (9), MTX265-456 (2), or MTX265-308 (O) for 10 min at room temperature. Modification of SBTI by MTX30-264 alone was set to 100%. The equimolar concentration of MTX 30-264 and inhibitory fragments is indicated in the diagram. Data are given as means ( SE (n ) 3). The inset illustrates the phosphorimager data of one representative experiment (enzyme activity inhibition induced by MTX265-870 compared to MTX265-308). (B) Influence of the 70-kDa C-terminal fragment MTX265-870 on the time course of ADP-ribosylation of SBTI by MTX30-264. SBTI (10 µM) was ADP-ribosylated by MTX30-264 (100 nM) alone (b) or in the presence of 100 nM MTX265-870. At time 0, reaction was started by addition of [32P]NAD (100 µM final concentration). MTX265-870 was either preincubated for 5 min with MTX30-264 (2) or added 10 min after the reaction was started (9). The arrow indicates the addition of MTX265-870.
was less than 10%. In all experiments, the concentration of activated MTX30-308 was 100 nM, and the incubation time was 5 min. For inhibition kinetics, three different concentrations of MTX265-285 or MTX285-870 were used: 5, 10, and 15 µM for MTX265-285 and 0.5, 1, and 5 µM for MTX285-870. Data were quantified by the phosphorimager with the help of the ImageQuant software (Amersham Pharmacia Biotech, Freiburg, Germany). Kinetic parameters were derived from transformation of data by the Lineweaver-Burk plot. MTX Pull-Down Assays. GST and GST-MTX30-308 cleaved by chymotrypsin (corresponding to GST-MTX30-264)
were bound to glutathione-Sepharose beads and incubated with HeLa cytosol (0.2 µg/µL) for 30 min at 4 °C to block unspecific binding sites. Beads were washed twice with buffer (10% glycerol, 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% Nonidet, and 2 mM MgCl2) and once with thrombin cleavage buffer. The beads were then incubated with the indicated amounts of MTX265-308, MTX265-870, MTX276-870, MTX285-870, MTX265-870_D273A,D375A, MTX265-750, or MTX265-840 for 1 h at room temperature. Some experiments were done in the presence of a 100-fold excess of MTX265-308. Beads were washed as before and
Autoinhibition of MTX
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subjected to SDS-PAGE. Proteins were detected with Coomassie blue. RESULTS Expression of the MTX Truncations. At first, we constructed several C-terminal fragments, which all started with residue 265 of MTX (Figure 1A). The expressed MTX proteins were analyzed by SDS-PAGE (Figure 1B). We compared the inhibitory potencies of these fragments with the putative binding component MTX265-870, which potently blocks the enzyme activity of MTX30-264. Because expression of MTX30-264 was not possible, the active MTX enzyme domain was obtained by chymotryptic cleavage of MTX30-308 (10). Figure 2A shows the inhibitory potency of the various constructs. MTX265-870 was by far the most potent inhibitor of the enzyme activity. Figure 2B shows the influence of this 70-kDa fragment on the time course of the ADPribosylation of SBTI by MTX30-264. Complete inhibition was achieved within 5 min, when MTX30-264 was added during ADP-ribosylation. The IC50 of all other fragments were shifted to the right by about 2 orders of magnitude as compared to MTX265-870 (Figure 2A). Nevertheless, inhibition of the ADP-ribosylation activity of MTX30-264 by these fragments was specific, because we did not observe any inhibitory effect of the peptides on ADP-ribosylation of RhoA by C3, of actin by C2 toxin, or of SBTI by ExoS (not shown). Remarkably, the small fragment MTX265-308 also exhibited the same inhibitory potency as the much larger fragments MTX265-456 and MTX265-605, all sharing an IC50 of about 7.5 µM. Therefore, we studied the structural determinants for inhibition of the ADP-ribosyltransferase activity of MTX30-264 in more detail. Previous studies (10) showed that, after cleavage of MTX30-870, the 27- and 70-kDa fragments remain tightly bound to each other due to strong noncovalent interactions. Therefore, we wanted to study whether coprecipitation of the small peptide MTX265-308 by GST-MTX30-264 bound to glutathione-Sepharose beads was possible. After incubation of GST-MTX30-264 beads with the toxin fragments MTX265-870 or MTX265-308 and washing, the beads were subjected to SDS-PAGE. As shown in Figure 3, MTX265-870 and MTX265-308 were both coprecipitated with GSTMTX30-264 but not with GST alone. However, MTX265-308 was coprecipitated to a much lesser extent than MTX265-870, indicating a reduced affinity of MTX265-308 to MTX30-264 compared to MTX265-870. Note that, on a molar basis, 100 times more MTX265-308 than MTX265-870 was used to detect the coprecipitated protein. These results are also in line with the inhibitory potencies of these fragments. Acidic Residues of MTX265-308 Are Essential for Inhibition of MTX30-264. To identify residues in MTX265-308 which are responsible for inhibition of MTX30-264, we tested the effect of a peptide consisting of the first 21 amino acid residues (MTX265-285). This peptide was commercially synthesized and its inhibitory effect studied in comparison with MTX265-308. As shown in Figure 4A, the peptide MTX265-285 inhibited the enzyme activity of MTX30-264 with a concentration-effect curve similar to that of MTX265-308. We also constructed and expressed MTX30-285 to test whether the additional 21 amino acid residues are enough to block the enzyme activity of MTX. Figure 4B shows that activation
FIGURE 3: Direct interaction of MTX fragments. Binding of MTX265-308 and MTX265-870 to GST-MTX30-264. GlutathioneSepharose beads (20 µL) were loaded with 10 µg of GST or with 10 µg of GST-MTX30-308. GST-MTX30-308 beads were subsequently incubated with chymotrypsin (protease/toxin ) 1/100) for 2 h at room temperature. The beads were incubated with 100 µg of MTX265-308 or with 10 µg of MTX265-870 for 1 h at room temperature. For controls, the same amounts of MTX265-308 and MTX265-870 were run on the two last lanes of the gel, enabling an estimation of the amount of coprecipitated protein. Thereafter, the beads were washed as described in the Experimental Procedures section and loaded on SDS-PAGE. The Coomassie-stained gel is shown. Arrows indicate precipitated MTX265-308 and MTX265-870, respectively.
of MTX30-285 by chymotrypsin largely increased the enzyme activity compared with uncleaved MTX30-285, indicating that the 21 additional residues are sufficient to block the ADPribosyltransferase activity of MTX30-264. Next, we studied the role of the peptide 265-285 in the most potent inhibitory fragment MTX265-870. Therefore, a deletion was constructed and expressed, which covered residues 285-870 of MTX. Subsequently, the inhibitory potency of this fragment (MTX285-870) was studied. Figure 5 shows that the inhibition curve of the ADP-ribosyltransferase activity of MTX30-264 by MTX285-870 was shifted to the right as compared to MTX265-870. Complete inhibition of the enzyme activity was not observed even at the highest peptide concentration studied. We checked the stability of this peptide by comparing its trypsin digestion pattern to that of MTX265-870 and did not find major changes in stability (not shown). All of these findings suggested that the N-terminal part of fragment MTX265-870 is of major importance for inhibition of the enzyme activity of MTX30-264. We attempted to recover the complete inhibition induced by MTX265-870 by adding equimolar amounts of MTX265-285 and MTX285-870, but no additive inhibitory effect was observed (not shown). Next, we attempted to identify the amino acid residues responsible for this inhibition. The 21 amino acid peptide (MTX265-285), which was essential for inhibition of ADPribosyltransferase activity of MTX, is characterized by several acidic residues (Figure 4A). Therefore, we changed the aspartate residues at positions 267, 269, 273, and 275 to alanine in MTX265-870. MTX265-870D267A,D269A,D273A, D275A exhibited reduced inhibition potency on the ADPribosyltransferase activity of MTX30-264 (not shown). Even
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FIGURE 4: Inhibitory potency of residues 265-285 on ADP-ribosyltransferase activity of MTX30-264. (A) SBTI was ADP-ribosylated by MTX30-264 (100 nM) in the presence of [32P]NAD and the indicated concentrations of MTX265-308 (b) or MTX265-285 (O) for 10 min at room temperature. Modification of SBTI by MTX30-264 was set to 100%. The equimolar concentration of MTX30-264 and inhibitory fragments is indicated in the diagram. Data are given as means ( SE (n ) 3). The amino acid sequence of the peptide MTX265-285 is shown. Aspartic acid residues are marked in gray. (B) SBTI (10 µM) was incubated with MTX30-285 previously incubated with and without chymotrypsin for 1 h in the presence of [32P]NAD for 10 min at room temperature. Labeled proteins were analyzed by SDS-PAGE and phosphorimaging (shown).
FIGURE 5: Inhibition of ADP-ribosyltransferase activity by MTX265-870 N-terminal deletions and mutants of MTX265-870. Experiments were performed as described in Figure 2, using MTX265-870 ([), MTX265-870_D273A,D275A (4), MTX276-870 (9), MTX285-870 (b), and MTX265-456_D273A,D275A (O) as inhibitors. The equimolar concentration of MTX30-264 and inhibitory fragments is indicated in the diagram. Data are given as means ( SE (n ) 3).
exchange of only two aspartate residues (MTX265-870D273A, D275A) reduced the inhibiting potency. Accordingly, the slightly shortened peptide MTX276-870 showed much less inhibition of MTX30-264 than MTX265-870 (Figure 5). However, MTX276-870 and MTX265-870D273A,D275A exhibited a stronger inhibitory effect than MTX285-870, suggesting that the region between residues 276 and 285 participates in the inhibiting effect. Noteworthy, the fragment MTX265-456, harboring the amino acid exchanges D273A and D275A, did
not show any inhibiting effect, emphasizing the crucial role of these residues in the inhibition of enzyme activity. To study whether shortened and mutant inhibiting peptides still bind with MTX30-264, we performed pull-down assays. As shown in Figure 6, MTX265-870, the mutant MTX265-870_D273A,D275A, and the deletions MTX276-870 and MTX285-870 were similarly precipitated by GSTMTX30-264. In addition, we studied the stability of these peptides toward trypsin treatment (not shown). Because the
Autoinhibition of MTX
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FIGURE 6: Interaction of MTX265-870 N-terminal deletions and mutants with the enzyme component. Comparison of the binding of MTX265-870, MTX265-870_D273A,D275A, MTX276-870, and MTX285-870 to GST-MTX30-264. Glutathione-Sepharose beads (20 µL) were loaded with 5 µg of GST-MTX30-308 and cleaved with chymotrypsin (protease/toxin ) 1/100) for 2 h at room temperature. The beads were incubated with 5 µg of each truncation for 1 h at room temperature. Washed beads were loaded on SDS-PAGE. For controls, the same amounts of the truncations were run on lanes 6-9 of the gel. The Coomassie-stained gel is shown.
cleavage pattern of the mutant peptides was very similar to that of MTX265-870, we suggested that exchange of the acidic residues or deletion of the very N-terminus in the 70-kDa inhibitory component did not grossly change the overall structure of the mutants. All of these data indicated that the N-terminal part of MTX265-870 and most likely the acidic residues within this area are involved in inhibition of the enzyme activity of MTX30-264 by MTX265-870. On the other hand, it is obvious from the inhibition curves that the N-terminal part of MTX265-870 is not exclusively responsible for the inhibition of the enzyme activity of MTX30-264. The Complete Fourth Ricin-like (QxW)3 Domain of MTX265-870 Is Necessary for Inhibition of MTX Enzyme ActiVity. The C-terminal part of MTX consists of four ricinlike domains; each of them is characterized by three QxW internal repeats (12, 14). To study the role of the C-terminal ricin-like domains in inhibition of ADP-ribosyltransferase activity of MTX30-264, two other C-terminal truncations were designed, covering amino acids 265-840 (MTX265-840) and 265-750 (MTX265-750), and their inhibitory potencies were analyzed. Figure 7A shows that MTX265-750 has no greater inhibitory activity than the small peptide MTX265-285, while MTX265-840 exhibited only 1 order of magnitude less inhibition than MTX265-870. This localizes the second inhibitory site between residues 750 and 870, corresponding to the fourth ricin-like domain, which covers residues 739870. To confirm these findings, we performed coprecipitation assays. Figure 7B shows a reduced pull-down assay of MTX265-840 with GST-MTX30-264 compared to MTX265-870. By contrast, no coprecipitation of MTX265-750 with GSTMTX30-264 was observed under these experimental conditions (not shown). Next, we studied whether the peptide MTX265-308 has any influence on the precipitation of MTX265-840. As shown in Figure 7B, a 100-fold excess of MTX265-308 did not influence the binding of the full-length 70-kDa inhibitory component (MTX265-870) but reduced the precipitation of the C-terminal truncation, again suggesting a lowered affinity
of MTX265-840 as compared to MTX265-870. Thus, deletion of only one part of the fourth ricin-like domain (MTX265-840) decreased binding, whereas loss of this domain (MTX265-750) drastically reduced coprecipitation of the truncated protein with the enzyme domain. All of these results suggested that the very C-terminus of MTX is crucial for the interaction with the enzyme domain. Kinetics ReVeal Different Types of Inhibition by Fragment MTX265-870. To further characterize the inhibition of the ADPribosyltransferase activity of MTX30-264, we performed kinetic studies. At first we studied the NAD dependency of the inhibition of ADP-ribosyltransferase activity of MTX30-264 by the 21-residue peptide MTX265-285. As shown in Figure 8A, increase in the concentration of NAD compensated the inhibitory effect of the 21-residue peptide. Transformation of the data (Lineweaver-Burk plot, Figure 8B) revealed a competitive inhibition with no change in Vmax but increase in Km by addition of the inhibitory peptide. We compared these kinetics with the kinetics of inhibition induced by the large inhibiting fragment (MTX285-870), which was deleted of the N-terminal 21 amino acid residues. As shown in Figure 9, the type of inhibition by MTX285-870 was different from the inhibition caused by MTX265-285. The peptide (MTX285-870) reduced the activity of MTX30-264 in a manner comparable to a noncompetitive type of inhibition. The Km was not affected; however, Vmax was decreased. These findings suggest that MTX265-870 inhibits the enzyme activity of MTX30-264 by two different mechanisms. DISCUSSION MTX is an ADP-ribosyltransferase, which is activated by proteolytic cleavage, thereby releasing an ∼27-kDa enzyme fragment and an ∼70-kDa putative binding component. In vitro the major split fragments remain complexed, resulting in inhibition of enzyme activity. Here, we studied the structural basis for inhibition of the ADP-ribosyltransferase activity of MTX by the 70-kDa fragment. Studies with MTX
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FIGURE 7: Complete or partial deletions of the fourth (QxW)3 domain of MTX. (A) Inhibition of ADP-ribosyltransferase activity of MTX30-264 by MTX265-870 ([), MTX265-840 (0), MTX265-750 (b), and MTX265-285 (4) was studied, and experiments were performed as described in Figure 2. The equimolar concentration of MTX30-264 and inhibitory fragments is indicated in the diagram. Data are given as means ( SE (n ) 3). (B) Binding of MTX265-840 and MTX265-870 to GST-MTX30-264 was performed as described in Figure 3. The GST-MTX30-264 beads were incubated with 5 µg of MTX265-870 or MTX265-840 for 1 h at room temperature in the absence or in the presence of a 100-fold excess of MTX265-308. After washing, the beads were loaded on SDS-PAGE. The Coomassie-stained gel is shown.
are hampered by poor expression of the active fragment in E. coli likely due to toxic effects of the active transferase. Therefore, we started with MTX30-308, which is highly expressed and can be fully activated by chymotrypsin cleavage. We constructed several deletions of the 70-kDa inhibiting MTX265-870 fragment and studied their effects on the active enzyme. Whereas the 70-kDa fragment blocked the enzyme activity with high potency, most of C-terminal deletions were about 2 orders of magnitude less potent. The same inhibiting potency was also observed with a peptide of 21 residues covering MTX265-285. Because this peptide is characterized by several aspartic acid residues, we tested the role of the acidic residues in the large inhibiting peptide. Exchange of aspartate in positions 267, 269, 273, and 275 with alanine strongly reduced the potency of the inhibiting peptide. Even the exchange of Asp272 and Asp275 with
alanine shifted the concentration inhibition curve of the inhibiting MTX265-870 to the right (see also Figure 10). These data indicate that these aspartic residues are essential for inhibition of the enzyme activity of MTX30-264 by MTX265-870. We verified the interaction of these inhibiting peptides (mutants and deletions) with the enzyme domain by precipitation experiments with GST-MTX30-264 and found that they all interact with the enzyme domain in the same way as MTX265-870 does. This and the finding that the trypsin cleavage of the mutant 70-kDa peptide was not changed indicated that exchange of the acidic amino acid residues had no major consequences for the overall structure of the large peptide. No additive inhibitory effect was observed when equimolar amounts of MTX265-285 and MTX285-780 were added together to MTX30-264, implying the necessity of the intact structure of MTX265-870 for potent inhibition.
Autoinhibition of MTX
FIGURE 8: Kinetics of MTX30-264 ADP-ribosyltransferase activity and enzyme inhibition by MTX265-285. (A) ADP-ribosylation of SBTI by MTX30-264 (100 nM) in the absence (b) or in the presence of 5 µM (]), 10 µM (2), or 15 µM (0) MTX265-285 at the indicated [32P]NAD concentrations. Labeled proteins were analyzed by SDSPAGE and phosphorimaging. [32P]ADP-ribose incorporated is given in picomoles of SBTI modified per picomole of enzyme per minute. (B) Lineweaver-Burk plot of data and resulting kinetic values. Data are given as means ( SE (n ) 3). Km for NAD in the absence and presence of the highest inhibiting concentration, 15 µM MTX265-285, were 40.3 ( 6.5 and 1133 ( 617 µM, respectively. The corresponding kcat values were 6.2 ( 1.0 and 10.6 ( 5.5 min-1, respectively.
To get more insight into the mechanisms underlying inhibition of enzyme activity of MTX by its 70-kDa C-terminal fragment, we performed kinetic studies. These data revealed that the small peptide, covering residues 265285, caused a competitive inhibition of MTX30-264. By contrast, the large inhibiting peptide, which was deleted of its N-terminal 21 residues (MTX285-870), behaved like a noncompetitive inhibitor. Further deletions (e.g., MTX265-750) revealed that the C-terminus of MTX contains a region which is responsible for the noncompetitive inhibition. The data obtained can be interpreted by a two-site binding model for the interaction of MTX265-870 with MTX30-264. It appears that the N-terminal part of the 70-kDa fragment of MTX (e.g., residues 265-285) interacts with the catalytic cleft of the ADP-ribosyltransferase, thereby eliciting competitive inhibition of NAD. By contrast, the C-terminal part of MTX interacts in an allosterical manner with the enzyme domain, thereby decreasing the ADP-ribosyltransferase activity. In line with this hypothesis, we observed that the peptide MTX265-456_D273A,D275A, was not able to inhibit the ADPribosyltransferase activity at any concentration tested. This
Biochemistry, Vol. 43, No. 38, 2004 12017
FIGURE 9: Kinetics of MTX30-264 ADP-ribosyltransferase activity and enzyme inhibition by MTX285-870. (A) ADP-ribosylation of SBTI by MTX30-264 (100 nM) in the absence (b) or in the presence of 0.5 µM (]), 1 µM (2), or 5 µM (0) MTX285-870 at the indicated [32P]NAD concentrations. Labeled proteins are analyzed by SDSPAGE and phosphorimaging. (B) Lineweaver-Burk plot of data and resulting kinetic values. Data are given as means ( SE (n ) 3). Km for NAD in the absence and presence of the highest inhibiting concentration, 5 µM MTX285-87,0 were 41.1 ( 2.0 and 49.3 ( 7.5 µM, respectively. The corresponding kcat values were 6.5 ( 0.7 and 3.0 ( 0.3 min-1, respectively.
peptide has amino acid exchanges of the aspartic residues likely to be crucial for competitive inhibition and is Cterminally deleted of its noncompetitively inhibiting part. So far, the precise binding site of the inhibiting small peptide (MTX265-285) is not known. However, one can speculate that basic residues are involved in the interaction, because the aspartate residues of the peptide are essential for its inhibiting function. In this respect, it is of interest that several conserved arginine residues are involved in NAD binding in various ADP-ribosyltransferases (15-19). These residues might be involved in the interaction with the inhibiting peptide in MTX. Because mutation of these residues blocks NAD binding and inhibits ADP-ribosyltransferase activity, it is difficult to study their putative role in the inhibiting effects of the small MTX265-285 peptide. Recently, it was recognized that the putative binding/ translocation part of MTX (MTX265-870) possesses sequence similarity with the lectin-like binding component of ricin (ricin-B) and belongs to the (QxW)3 family, which is characterized by 3-fold internal repeats involved in galactose binding (12, 14). MTX consists of four (QxW)3 repeats, whereas ricin possesses two (QxW)3 repeats. The ricin-like repeats are localized at the C-terminal end of MTX covering
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Carpusca et al.
FIGURE 10: Schematic drawing of the structure of MTX. Residues 30-264 (red) cover the active domain of MTX. 195EDE197 indicates the active site of the ADP-ribosyltransferase. The putative binding and autoinhibiting domain covers residues 265-870. This binding domain consists of four ricin-like domains (domain 1, 303-457; domain 2, 458-598; domain 3, 599-739; domain 4, 740-870), each of which are characterized by their QxW repeats. The various MTX truncations studied are indicated by arrows. Residues D273 and D275 in the peptide 265-285 (dark blue) are essential for the potent inhibition of the catalytic site of MTX30-264 by MTX265-870. The C-terminal residues 750-870, which are part of the fourth ricin-like domain, are involved in the second site inhibition by MTX265-870 (dark green).
residues 303-870 (Figure 10). Because C-terminal deletions of the 70-kDa MTX fragment resulted in a significant decrease of its inhibiting potency, we assumed that the intact ricin-like structure is important for the autoinhibition. Therefore, we constructed the C-terminal deletion MTX265-840, which has lost one of the three QxW motifs of the last ricinlike domain. This deletion caused a significant decrease in the capacity of the C-terminus of MTX to inhibit the enzyme activity. Moreover, deletion of all three QxW motifs of the most C-terminal ricin-like domain prevented the inhibiting effect on enzyme activity. Thus, all of these data suggest that the intact structure of the last ricin-like domain is essential for high-affinity inhibition of MTX 265-870. A similar primary structure to that of MTX was reported for the 98-kDa cabbage butterfly toxins called pierisins (20, 21). The amino acid sequence of pierisin-1 is about 32% identical to that of MTX. Also, pierisins possess ADPribosyltransferase activity. However, pierisin-1 modifies DNA and not proteins (22). Recently, it was shown that the C-terminal part of pierisin-1 binds to carbohydrates most likely to the glycosphingolipids Gb3 and Gb4. Interestingly, amino acid changes in the (QxW)3 repeats of pierisin blocked its cytotoxicity, suggesting that a ricin-like structure is responsible for receptor interaction and subsequent toxin uptake (23). The ricin-like sequence similarities between MTX and pierisin start with residues 300 of MTX (residue 267 of pierisin-1). We have shown that for full enzyme activity of MTX an N-terminal fragment, covering residues 30-264, is sufficient (10). The respective catalytic domain of pierisin, which is very similar to that of MTX, ends at residue 233. The region between the catalytic and the ricinlike domains is covered by about 35 residues in MTX and pierisin. Our data reported in this paper show that this region of MTX is involved in autoinhibition of the catalytic activity. MTX and pierisin differ in this region between each other. Recently, it was reported that pierisin is also autoinhibited under specific conditions (24). It would be of interest to study whether the autoinhibition of pierisin is also caused via a two-site mechanism.
Taken together, our data indicate a novel two-site model for the autoinhibition of MTX. Whereas a small peptide, covering residues 265-285 of MTX, inhibits the enzyme activity by interaction with the active site of the catalytic domain, the C-terminal part of MTX causes inhibition of the enzyme activity through a different site. Both sites are essential for the high inhibiting potency of the 70-kDa fragment of MTX on ADP-ribosyltransferase activity of MTX30-264 observed after cleavage of the holotoxin. REFERENCES 1. Charles, J.-F., Nielsen-LeRoux, C., and Dele´cluse, A. (1996) Bacillus sphaericus toxins: Molecular biology and mode of action, Annu. ReV. Entomol. 41, 451-472. 2. Baumann, P., Clark, M. A., Baumann, L., and Broadwell, A. H. (1991) Bacillus sphaericus as a mosquito pathogen: properties of the organism and its toxin, Microbiol. ReV. 55, 425-436. 3. Liu, J. W., Porter, A. G., Wee, B. Y., and Thanabalu, T. (1996) New gene from nine Bacillus sphaericus strains encoding highly conserved 35.8-kilodalton mosquitocidal toxins, Appl. EnViron. Microbiol. 62, 2174-2176. 4. Chan, S. W., Thanabalu, T., Wee, B. Y., and Porter, A. G. (1996) Unusual amino acid determinations of host range in the Mtx2 family of mosquitocidal toxins, J. Biol. Chem. 271, 14183-14187. 5. Thanabalu, T., Hindley, J., Jackson-Yap, J., and Berry, C. (1991) Cloning, sequencing, and expression of a gene encoding a 100kilodalton mosquitocidal toxin from Bacillus sphaericus SSII-1, J. Bacteriol. 173, 2776-2785. 6. Thanabalu, T., Berry, C., and Hindley, J. (1993) Cytotoxicity and ADP-ribosylating activity of the mosquitocidal toxin from Bacillus sphaericus SSII-1: Possible roles of the 27- and 70-kilodalton peptides, J. Bacteriol. 175, 2314-2320. 7. Priest, F. G., Ebdrup, L., Zahner, V., and Carter, P. E. (1997) Distribution and characterization of mosquitocidal toxin genes in some strains of Bacillus sphaericus, Appl. EnViron. Microbiol. 63, 1195-1198. 8. Davidson, E. W. (1995) Biochemistry and mode of action of the Bacillus sphaericus toxins, Mem. Inst. Oswaldo Cruz 90, 81-86. 9. Thanabalu, T., Hindley, J., and Berry, C. (1992) Proteolytic processing of the mosquitocidal toxin from Bacillus spaericus SSII-1, J. Bacteriol. 174, 5051-5056. 10. Schirmer, J., Just, I., and Aktories, K. (2002) The ADP-ribosylating mosquitocidal toxin (MTX) from Bacillus sphaericussproteolytic activation, enzyme activity and cytotoxic effects, J. Biol. Chem. 277, 11941-11948.
Autoinhibition of MTX 11. Schirmer, J., Wieden, H. J., Rodnina, M. V., and Aktories, K. (2002) Inactivation of the elongation factor Tu by mosquitocidal toxin-catalyzed mono-ADP-ribosylation, Appl. EnViron. Microbiol. 68, 4894-4899. 12. Hazes, B., and Read, R. J. (1995) A mosquitocidal toxin with a ricin-like cell-binding domain, Nat. Struct. Biol. 2, 358-359. 13. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227, 680685. 14. Hazes, B. (1996) The (QxW)3 domain: a flexible lectin scaffold, Protein Sci. 5, 1490-1501. 15. Domenighini, M., Magagnoli, C., Pizza, M., and Rappuoli, R. (1994) Common features of the NAD-binding and catalytic site of ADP-ribosylating toxins, Mol. Microbiol. 14, 41-50. 16. Takada, T., Iida, K., and Moss, J. (1995) Conservation of a common motif in enzymes catalyzing ADP-ribose transfer, J. Biol. Chem. 270, 541-544. 17. Wilde, C., Just, I., and Aktories, K. (2002) Structure-function analysis of the Rho-ADP-ribosylating exoenzyme C3stau2 from Staphylococcus aureus, Biochemistry 41, 1539-1544. 18. Ritter, H., Koch-Nolte, F., Marquez, V. E., and Schulz, G. E. (2003) Substrate binding and catalysis of ecto-ADP-ribosyltransferase 2.2 from rat, Biochemistry 42, 10155-10162. 19. Han, S., Arvai, A. S., Clancy, S. B., and Tainer, J. A. (2001) Crystal structure and novel recognition motif of Rho ADPribosylating C3 exoenzyme from Clostridium botulinum: Structural insights for recognition specificity and catalysis, J. Mol. Biol. 305, 95-107.
Biochemistry, Vol. 43, No. 38, 2004 12019 20. Matsushima-Hibiya, Y., Watanabe, M., Kono, T., Kanazawa, T., Koyama, K., Sugimura, T., and Wakabayashi, K. (2000) Purification and cloning of pierisin-2, an apoptosis-inducing protein from the cabbage butterfly, Pieris brassicae, Eur. J. Biochem. 267, 5742-5750. 21. Watanabe, M., Kono, T., Matsushima-Hibiya, Y., Kanazawa, T., Nishisaka, N., Kishimoto, T., Koyama, K., Sugimura, T., and Wakabayashi, K. (1999) Molecular cloning of an apoptosisinducing protein, pierisin, from cabbage butterfly: Possible involvement of ADP-ribosylation in its activity, Proc. Natl. Acad. Sci. U.S.A. 96, 10608-10613. 22. Takamura-Enya, T., Watanabe, M., Totsuka, Y., Kanazawa, T., Matsushima-Hibiya, Y., Koyama, K., Sugimura, T., and Wakabayashi, K. (2001) Mono(ADP-ribosyl)ation of 2′-deoxyguanosine residue in DNA by an apoptosis-inducing protein, pierisin-1, from cabbage butterfly, Proc. Natl. Acad. Sci. U.S.A. 98, 12414-12419. 23. Matsushima-Hibiya, Y., Watanabe, M., Hidari, K. I. P. J., Miyamoto, D., Suzuki, Y., Kasama, T., Kanazawa, T., Koyama, K., Sugimura, T., and Wakabayashi, K. (2003) Identification of glycosphingolipid receptors for pierisin-1, a guanine-specific ADPribosylating toxin from the cabbage butterfly, J. Biol. Chem. 278, 9972-9978. 24. Watanabe, M., Enomoto, S., Takamura-Enya, T., Nakano, T., Koyama, K., Sugimura, T., and Wakabayashi, K. (2004) Enzymatic properties of pierisin-1 and its N-terminal domain, a guanine-specific ADP-ribosyltransferase from the cabbage butterfly, J. Biochem. (Tokyo) 135, 471-477. BI048729Y
