Centre de Recherche sur la Fonction, Structure et Ingénierie des Protéines, Faculté de Médecine, Pavillon Charles-Eugène-Marchand, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4
Received 25 August 2004; returned 20 October 2004; revised 10 November 2004; accepted 24 November 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: The L-1 metalloenzyme from Stenotrophomonas maltophilia was cloned, over-expressed, purified to homogeneity and used in screening of peptide libraries by phage display with a selective and competitive biopanning assay. This was based upon the high affinity of L-1 for cefoxitin and its slow hydrolysis.
Results: From six peptides, the consensus sequence Cys-Val-His-Ser-Pro-Asn-Arg-Glu-Cys was identified as a promising inhibitor of L-1 hydrolytic activity. This peptide showed a mixed inhibition of L-1 with a Ki competitive of 16 ± 4 µM and a Ki uncompetitive of 9 ± 1 µM. The same peptide was prepared without flanking Cys residues and demonstrated no detectable inhibition of L-1 hydrolytic activity with nitrocefin as a substrate. These data confirmed the importance of the peptide conformation for the inhibition of L-1 hydrolytic activity. Further analysis revealed rescue by Zn2+ ions. The mixed inhibition indicated peptide binding near the active site of L-1 and blocking of zinc atoms for optimal conformation in the pocket of the active site.
Conclusion: This is the first report of a peptide inhibitor for Class B metallo-ß-lactamases. It will be used as a lead to identify more potent small molecule inhibitors via peptidomimetics.
Keywords: L-1 ß-lactamase , selective biopanning , ß-lactamase inhibitors
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we used a phage-display technique coupled to a selective biopanning protocol for screening and for identification of peptides having significant inhibition of L-1 metallo-ß-lactamase hydrolytic activity.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Escherichia coli Novablue (endA1 hsdR17(r12K m12K+) supE44 thi-1 recA1 gyrA96 relA1 lac [F'proA + B+ lacIqZM15::Tn10 (TcR)] (Novagen, Madison, WI, USA) was the recipient strain for construction and production of DNA for sequencing. E. coli BL21(
DE3) (FompT hsdSB (rb mb) gal dcm (DE3) (Novagen) was the recipient strain for protein expression and purification. The plasmid pMON13 containing blaS coding for class B ß-lactamase L-1 from Stenotrophomonas maltophilia GN128734
was used to perform PCR cloning. PCR primers containing NdeI-XhoI restriction sites in plasmid pET30a were used to give the plasmid pMON19 coding for a fusion protein L-1 having six histidine residues at the C-terminus. Preparation of DNA and related techniques was by standard methods.5
Protein expression and purification
L-1 ß-lactamase expression and purification was carried out with a B-PER 6x His Fusion Protein Purification Kit, as recommended by the manufacturer (Pierce, Rockford, IL, USA), except that lysis of E. coli BL21 was performed by osmotic shock.6
Phage display
Purified L-1 was used to screen for metallo-ß-lactamase peptide inhibitors by phage display using a PH.D.-C-7-C library (New England Biolabs, Mississauga, Ont., Canada) containing 3.7 x 109 C-7-C mer random peptide sequences. Biopanning was carried out as described previously2
except for the following modifications: L-1 was coated on the bottom of a 96-well plate, four rounds of biopanning were performed instead of three and elution in rounds three and four was conducted in two stepsone elution of 5 min followed by a second elution of 30 min, both with 1 mM cefoxitin (Sigma, Oakville, Ont., Canada). Phage recovered after the second elution was used as input for the fourth biopanning. Phage titration, DNA preparation and sequencing of eluted phage after four rounds of biopanning were performed as described previously.2
Peptide sequences deduced were aligned and a consensus peptide was selected for synthesis.2
Peptides were synthesized on an ABI 433A Peptide Synthesizer using FastMoc chemistry and purified on a Vydak 22 x 250 mm C-18 reverse-phase HPLC column using a 0.1% TFA/acetonitrile gradient at 10 mL/min.
The dissociation constants (Ki competitive and Ki uncompetitive) for the consensus peptide were determined at 30 °C in 50 mM TrisHCl buffer, pH 8.0, in a 1 mL cuvette reaction volume in a Cary 1 spectrophotometer (Varian, Mississauga, Ont., Canada). Hydrolysis for nitrocefin (Oxford, Mississauga, Ont., Canada) (=17400 M1 cm1) was monitored at 485 nm. Kinetic constants Vmax and Km were determined by rates of hydrolysis calculated from the initial velocity in the linear portion, with the same cuvette and a least-squares calculation. The concentration of L-1 was 20 nM and the five concentrations of nitrocefin tested were 2, 10, 25, 50 and 100 µM with three concentrations of C-7-C peptide at 5, 20, 50 µM, respectively. Hydrolytic activity was measured immediately after mixing reagents. All experiments were carried out in triplicate. Analysis of enzyme kinetic data was carried out using the Leonora software for robust regression analysis of enzyme data and a biweighting regression system.7
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The purified L-1 enzyme was coated on a microtitre plate and incubated with a phage-display library expressing randomized Cys-(7 amino acids)-Cys peptides. Screening was conducted using four rounds of biopanning; each step included increasingly restrictive conditions of washing and limited time of contact. Competitive elution with cefoxitin was envisaged as a potential tool to identify specific peptides with high affinity for the L-1 active site. After a fourth round of competitive biopanning, DNA sequencing of 17 randomly selected phages and alignment of peptide sequences identified a consensus, repeated eight times, as depicted in Figure 1. We noted the specific motifSer-Pro-Asnin the first two peptides, and in the third sequence in the reverse orientation for a total of 13 among 17 peptide sequences.
|
|
To assess the importance of peptide conformation, the peptide Val-His-Ser-Pro-Asn-Arg-Glu was synthesized without Cys residues and had no significant inhibition of nitrocefin hydrolysis by L-1, even in 1000-fold excess. Hydrolysis of nitrocefin with 20 µM of peptide incubated with L-1 is not affected by the addition of 20 µM ZnCl2; however, we noted that 1000 µM of ZnCl2 rescued the hydrolytic activity from the peptide inhibitor.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Cys-Val-His-Ser-Pro-Asn-Arg-Glu-Cys peptide gave a mixed inhibition. The apparent affinity of the peptide is almost equal for the enzyme and the enzymesubstrate complex (Ki competitive Ki uncompetitive) at µM concentrations indicating that the behaviour of inhibition is almost purely non-competitive.10
The peptide Val-His-Ser-Pro-Asn-Arg-Glu had no inhibitory activity suggesting that flanking Cys residues were essential. The TEM-1 consensus peptide, His-Ser-Ala-Cys-Asp-Thr-Arg-Arg-Gly-Asp-Cys-Gly, obtained by phage display was initially found to inhibit very weakly TEM-1 ß-lactamase with a Km of
3.5 mM.1
Compared with the L-1 peptide inhibitor, this two-log difference can be explained by differences in biopanning where we used a competitive elution with cefoxitin. We entertain the possibility that competitive biopanning, such as using cefoxitin, is a more promising approach for finding ß-lactamase inhibitors. Huang and collaborators1
suggested that the disulphide bond consensus peptide constrained its conformation and was not optimal for binding and inhibition of TEM-1. For the L-1 peptide inhibitor, we noted that the disulphide bond was essential for its binding and blocking hydrolytic activity. Specific inhibition of Class B L-1 versus Class A TEM-1 and PSE-4, Class C P-99, is not surprising because these enzymes share low sequence identity and have major differences in their active site. Metallo-ß-lactamases require zinc, whereas other ß-lactamases have a serine protease mechanism. The kinetic data suggested binding of the enzyme and enzymesubstrate complex. The consensus peptide probably binds in the active site region, presumably displacing one of the two Zn atoms essential in the active site for maintaining hydrolytic activity.11
The peptide inhibitor described here will now be used as a lead compound in peptidomimetics to screen, identify and develop small molecules as inhibitors of class B metallo-ß-lactamases.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2
.
El Zoeiby, A., Sanschagrin, F., Darveau, A. et al. (2003). Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries. Journal of Antimicrobial Chemotherapy 51, 53143.
3
.
Paradis-Bleau, C., Sanschagrin, F. & Levesque, R. C. (2004). Identification of Pseudomonas aeruginosa FtsZ peptide inhibitors as a tool for development of novel antimicrobials. Journal of Antimicrobial Chemotherapy 54, 27880.
4
.
Sanschagrin, F., Dufresne, J. & Levesque, R. C. (1998). Molecular heterogeneity of the L-1 metallo-ß-lactamase family from Stenotrophomonas maltophilia. Antimicrobial Agents and Chemotherapy 42, 12458.
5 . Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
6
.
Neu, H. C. & Heppel, L. A. (1965). The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. Journal of Biological Chemistry 240, 368592.
7 . Cornish-Bowden, A. (1995). Analysis of Kinetic Data. Oxford Science Publication, Oxford, UK.
8
.
Garau, G., Garcia-Saez, I., Bebrone, C. et al. (2004). Update of the standard numbering scheme for class B ß-lactamases. Antimicrobial Agents and Chemotherapy 48, 23479.
9
.
Crowder, M. W., Walsh, T. R., Banovic, L. et al. (1998). Overexpression, purification, and characterization of the cloned metallo-ß-lactamase L1 from Stenotrophomonas maltophilia. Antimicrobial Agents and Chemotherapy 42, 9216.
10 . Engel, P. C. (1996). Enzymology Labfax. BIOS Scientific, Oxford, UK.
11 . Ullah, J. H., Walsh, T. R., Taylor, I. A. et al. (1998). The crystal structure of the L1 metallo-ß-lactamase from Stenotrophomonas maltophilia at 1.7Å resolution. Journal of Molecular Biology 284, 12536.[CrossRef][ISI][Medline]