Institut de Génétique et Microbiologie, Laboratoire de Pathogenèse Comparée, CNRS UMR 8621, Bâtiment 360, Université Paris XI, 91405 Orsay Cedex, France1
Author for correspondence: Mark A. Blight. Tel: +33 1 6915 8168. Fax: +33 1 6915 6334. e-mail: mark.blight{at}igmors.u-psud.fr
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ABSTRACT |
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Keywords: protease inhibitor, entomopathogen, protein purification
Abbreviations: APR, alkaline protease; APRin, alkaline protease inhibitor; Inh, inhibitor protein
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INTRODUCTION |
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A class of metzincin metalloendopeptidases that have been studied extensively are those that belong to the repeats-in-toxin (RTX) family (Welch, 1991 ). This family of zinc metalloproteases are secreted to the external medium via a Type I pathway, and members of this family have been identified in Erwinia chrysanthemi (Dahler et al., 1990
; Letoffe et al., 1990
), Erwinia amylovora (Zhang et al., 1999
), Erwinia carotovora (Marits et al., 1999
), Pseudomonas aeruginosa (Duong et al., 1992
; Guzzo et al., 1990
, 1991a
, b
), Pseudomonas fluorescens (Ahn et al., 1999
; Kawai et al., 1999
; Liao & McCallus, 1998
), Serratia marcescens (Braunagel & Benedik, 1990
; Letoffe et al., 1991
; Nakahama et al., 1986
), Pseudomonas brassicacearum (Chabeaud et al., 2001
) and Photorhabdus luminescens (M. Valens, A.-C. Broutelle, M. Lefebvre, M. A. Blight, D. Bowen and R. ffrench-Constant, unpublished data). The genetic organization of the operons of members of the RTX family varies between organisms, but essentially includes the structural gene for the RTX zinc metalloprotease(s) and an associated type I secretion apparatus comprising an inner-membrane ATP-binding cassette, a membrane-fusion protein and an outer-membrane protein. Moreover, in all cases a gene encoding a specific protease inhibitor is also present. The majority of investigations into these operons have concentrated either on the protease or on its secretion mechanism. Few studies have reported data for the protease inhibitor. The Erwinia chrysanthemi inh gene has been cloned and expressed in Escherichia coli (Letoffe et al., 1989
), and the inhibitor protein (Inh) has been shown to be a heat-stable, low-molecular-mass periplasmic enzyme. Furthermore, the inhibitor of Pseudomonas aeruginosa alkaline protease (APR), APRin, has also been characterized with respect to its binding to APR, and it has been shown to require the N-terminal five amino acids for its inhibition activity (Feltzer et al., 2000
). In addition, a 12 kDa broad-spectrum protease inhibitor has been described in Photorhabdus luminescens (Wee et al., 2000
), which was secreted into the culture medium by phase II phenotypic-variant cells.
Photorhabdus luminescens is a Gram-negative entomopathogenic member of the Enterobacteriaceae (Fischer-Le Saux et al., 1999 ). It exists in a symbiotic relationship with entomophagous nematodes of the family Heterorhabditiae [see reviews by Forst et al. (1997)
and Forst & Nealson (1996)
]. The bacteria occupy the intestinal tract of the infective stage of the nematode (the infective juvenile). On finding a host, the nematode enters the larva and releases the bacteria into the insect haemocoel (body cavity). The bacteria rapidly divide, producing a wide range of toxins and hydrolytic exoenzymes that are responsible for the death and subsequent bioconversion of the insect larva. These conditions are ideal for nematode growth and development.
Here, we describe the cloning and expression of the Photorhabdus luminescens W14 inh gene in Escherichia coli, together with the purification of Inh from the periplasm of Escherichia coli. We also demonstrate the in vitro inhibition activity of Inh on its purified cognate protease, PrtA.
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METHODS |
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Molecular biology techniques.
All molecular biology techniques were done as described by Maniatis et al. (1982) , unless stated otherwise. Restriction endonuclease (Promega) digestions were performed according to the manufacturers instructions. Photorhabdus luminescens W14 genomic DNA was isolated as follows. A sample (1·5 ml) of a stationary phase culture of Photorhabdus luminescens W14, grown in LB medium at 30 °C with agitation, was centrifuged at 8000 g (4 °C); the cell pellet was resuspended in 567 µl TE (10 mM Tris/HCl, 0·1 mM EDTA, pH 8·0). Following the addition of 30 µl of 10% (w/v) SDS and 3 µl proteinase K (20 mg ml-1), the cells were incubated at 37 °C for 1 h. The lysate was mixed thoroughly with 100 µl of 5 M NaCl and 80 µl CTAB buffer [10% (w/v) hexadecyltrimethyl ammonium bromide (Sigma) in 0·7 M NaCl] and incubated at 65 °C for 10 min. Upon completion of the incubation, an equal volume of chloroform/isoamyl alcohol (24:1, v/v) was added to the solution and it was mixed thoroughly. The mixture was centrifuged at 8000 g (4 °C) for 5 min. The aqueous phase was extracted once with phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v) and centrifuged as before. The aqueous supernatant was then mixed with 0·6 vols of 2-propanol; the precipitated genomic DNA was spooled and washed with 70% (v/v) ethanol prior to air-drying and resuspension in 100 µl TE buffer.
PCR amplification of inh was achieved by using two oligonucleotide primers (Inh-W14-5', 5'-ATATCATATGGTTTTTGCAGCTTGGTATCTG-3', and Inh-W14-3', 5'-ATATAAGCTTTTATTCATTTTTCTTATTAGTC-3') that provided 5' and 3' NdeI and HindIII restriction sites (shown in bold), respectively, and which amplified the Photorhabdus luminescens W14 inh gene from its initiation to termination codons. Amplification was performed with the following protocol: 5 cycles at 96 °C for 1 min, 40 °C for 1 min and 72 °C for 30 s, followed by 25 cycles at 95 °C for 1 min, 55 °C for 1 min and 72 °C for 30 s. This amplification resulted in a single PCR product of 422 bp in size. Overexpression of Inh was achieved by cloning the PCR product into the plasmid vector pBAD33-GFPuv (Dr H. Benabdelhak, University Paris Sud, France), which consists of pBAD33 containing the gene encoding GFPuv (Clontech) on an NdeIHindIII restriction fragment. Both the vector pBAD33-GFPuv and the inh PCR product were digested with NdeI and HindIII. Digested plasmid DNA yielded four fragments of 5338, 540, 125 and 109 bp in size. The 5338 bp NdeIHindIII fragment was isolated following electrophoresis of the fragments through a 0·7% agarose/TAE (40 mM Tris acetate, 2 mM EDTA) gel. It was then ligated with the 407 bp digested PCR product using T4 DNA ligase (Promega). Following their transformation with the ligation product, cells of electrocompetent Escherichia coli DH5 were plated onto LB agar containing 12·5 µg chloramphenicol ml-1 and incubated at 30 °C. The sequence of the resulting recombinant clone, pINH-1, was confirmed by using an ABI model 373 automated DNA sequencer (Applied Biosystems).
Purification of Inh from the periplasm of Escherichia coli DH5.
A portion of an overnight culture of Escherichia coli DH5(pINH-1) that had been grown in LB broth supplemented with 12·5 µg chloramphenicol ml-1 was inoculated into 1 l fresh medium to an OD450 of 0·1. This new culture was incubated at 37 °C until growth reached an OD450 of 0·6. Induction of inh expression, from the pBAD promoter, was achieved by the addition of L-arabinose (to a final concentration of 0·1%, w/v) to the culture and a further 2 h incubation. After this time, the bacterial cells were pelleted by centrifugation at 6000 g (4 °C) for 15 min and the cell pellets were resuspended in 10 ml of 30 mM Tris/HCl containing 20% (w/v) sucrose (pH 8·0). Following the addition of 20 µl of 0·5 M EDTA (1 mM final concentration) to the suspension, the cells were incubated at room temperature for 10 min, with gentle stirring. The cells were pelleted by centrifugation at 8000 g (4 °C) for 10 min and resuspended in 10 ml ice-cold 5 mM MgSO4. They were then incubated on ice for 10 min, with gentle stirring. After this time, the cells were centrifuged as above; the supernatant containing the osmotically shocked periplasmic fraction was retained.
Inh was purified from the periplasmic fraction of the supernatant by anion-exchange and size-exclusion chromatography using an ÄKTA FPLC system (Amersham-Pharmacia Biotech) at 10 °C as follows. A MonoQ HR 5/5 anion-exchange column (Amersham-Pharmacia Biotech) that had been equilibrated in Buffer A (20 mM Tris/HCl, pH 7·5) was loaded with the periplasmic fraction (approx. 4070 µg total protein ml-1) at a flow rate of 1·0 ml min-1. Following washing of the column with Buffer A for 2 column volumes, proteins were eluted with a linear gradient of Buffer B (035%; Buffer A+1 M NaCl) over 20 column volumes at a flow rate of 1·0 ml min-1. Inh eluted at approximately 250 mM NaCl and was further purified by size-exclusion chromatography on a Superdex HR75 column (Amersham-Pharmacia Biotech) using Buffer A as the mobile phase. Eluted Inh was supplemented with 10% (v/v) glycerol and stored at -20 °C prior to further analysis.
Purification of PrtA.
Photorhabdus luminescens W14 phase I cells were grown in 1 l of LB medium at 30 °C (agitation at 250 r.p.m.) to the late stationary phase of growth over 48 h. The culture supernatant was retained following centrifugation of the culture at 8000 g (4 °C) for 30 min. Solid ammonium sulphate was added to the supernatant to a final saturation of 80%, and proteins were precipitated at 4 °C for 2 h with gentle stirring. Precipitated material was collected by centrifugation at 10000 g (4 °C) for 30 min, and the pellets were combined and solubilized in a total volume of 80 ml of a solution containing 1 M ammonium sulphate and 50 mM sodium phosphate (pH 8·0). Chromatographic procedures were performed on an ÄKTA FPLC system at 20 °C. Following a final clarification of the solubilized supernatant by centrifugation at 10000 g (4 °C) for 30 min, the solubilized proteins were loaded onto 30 ml of Octyl Sepharose 4 Fast Flow hydrophobic interaction resin packed in an XK16/20 column (Amersham-Pharmacia Biotech) at a flow rate of 1 ml min-1. Elution of the proteins was performed with a linear gradient over 20 column volumes into 5 mM sodium phosphate (pH 8·0); fractions of 5 ml were collected. The protease activity of the fractions was determined by spectrophotometric determination of the Coomassie blue released at 595 nm from Blue Hide Azure powder (Sigma) as follows. Blue Hide Azure Powder was resuspended to 30 mg ml-1 in a solution containing 20 mM Tris/HCl and 5 mM CaCl2 (pH 8·0). Aliquots (5 µl) of each fraction were assayed for 30 min at 37 °C with 0·5 ml of the Blue Hide Azure suspension in 1·5 ml Eppendorf tubes, with constant agitation. Following incubation, the assays were centrifuged and the absorption value at 595 nm was determined for the supernatants. One unit of protease activity was defined as 0·01 A595 units released min-1 (ml fraction assayed)-1. Fractions containing protease activity were pooled and applied directly to a MonoQ HR5/5 column (Amersham-Pharmacia Biotech) that had been equilibrated with 5 mM sodium phosphate (pH 8·0). Fractions of 0·5 ml were collected during elution with a solution containing 5 mM sodium phosphate and 1 M NaCl (pH 8·0) over 10 column volumes. Protease-containing fractions were assayed as described above and eluted at approximately 300 mM NaCl. These were then analysed by SDS-PAGE.
Interactions between the protease (PrtA) and the inhibitor (Inh).
The demonstration of stoichiometric interactions between PrtA and its cognate inhibitor (Inh) was done by anion-exchange chromatography. Purified PrtA and Inh (both at a concentration of 1 mg ml-1) were diluted in 1 ml of Buffer IEX-A (20 mM Tris/HCl, 1 mM CaCl2, pH 8·0), either individually or together to a final concentration of 93 µM this required 50 µg of PrtA and 11 µg of Inh. Samples were analysed by anion-exchange chromatography on a MonoQ HR 10/10 anion-exchange column (Amersham-Pharmacia Biotech) using an ÄKTA FPLC system. Samples were loaded in Buffer IEX-A and eluted with a linear gradient of Buffer IEX-B (Buffer IEX-A+1 M NaCl) over 10 column volumes to a final concentration of 0·5 M NaCl.
Protein analysis.
Protein concentrations were measured using the Coomassie Plus Protein Assay Reagent (Pierce) and denaturing SDS-PAGE (15%, w/v, acrylamide), performed as described by Laemmli (1970) . Proteins were stained with Coomassie Brilliant Blue R250 (Sigma).
Protease inhibition assay.
Purified Photorhabdus luminescens W14 PrtA was prepared as described above. The protease activity of PrtA was measured as the increase of BODIPY-FL fluorescence released from cleaved BODIPY-FL-labelled casein using the EnzChek Protease Assay Kit (Molecular Probes). Measurements of fluorescence were made in a Biolumin Microplate Fluorescence Spectrophotometer (Molecular Dynamics), with excitation and emission wavelengths of 485 and 530 nm, respectively. Purified PrtA dissolved in 20 mM Tris/HCl containing 5 mM CaCl2 (pH 8·0) was assayed in a total volume of 200 µl, with BODIPY-FL casein added to a final concentration of 5 µg ml-1. Alternatively, assays were conducted as described above but in a final volume of 1 ml and using an SFM25 spectrofluorometer (Biotek Kontron) with the same excitation and emission wavelengths. All kinetic experiments were repeated in triplicate and Figs 1 and 46
represent the mean data with a typical SE of between 2 and 5%.
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RESULTS |
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Inh production by Escherichia coli DH5(pINH-1) was induced by incubating a culture of the cells for 2 h in the presence of 0·1% (w/v) L-arabinose. After this incubation, Inh was purified from the isolated periplasmic fraction of Escherichia coli DH5
(pINH-1) in two chromatographic steps. Fig. 1
illustrates the chromatograms produced following anion-exchange (Fig. 1a
) and size-exclusion (Fig. 1b
) chromatography, together with SDS-PAGE analysis of the fractions produced by these two methods (Fig. 1c
).
Following the induction of its expression, Inh could be seen as a low-molecular-mass species (11 kDa) that accumulated in whole cells of Escherichia coli DH5(pINH-1) (Fig. 1c
, lane B). Fractionation of the cells into a periplasmic fraction (Fig. 1c
, lane D) and a residual whole-cell fraction (Fig. 1c
, lane C) demonstrated that the majority of Inh was localized in the periplasmic fraction, as expected. This resulted in a high enrichment of Inh, with Inh representing approximately 65% of the total periplasmic protein as estimated from the peak area in the anion-exchange chromatogram (Fig. 1a
). SDS-PAGE analysis of the chromatographic fractions clearly showed that all of the Inh bound to the MonoQ resin (absence of Inh in flow-through fractions 1 and 2, Fig. 1c
) and that it eluted in a compact peak, with minor contaminating proteins appearing at approximately 42 and 47 kDa (Fig. 1c
, lane 3+4). The material from fractions 3+4 was further purified by size-exclusion chromatography on a Superdex HR75 column (Fig. 1b
), resulting in a single migrating 11 kDa form of Inh that was estimated to be 99% pure (Fig. 1c
, lane 10).
Inhibition of metzincin metalloendopeptidases by the Inh family minimally requires the N-terminal five amino acid residues of the Inh proteins (Feltzer et al., 2000 ). Consequently, it was important to determine whether the N terminus of the recombinant Inh purified from the periplasm of Escherichia coli DH5
(pINH-1) was identical to that of wild-type Inh from Photorhabdus luminescens W14. Hence, we determined the N-terminal-amino-acid sequence of the wild-type Inh purified from Photorhabdus luminescens W14 and of the recombinant Inh purified from Escherichia coli DH5
(pINH-1). For both the wild-type and the recombinant protein the N-terminal sequence was SSLVL, indicating that leader peptidase processing was identical in both organisms. Furthermore, matrix-assisted laser desorption ionization/time of flight (MALDI/TOF) mass spectrometry indicated a molecular mass of 11951 Da for Inh, in excellent agreement with the predicted mass of 11953 Da for mature Inh. Therefore, recombinant Inh, which could be purified in substantial amounts from Escherichia coli DH5
(pINH-1), was used for PrtA inhibition studies.
Comparison of Photorhabdus luminescens W14 Inh with other zinc metalloprotease inhibitors
A multiple-amino-acid-sequence alignment of the 13 available Inh proteins (Fig. 2) showed that there are two highly conserved domains between residues 25 and 41 and residues 67 and 100 (Photorhabdus luminescens W14 Inh co-ordinates) of the Inh proteins. Domain 1 encompasses the predicted leader peptidase cleavage site with the consensus sequence MA/SSL, resulting in the mature polypeptide possessing a strictly conserved Ser at position 2. The crystal structure of the complex between the S. marcescens metalloprotease PrtSM and the Erwinia chrysanthemi inhibitor (Baumann et al., 1995
) (Protein database accession no. 1SMP; http://www.rcsb.org/pdb/) and of the complex between Pseudomonas aeruginosa APR and APRin (Hege et al., 2001
) indicates that both Inh and APRin have a compact globular structure with an unstructured N terminus in direct interaction with the zinc-binding pocket of the protease active site. The first amino acid of Inh is not strictly conserved as it is the main chain atoms that form the inhibitory hydrogen bonds, although Ser1 of the Pseudomonas aeruginosa APRin forms a co-ordinate bond with the zinc ion (Hege et al., 2001
). The strictly conserved Ser2 residue bonds the catalytic Glu of the protease and Leu3 is accommodated in a hydrophobic pocket. The second highly conserved domain within the Inh family Domain 2 is characterized by a ß-sheet being bent into a loop by the strictly conserved Pro62 (mature protein co-ordinates). This loop is in close proximity to the extended N terminus, with a closest distance of 3·52
between the Pro62 and Arg4 side chains. Domain 2 may stabilize the N-terminal of Domain 1 via the Pro62 loop, enabling efficient interaction and insertion of the Inh N terminus into the protease active site. Other than Domains 1 and 2, the intervening sequences of Inh are poorly conserved and result in percentage identity and similarity values that vary from 89·2 and 85·8% identity and similarity, respectively, between Erwinia chrysanthemi B374 and Erwinia chrysanthemi E16 to only 20·8% identity between Pseudomonas brassicacearum NFM421 and S. marcescens SM6 and 18·0% similarity between Pseudomonas tolaasii 1116S and S. marcescens ATCC 27117 (Fig. 3
).
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Inhibition of PrtA by Inh
Casein hydrolysis by purified Photorhabdus luminescens W14 PrtA in the presence of increasing concentrations of Inh was measured. PrtA was diluted to a concentration of 1 nM (see Methods) and Inh was added to give molar ratios of PrtA/Inh ranging from 1:0 to 1:10. Proteins were incubated for 2 min at 25 °C followed by the addition of the PrtA substrate, BODIPY-FL-labelled casein. The increase in fluorescence was measured (Fig. 5a) and the slope of steady-state hydrolysis was calculated between 120 and 540 s. A bar graph of the substrate-hydrolysis rate for each PrtA/Inh molar ratio (Fig. 5b
) clearly demonstrated that little significant inhibition of PrtA by Inh occurred until a molar ratio of 1:0·5 PrtA/Inh was used; total inhibition was observed at molar ratios of 1:1 PrtA/Inh and above. These data are consistent with the unimolecular stoichiometry of inhibition described above and are indicative of a strong interaction between the two proteins that results in the efficient inhibition of PrtA by Inh. To further characterize the interaction between PrtA and Inh, the association constant for the PrtAInh complex was determined.
Kinetics of PrtA inhibition by Inh
Many protease(P)inhibitor(I) interactions are characterized by classical slow-binding kinetics with a typical equilibrium characterized by:
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Not all proteaseinhibitor interactions result in covalent bonding (i.e. an irreversible reaction), and frequently inhibitor binding is so tight that one can not determine a binding constant. Under these conditions the reaction is classified as pseudo-irreversible and the most relevant quantity to measure is the apparent rate of inhibition (or association), kass, where:
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The inhibition curves for purified Photorhabdus luminescens W14 PrtA were determined using BODIPY-FL-labelled casein with 1·0 nM PrtA and either a 1:1 or 1:10 PrtA/Inh molar ratio (Fig. 6). Following the addition of Inh to the PrtA/casein-hydrolysis reaction, a rapid decline in PrtA activity was observed as detected by the reduced rate of change of fluorescence due to liberated BODIPY-FL-labelled peptides. At a molar ratio of 1:10 PrtA/Inh (and higher ratios; data not shown) inhibition was sufficiently rapid that a pseudo-first-order kinetic analysis was not possible. Therefore, to determine a reasonable estimate of kass, a second-order analysis was performed at a molar ratio of 1:1 analysing the data from the addition of Inh (t=120 s) to stable inhibition at t=240 s. A plot of the reciprocal residual enzyme concentration against time (Fig. 6
, inset) yielded a straight line with slope kass=1·34x107 M s-1 and an intercept (9·98x108 M-1) equal to the reciprocal of the starting enzyme concentration. The latter is in good agreement with the initial enzyme concentration of 1·0 nM. The large value for kass indicates that the PrtAInh complex is highly stable and that if an intermediate complex exists it is rapidly converted to the stable inhibited form. Since PrtAInh can be dissociated by the addition of SDS, no covalent modification of the complex occurs; therefore, we can consider the PrtAInh interaction to be pseudo-irreversible. Thus, it is possible to calculate the half-life of protease inhibition from:
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For the Photorhabdus luminescens W14 PrtAInh complex, the protease inhibition t1/2=51·7 s. Studies of PrtA homologues and their kinetic interactions with their cognate Inh homologues will yield comparative inhibition data for that presented above.
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DISCUSSION |
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The zinc metalloprotease inhibitor of Photorhabdus luminescens presented in this work has a high specificity and affinity for the extracellular PrtA protease. Analysis of the complexes formed between the S. marcescens metalloprotease (PrtSM) and the Erwinia chrysanthemi inhibitor (Baumann et al., 1995 ) and between Pseudomonas aeruginosa APR and APRin (Hege et al., 2001
) indicate that the mechanism of inhibition of this class of proteases is unique and involves the extreme N terminus of the inhibitor molecule. The N-terminal five amino acid residues of the inhibitor protein are an absolute requirement for inhibition, as demonstrated by deletion studies on APRin (Feltzer et al., 2000
) and on S. marcescens SMPI (Bae et al., 1998
), where serial deletion of residues 15 of APRin resulted in a progressive reduction of its affinity for APR and deletion of the N-terminal three residues of SMPI resulted in reduced protease inhibition, respectively. However, studies have demonstrated that there are several important differences in the apparent role of the extreme N terminus of inhibition proteins of different bacterial species, and these suggest possible subtle differences in the mechanism of action for the different protein inhibitors. The deletion of Gly1 and Ser2 from S. marcescens SMPI had little effect upon SMP inhibition, whereas the deletion of Leu3 resulted in severely impaired function (Bae et al., 1998
). Furthermore, mutation of SMPI Leu3 indicated a requirement for a hydrophobic side chain for efficient inhibition (Bae et al., 1998
). Conversely, for both Pseudomonas aeruginosa APRin and Erwinia chrysanthemi Inh, the extreme N-terminal residues appear to be absolutely necessary for inhibition (Feltzer et al., 2000
; Letoffe et al., 1989
). Circular dichroism studies of APRin also indicate that there may be structural differences between different inhibitor molecules, when compared with Erwinia chrysanthemi Inh (Feltzer et al., 2000
). These data may, in part, contribute to the observations of highly varied affinities between inhibitors and their cognate metzincin proteases. APRin binds to APR with a KD of approximately 4 pM (Feltzer et al., 2000
), whereas KD values for S. marcescens SMPI and SMP (Bae et al., 1998
) and for Erwinia chrysanthemi PrtA, PrtB and PrtC and their cognate Inh (Letoffe et al., 1989
) are reported as 0·7 µM and 110 µM, respectively. The data presented here for the inhibition of PrtA by Photorhabdus luminescens Inh also indicate a strong and highly stable interaction (kass=1·34x107 M s-1); they also indicate that if an intermediate complex exists it is rapidly converted to the stable inhibited form. We were unable to estimate a KD value for the association of Photorhabdus luminescens PrtA and Inh as (unlike for the Pseudomonas aeruginosa and Erwinia chrysanthemi proteases) we have not yet been able to demonstrate cleavage of chromogenic substrates and have, therefore, been limited to the use of a multiply-cleaved proteinaceous substrate, BODIPY-FL-labelled casein, in our studies.
Here, we have presented the purification and initial characterization of the interaction of the Photorhabdus luminescens W14 inhibitor, Inh, with its cognate protease, PrtA. Further studies of the specific interactions between proteases of the repeats-in-toxin (RTX) zinc metalloprotease family and their cognate, or otherwise, inhibitors will provide more information about this unique mode of inhibition. Co-crystallization and structural analyses of the Photorhabdus luminescens W14 PrtAInh complex are currently being performed and these data will be reported elsewhere.
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ACKNOWLEDGEMENTS |
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Received 11 February 2002;
revised 16 April 2002;
accepted 22 April 2002.