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X174 with bacterial translocase MraY and peptidyl-prolyl isomerase SlyD
Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK
Correspondence
Timothy D. H. Bugg
T.D.Bugg{at}warwick.ac.uk
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ABSTRACT |
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X174, responsible for host cell lysis, is known to be the enzyme phospho-MurNAc-pentapeptide translocase (MraY), an integral membrane protein involved in bacterial cell wall peptidoglycan biosynthesis, with an essential role being played by peptidyl-prolyl isomerase SlyD. A synthetic 37 aa peptide Epep, containing the N-terminal transmembrane 
-helix of E, was found to be bacteriolytic against Bacillus licheniformis, and inhibited membrane-bound MraY. The solution conformation of Epep was found by circular dichroism (CD) spectroscopy to be 100 % -helical. No change in the CD spectrum was observed upon addition of purified Escherichia coli SlyD, implying that SlyD does not catalyse prolyl isomerization upon E. However, Epep was found to be a potent inhibitor of SlyD-catalysed peptidyl-prolyl isomerization (IC50 0.15 µM), implying a strong interaction between E and SlyD. Epep was found to inhibit E. coli MraY activity when assayed in membranes (IC50 0.8 µM); however, no inhibition of solubilized MraY was observed, unlike nucleoside natural product inhibitor tunicamycin. These results imply that the interaction of E with MraY is not at the MraY active site, and suggest that a protein–protein interaction is formed between E and MraY at a site within the transmembrane region.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) produce a muralytic enzyme and a holin permease protein; while ssRNA and ssDNA phages produce a single lytic protein, which does not act as a muralytic enzyme (Young, 1992
; Young et al., 2000). The latter strategy is used by bacteriophage X174, in which a single E gene mediates host-cell lysis. The encoded 91 aa E protein causes cell lysis at concentrations of 100–300 molecules per cell (Young & Young, 1982). Overexpression of the cloned E gene is sufficient to cause cell lysis. Analysis of the amino acid sequence reveals a hydrophobic transmembrane domain close to the N terminus (residues 9–32), followed by a positively charged soluble domain. A mutant E protein, in which the C-terminal region is replaced by the lacZ gene product, is still capable of cell lysis, indicating that the soluble domain is not of primary importance for lytic action (Blasi & Lubitz, 1985; Maratea et al., 1985).
Genetic studies by Young and co-workers have implicated the SlyD and MraY gene products in the mechanism of action of the antibacterial E protein. Mutations in the slyD locus have been found to confer resistance to E (Roof et al., 1994). Sequencing of the slyD gene has revealed that the encoded protein shares sequence similarity with the peptidyl-prolyl cis-trans isomerase family of enzymes, such as FK506-binding protein (FKBP) (Hottenrott et al., 1997; Roof et al., 1997). However, subsequent experiments imply that SlyD is not the lethal target for E (Bernhardt et al., 2000). An Epos mutant of the E gene has been isolated, containing two point mutations R3H and L19F, which is bacteriolytic even in the absence of an active slyD gene, suggesting that SlyD acts as an accessory protein during cell lysis, rather than acting as the cellular target. Using the cloned Epos gene, searches have been made for further gene loci where mutations could confer resistance to Epos, and a mutation has been found at minute 2 of Escherichia coli which maps to the mraY gene (F288L point mutation) encoding phospho-MurNAc-pentapeptide translocase (MraY) (Bernhardt et al., 2000).
MraY catalyses the first step of the intramembrane cycle of reactions involved in bacterial peptidoglycan biosynthesis, namely the reaction of the cytoplasmic precursor UDPMurNAc-pentapeptide with the lipid carrier undecaprenyl phosphate, to give a lipid-linked intermediate undecaprenyl-P-P-MurNAc-pentapeptide and UMP (Bugg, 1999), as shown in Fig. 1. This enzyme, an integral membrane protein, is known to be the site of action of three nucleoside natural product antibiotics: tunicamycin, mureidomycin A and liposidomycin B (Brandish et al., 1996a, b). The inhibition kinetics for these inhibitors show that they compete for the UDPMurNAc-pentapeptide and undecaprenyl phosphate binding sites at the MraY active site, and in the case of mureidomycin A, compete for the Mg2+ cofactor binding site (Howard & Bugg, 2003).
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to lead to accumulation of the UDPMurNAc-pentapeptide peptidoglycan precursor, and MraY activity in membranes containing expressed Emyc protein was found to be reduced by 75 %, using a radiochemical exchange assay, consistent with inhibition of MraY by the E protein. However, it was not certain from these earlier results whether the interaction of E with MraY was at the MraY active site, or through a site in the transmembrane region; nor was the precise role of prolyl isomerase SlyD certain, although it was possible that the enzyme catalysed prolyl isomerization of E (Witte et al., 1997).
In view of our previous studies on the inhibition of MraY, and its structure and mechanism (Lloyd et al., 2004), we wished to investigate the molecular basis for the interaction of E with MraY. In this paper we report the interaction in vitro of purified E. coli SlyD and overexpressed E. coli MraY with a synthetic peptide containing the transmembrane domain of E.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-D-glu-m-dap(n-dansyl)-D-Ala-D-Ala was prepared as previously described by Brandish et al. (1996a). Undecaprenyl phosphate was purchased from Larodan Fine Chemicals. Peptides phospholamman and ERB2 were a gift from Dr A. Beevers (University of Warwick, UK). Peptide substrates for SlyD and other biochemicals were purchased from Sigma-Aldrich.
Cloning of N- and C-terminal domains of E.
DNA constructs containing the N- and C-terminal domains of E were amplified by PCR, using X174 DNA (Pharmacia) as a template, with the following primers: Ememb1 (containing the Nde1 site), 5'-ACGTACCATATGGTACGTGGACTTT-3'; Ememb2 (containing the Sap1 site), 5-TGATCGGCTCTTCTGCAGAGACAGGCCGTTTGAATG-3'; Ecyt1 (containing the Sap1 site and N-terminal Cys), 5'-TATACCGCTCTTCTAACTGCTGGAAGGCGCTGAATT-3'; and Ecyt2 (containing a PstI site), 5'-TAATGCACGCCTTCCTCACTGACGTCACATCA-3'.
The amplified DNA fragments were digested with Sap1/Nde1 (Ememb) or Sap1/PstI (Ecyt), and cloned into the pTwin2 vector (New England Biolabs), according to the manufacturer's instructions. Transformation of the Ememb–intein 1–CBD construct into E. coli ER2566 gave no viable colonies, indicating high toxicity. Transformation of CBD–intein 2–Ecyt gave the desired construct, containing an N-terminal S38C mutation. Expression of this construct, followed by purification on a chitin affinity column, gave the expected 31 kDa band by SDS-PAGE.
Antibacterial activity of E peptide.
Antibacterial activity was tested against E. coli K-12, Bacillus licheniformis, Pseudomonas putida, Leuconostoc mesenteroides and Arthrobacter globiformis. Activity on solid media was determined by agar diffusion assay. Solutions of Epep (0.1 mg ml–1 stock in 0.5 M Tris buffer, pH 7.5, containing 1 % SDS) were applied to a filter paper, which was placed onto a lawn of bacteria on an agar plate, and incubated for 24–48 h. Activity on liquid media was determined by addition of an aliquot of Epep (0.1–2.6 µg ml–1 final concentration) to a 100-fold dilution of an overnight culture in Luria Broth, followed by monitoring of OD600 in a microtitre plate reader over 6–8 h, in duplicate. Control experiments were carried out using buffer containing 0.025 % SDS, which showed <4 % growth inhibition, as measured by OD600.
Circular dichroism (CD) spectroscopy.
CD spectra were measured over the range 200–275 nm using a Jasco spectropolarimeter model J-175. Measurements were made using a 1 cm cell length and a protein concentration of 0.1 mg ml–1 in 50 mM Tris buffer, pH 7.5, at 20 °C. For the interaction of Epep with SlyD, 1.0 ml of each was mixed, with a 1 s response time and a scan rate of 100 nm min–1. Spectra were measured eight times and averaged. The data were analysed using K2D software (http://www.embl-heidelberg.de/
andrade/k2d/) to predict the percentage -helix and 
-sheet content.
Fluorescence measurements.
Fluorescence analysis of Epep and SlyD was carried out using a Perkin-Elmer LE5 fluorescence spectrophotometer, with fluorescence excitation at 280 nm, and recording fluorescence emission at 280–500 nm. Assays (total volume 500 µl) contained 50 mM Tris buffer, pH 7.5, and 0.1 % SDS, and solutions of Epep (0.1 mg ml–1, 22 µM) and SlyD (0.1 mg ml–1, 3.7 µM). Assays were carried out in triplicate.
Expression and purification of SlyD.
An overexpression plasmid containing the E. coli slyD gene cloned into the pQE30 vector, as described by Hottenrott et al. (1997), was a gift of Professor G. Fischer (Max Planck Institute Halle). The plasmid was transformed into E. coli strain BL21. SlyD was expressed in 1 l Luria Broth medium, containing 100 µg ampicillin ml–1, grown at 37 °C, and induced with 0.5 mM IPTG at OD600 0.6, followed by a further 3 h growth. Cell extract was purified on a Talon Metal Affinity Resins column (BD Biosciences), using a 0–200 mM imidazole gradient in 50 mM sodium phosphate, pH 7.0, containing 300 mM NaCl. Fractions containing SlyD protein were identified by SDS-PAGE, and were dialysed against 10 mM MOPS, pH 7.0, containing 1 mM DTT. Further purification was achieved by size-exclusion chromatography on an Ultrogel AcA44 column (Sigma) using the same buffer, to give homogeneous protein of Mr 27 000. Purified SlyD was then concentrated using an Amicon Ultra15 filter to a final concentration of 0.130 mg ml–1.
Assays of SlyD activity.
Assays of SlyD activity were based on the method of Hottenrott et al. (1997). Assays were carried out in 96-well microtitre plates using a Genios Tecan Plate Reader, with a total volume of 260 µl, monitoring at 400 nm over 5 min at 20 °C. Assays contained 50 mM Tris buffer, pH 7.5, 0.02 mg N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide ml–1 (32 µM; stock 10 mg ml–1 in DMSO), 0.3 µM -chymotrypsin and 0.1 mg SlyD ml–1. Under these conditions, a specific activity of 0.12 µmol min–1 (mg protein)–1 was observed, using fresh SlyD. Inhibition assays using Epep (0.1 mg ml–1 stock in 50 mM Tris buffer, pH 7.5, containing 1 % SDS) were carried out at 0–4 µM final concentration. Inhibition assays were also carried out using the following peptides: phospholamman (DYQSLQIGGLVIAGILFILGILIVLSRR, 28mer, Mr 3040); ERB2 (RASPVTFIIAIVEGVLLFLILVVVVGILIKRRR, 33mer, Mr 3672); insulin chain B, oxidized (FVNQHLCGSHLVEALYLVCGERGFFYTPKA, 30mer, Mr 3496, C represents cysteic acid); mast cell degranulating peptide HR1 (INLKAIAALVKKVL, 14mer, Mr 1493). Phospholamman and ERB2 were dissolved at 0.1 mg ml–1 in 50 mM Tris buffer, pH 7.5, containing 1 % SDS; and insulin chain B and mast cell peptide HR1 were dissolved at 1.0 mg ml–1 in 50 mM Tris buffer, pH 7.5. Control incubations using buffer containing 0.1 % SDS showed no inhibition of SlyD.
Preparation of E. coli membrane extracts containing overexpressed MraY activity.
A 500 ml culture of E. coli strain DH5/pJFY3c, containing the overexpressed mraY gene (Lloyd et al., 2004), was grown in Luria Broth with 100 µg ampicillin ml–1 at 37 °C, and overexpression was induced at OD600 0.6 by addition of 0.5 mM IPTG. Cultures were grown for a further 4 h, and cells were harvested by centrifugation at 4400 g for 10 min. Cells were stored overnight at –70 °C. Thawed cells were resuspended in buffer A (50 mM Tris, pH 7.5, 2 mM -mercaptoethanol, 1 mM MgCl2) at 2 ml buffer (g cells)–1. Lysozyme (5 mg) was added, and the cells were gently stirred at room temperature for 30 min, followed by sonication on ice. Whole cells and debris were removed by centrifugation at 12 000 g for 30 min, and membranes were collected from the supernatant by centrifugation at 60 000 g for 1 h. Membrane pellets were resuspended in buffer A plus 1 M NaCl, and stirred gently for 15 min at 4 °C. Salt-stripped membranes were collected by centrifugation at 60 000 g for 1 h, and resuspended in 400 µl buffer A, to give a protein concentration of 1.5–2.0 mg ml–1.
Solubilized MraY membranes.
The membranes were resuspended in extraction buffer plus 1.5 % CHAPS and 20 %, v/v, glycerol, and stirred for 1 h. Unsolubilized material was removed by centrifugation at 60 000 g for 1 h to give a protein concentration of 0.5 mg ml–1, and a specific activity of 1–2 nmol min–1 (mg protein)–1 (Brandish et al., 1996a).
MraY assays.
The fluorescence enhancement assay described by Brandish et al. (1996a) was used, in a 96-well microtitre plate format (excitation at 340 nm, emission at 535 nm). Incubation was carried out at 20 °C in a total volume of 100 µl. Assays contained 50 mM Tris buffer, pH 8.0, 1 µl (12 µM) undecaprenyl phosphate (stock 1 mg ml–1), 1 µl (8 µM) UDPMurNAc-L-ala--D-glu-m-dap(n-dansyl)-D-ala-D-Ala (stock 1 mg ml–1), and 10 µl MraY membranes (stock 2.0 mg protein ml–1). Measurements were taken over a 10 min interval with a Genios Tecan Plate Reader. Changes of 2000 fluorescence units were measured over a 10 min assay, relative to controls lacking enzyme. Inhibition was measured by adding different concentrations of Epep (0.1 mg ml–1 stock in 0.5 M Tris buffer, pH 7.5, containing 1 % SDS), to 1–9 µM final concentration, and tunicamycin at 1–50 µM final concentration. Control assays using buffer containing 
0.4 % SDS showed no inhibition of MraY activity.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Attempts to express E with a 45 kDa N-terminal maltose-binding-protein fusion still showed very high toxicity in E. coli, and attempts to express E in yeast were also unsuccessful (data not shown). DNA constructs encoding the N- (residues 1–37) and C-terminal (residues 38–91) domains, containing Cys in place of Ser-38, were generated by PCR, and expressed as intein fusions, with a view to using intein-mediated protein ligation to prepare full-length protein (see Fig. 2). It was observed that the N-terminal domain construct was still highly toxic towards E. coli, whereas the C-terminal domain construct could be transformed and expressed in E. coli. These observations imply that the toxicity of E lies in the N-terminal domain, consistent with the published observation that a fusion protein containing the N-terminal region of E fused to LacZ is bacteriolytic (Blasi & Lubitz, 1985). Therefore, a synthetic peptide (Epep) containing residues 1–37 of E (Mr 4333) was prepared by solid-phase peptide synthesis.
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). Epep showed no growth inhibition of the above strains on agar plates by zone diffusion assay, suggesting that Epep kills growing bacterial cells.
Interaction of Epep with E. coli SlyD
Peptidyl-prolyl isomerase SlyD from E. coli was expressed and purified as described by Hottenrott et al. (1997), to give homogeneous protein of Mr 27 000, which was catalytically active for the cis-trans isomerization of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, detected by cleavage with -chymotrypsin.
The sequence of E contains a proline residue close to the centre of the transmembrane -helix, Pro-21, which has been shown to be essential for the activity of E (Witte et al., 1997). Using the synthetic peptide Epep, it was possible to examine whether treatment with SlyD resulted in any change in conformation of Epep. A solution of 0.1 mg Epep ml–1 in 1 % SDS was examined using CD spectroscopy. A strong CD spectrum was observed, as shown in Fig. 3, showing a peak at 225 nm, diagnostic of an -helical conformation. Analysis of the CD spectrum using K2D software predicted 100 % -helical conformation for Epep. A 1 : 1 mixture of Epep and SlyD gave a CD spectrum with a peak at 226 nm, showing no significant change from that of Epep alone (see Fig. 3). These data imply that Epep readily adopts an -helical conformation, and that SlyD does not catalyse a conformational change upon Epep under these conditions.
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) showed an emission maximum at 345 nm (excitation at 280 nm), due to the presence of tryptophan residues at positions 4 and 7. Upon addition of 0.1 mg SlyD ml–1 in a 1 : 1 ratio, a 10 % increase in fluorescence emission intensity at 345 nm was observed, compared to the fluorescence spectrum of Epep, suggesting that there was an interaction between Epep and SlyD. There were no time-dependent changes in fluorescence emission following mixing of Epep and SlyD, over 5–10 min.
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. From the data in Fig. 5, an IC50 value of 0.15 µM for Epep was estimated. Control assays in the presence of 0.1 % SDS showed no inhibition of SlyD activity, indicating that inhibition was due to binding of Epep. Four other peptides were also assayed as inhibitors of SlyD: two soluble peptides, insulin chain B (30mer, Mr 3496) and mast cell degranulating peptide HR1 (14mer, Mr 1493); and two transmembrane peptides, phospholamman (28mer, Mr 3040) and ERB2 (33mer, Mr 3672). The soluble peptides showed no inhibition of SlyD, at concentrations up to 20 µM. As shown in Fig. 5, the two transmembrane peptides phospholamman and ERB2 showed inhibition of SlyD, with IC50 values of 1.1 and 0.8 µM, respectively, 5–8-fold higher than that of Epep. The sequence of ERB2 (see Methods) contains one proline residue, close to the N terminus, whereas the sequence of phospholamman contains no proline residues.
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, b). E. coli MraY has been overexpressed 30–40-fold and can be solubilized in Triton X-100 or CHAPS detergents in high yields, but it is refractory to further purification (Brandish et al., 1996a; Lloyd et al., 2004). Therefore, the overexpressed E. coli enzyme was used for inhibition studies, in particulate form (membranes suspended in 50 mM Tris, pH 8.0) and in detergent-solubilized form (solubilized in 1.5 % CHAPS). Assays were carried out using 8 µM fluorescent substrate UDPMurNAc-L-ala--D-glu-m-dap(
-dansyl)-D-ala-D-Ala and 12 µM undecaprenyl phosphate in 50 mM Tris buffer, pH 8.0.
Using MraY enzyme solubilized in 1.5 % CHAPS, addition of 1–10 µM Epep gave no MraY inhibition (see Fig. 6), with or without additional SlyD. In contrast, inhibition of detergent-solubilized MraY was observed using tunicamycin (IC50 2 µM), as reported previously (Brandish et al., 1996b).
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. From these data, an IC50 value of 0.8 µM was estimated, although complete inhibition was not observed at higher concentrations of Epep. Inhibition of membrane-bound MraY by tunicamycin, under the same conditions, gave an IC50 value of 30 µM, as shown in Fig. 6(B). Addition of SlyD to these assays had no additional effect; however, the E. coli membranes would have contained endogenous SlyD. Control MraY assays carried out in the presence of 0.4 % SDS showed no inhibition of MraY activity. Epep was also solubilized in buffer containing 1 % CHAPS, and assayed against membrane-bound MraY; similar (or slightly higher) levels of inhibition were observed, compared with Epep solubilized in 1 % SDS. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2001). However, it is unclear exactly how the E protein interacts with MraY, since the known MraY inhibitors are nucleoside natural products, whereas E is a 9 kDa transmembrane protein. The precise biochemical role of peptidyl-prolyl isomerase SlyD in this process is also unclear.
This present work has established that the N-terminal 37 aa transmembrane domain of E retains bacterial cytotoxicity upon expression in E. coli, and that a synthetic peptide Epep retains the ability to inhibit membrane-bound MraY, and possesses some antibacterial activity. The conclusion that the N-terminal domain is responsible for the biological activity of E is consistent with the earlier observation that a mutated form of E, in which the cytoplasmic domain was replaced by LacZ, is active (Blasi & Lubitz, 1985).
These studies have demonstrated experimentally that there is an interaction between Epep and peptidyl-prolyl isomerase SlyD, since Epep is a potent inhibitor of SlyD-catalysed peptidyl-prolyl isomerization (IC50 0.15 µM). However, there is no observable change in the secondary structure of Epep upon addition of SlyD, as shown by CD spectroscopy; therefore, under these conditions, SlyD does not appear to catalyse a prolyl isomerization upon Epep, as proposed by Witte et al. (1997). Bernhardt et al. (2002) have proposed that the role of SlyD is to protect E from proteolysis, which is consistent with our data. The results shown in Fig. 5 indicate that SlyD is also inhibited by two hydrophobic transmembrane peptides, but is not inhibited by two hydrophilic soluble peptides. These data appear to indicate that SlyD has an affinity for hydrophobic -helical peptides, which provides an explanation for why SlyD binds the nascent E protein in vivo.
One of the hydrophobic peptides, phospholamman, contains no proline residues; therefore, the presence of a proline residue is not a pre-requisite for binding to SlyD. Nevertheless, Epep does bind 5–8-fold more tightly to SlyD than to ERB2 and phospholamman, so there is some selectivity for the Epep sequence. Scholz et al. (2006) have recently reported that SlyD exhibits high chaperone activity upon unfolded proteins, and is inhibited strongly (Ki 0.2–2 µM) by several polypeptides lacking proline residues, with a preference for unstructured peptides. It is conceivable that there is an interaction between SlyD and MraY which assists the formation of a protein–protein interaction with E. We note that SlyD has been reported to co-purify with the integral membrane protein adenylate cyclase (Mitterauer et al., 1999). We have not detected any inhibition of MraY by SlyD alone; however, we were unable to exclude SlyD completely from the MraY inhibition experiments, since wild-type E. coli SlyD is present in membrane fractions. The precise cellular roles for the FKBP and cyclophilin families of peptidyl-prolyl isomerases are still uncertain (Ivery, 2000), but it is believed that they are able to stabilize certain peptide conformations found in partly folded proteins, and hence stabilize the formation of protein complexes (Schiene-Fischer & Yu, 2001).
The N-terminal synthetic peptide Epep was found to inhibit membrane-bound MraY, with an IC50 value of 0.8 µM, to the extent of 80–90 %, whereas no inhibition of detergent-solubilized MraY was observed. By comparison, using a radiochemical assay, Bernhardt et al. (2001) observed a 75 % reduction in MraY activity in cell membranes, after expression of Emyc. This behaviour is quite different to that shown by nucleoside inhibitors tunicamycin, mureidomycin A and liposidomycin B, which inhibit membrane-bound and solubilized MraY (Brandish et al., 1996a, b). These observations demonstrate that the mechanism of inhibition by Epep is quite different to that of small-molecule inhibitors, and imply that the site of inhibition by Epep is not at the MraY active site.
The secondary structure of MraY consists of ten transmembrane -helices, whose positions have been predicted by -lactamase fusion analysis (Bouhss et al., 1999) and by bioinformatic analysis (Lloyd et al., 2004). The active site of MraY is apparently formed by the five cytoplasmic loops, in particular loops 2 and 4, which bear catalytic residues Asp-115, Asp-116 and Asp-267 (Lloyd et al., 2004), as shown in Fig. 7(A). Two separate considerations imply that the interaction of Epep with MraY is at a site in the transmembrane region: (1) Epep consists of a transmembrane -helix alone, whose formation has been demonstrated herein using CD spectroscopy; and (2) the mutation in MraY known to cause resistance to E (Phe-288 to Leu) (Bernhardt et al., 2000) lies in transmembrane helix 9, close to the periplasmic face of the protein (see Fig. 7A). We propose that the inhibition of MraY by Epep is caused by a protein–protein interaction at an intramembrane site, and not by active site binding. Attempts to visualize by immunoprecipitation the binding of Epep to a (His)6–MraY fusion protein, prepared previously by Lloyd et al. (2004), were unsuccessful (data not shown), since the membrane-bound (His)6–MraY could not be visualized by immunoblotting.
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) is that there is a high-activity form of MraY in cell membranes, formed upon interaction with other membrane-bound proteins, and that the binding of E to MraY via a protein–protein interaction blocks the formation of this high-activity form, leading to inhibition. Detergent-solubilized MraY activity is not inhibited by Epep, presumably because the formation of a high-activity protein complex involving MraY is disrupted by detergent solubilization. There is some evidence for the formation of a multi-protein biosynthetic complex at cell division (Höltje, 1998), at which point a high turnover of cell wall synthesis is needed, and at which point the E protein causes cell lysis (Young, 1992). If MraY is involved in this complex, then it is quite feasible that the binding of E to MraY at a transmembrane site disrupts the formation of essential protein–protein interactions involving MraY (see Fig. 7B), leading to inhibition of high-activity turnover in vivo, and hence to cell lysis. It is therefore of interest to determine the precise site of interaction between E and MraY, as this site could be a novel antibacterial target. A recent precedent is the inhibition of the transmembrane enzyme -secretase by -helical peptides at a transmembrane site removed from the active site (Das et al., 2003). MraY from Bacillus subtilis has recently been purified on a small scale (Bouhss et al., 2004); thus, it may be possible in future to carry out studies using homogeneous MraY protein. The availability of a biologically active synthetic peptide now allows more detailed studies of the E–MraY interaction.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 16 December 2005;
revised 23 June 2006;
accepted 5 July 2006.
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