Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
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Abstract |
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Keywords: ß-lactamase/BLIP/drug design/protein minimization/protein/protein interactions
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Introduction |
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Another method to combat ß-lactam resistance has been the use of small molecule ß-lactamase inhibitors, such as clavulanic acid and sulbactam (English et al., 1978; Page and Laws, 1998
). These mechanism-based inhibitors protect the ß-lactam drug from hydrolysis by ß-lactamases and restore the therapeutic potential of the antibiotic (Charnas et al., 1978
; Fisher et al., 1978
; Therrien and Levesque, 2000
). Variants of ß-lactamase have now evolved, however, that resist these inhibitors while still maintaining the ability to hydrolyze ß-lactam antibiotics (Imtiaz et al., 1994
; Henquell et al., 1995
; Palzkill, 1998
).
Clavulanic acid was initially purified from the soil bacterium Streptomyces clavuligerus, which also produces a protein inhibitor of ß-lactamases called ß-lactamase inhibitor protein (BLIP) (Doran et al., 1990). BLIP is a 165 amino acid protein composed of two-domains of ~78 residues each and is a potent inhibitor of TEM-1 ß-lactamase (Ki = 0.10.6 nM) (Strynadka et al., 1994
; Petrosino et al., 1999
; Rudgers and Palzkill, 1999
). In addition, BLIP inhibits ß-lactamases from both Gram-positive and Gram-negative bacteria to varying degrees and also inhibits the cell-wall transpeptidase PBP5 from Enterococcus faecalis (Strynadka et al., 1994
).
The co-crystal structure of TEM-1 ß-lactamase and BLIP reveals that BLIP binds just outside the active site pocket of ß-lactamase and inserts two loops, one from each domain, into the active site of the enzyme (Figure 1) (Strynadka et al., 1996
). The majority of interactions with TEM-1 ß-lactamase arise from domain-1 of BLIP, with Asp49 from the loop of domain-1 making four critical hydrogen bonds to four conserved residues in the active site pocket of the enzyme (Strynadka et al., 1996
). Together with Phe142 from the loop in domain 2, the residues from the two turns of BLIP mimic the binding of penicillin G in the active site pocket of the enzyme (Strynadka et al., 1996
).
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Phage display is an effective method for sorting large, randomized libraries of mutants for specific proteins or peptides that bind a target molecule with high affinity (Lowman et al., 1991; Dennis et al., 1995
; Rudgers and Palzkill, 1999
; Huang et al., 2000
). To aid in the development of BLIP-based peptide inhibitors of ß-lactamases, we have expanded on this property of phage display to select small, contiguous regions of BLIP that retain the ability to bind and inhibit TEM-1 ß-lactamase from a large pool of randomly fragmented BLIP peptides. The results obtained suggest that random fragmentation of a gene followed by phage display selection of fragments that encode peptides that retain binding function is an efficient method to identify peptide mimics of a proteinprotein interaction.
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Materials and methods |
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Escherichia coli XL1-Blue (Bullock et al., 1987) (Stratagene) was used for transformation of the ligation reaction of the pGR48 library and to generate the initial unpanned bacteriophage stocks of the pGR48 library. E.coli TG1 (Gibson, 1984
) was used for all other transformations of ligation reactions and also for production, amplification and determination of the titer of bacteriophage stocks. The bli gene was PCR amplified from the pTP154 plasmid that has been described previously (Huang et al., 2000
). To display randomized peptide libraries on the surface of M13 bacteriophage, the pTP167 phagemid was used (T.Palzkill, unpublished data). This vector expresses proteins under the control of the constitutive ß-lactamase promoter as fusions to the His6-tag at their N-terminus and the M13 gVIIIp at their C-terminus. The vector encodes chloramphenicol resistance allowing for the selection of bacteria harboring the phagemid on medium containing chloramphenicol.
Construction of minimized BLIP phage-display library
To construct phage display libraries of randomly fragmented BLIP peptides, bli was fragmented using a nebulizer as described (http://dna1.chem.ou.edu/protocol_book/protocol_partII.html). The BLIP gene was PCR amplified from pTP154 using the top strand primers PD-bla1 (5'-CGGGGGAGCTCGTTTCTTAGACGTCAGGTGGC-3') and the bottom strand primer BLIP-Xba (5'-CGGGCCGTCTAGAATACAAGGTCCCACTGCCGCTTG-3'). Since the PCR primers used to amplify bli are complementary to regions outside the gene of interest, an additional 251 nucleotides upstream and eight nucleotides downstream of bli were also amplified, generating a 760 bp PCR product. Approximately 20 µg of the PCR product in 2 ml of ice-cold sterile 40% glycerol, 10 mM Tris, pH 7.5 was added to a nebulizer cup (IPI Medical Products, Part No. 4207) and placed on ice. The nebulizer was modified as described (http://dna1.chem.ou.edu/protocol_book/protocol_partII.html). The DNA was fragmented by applying 45 p.s.i. of nitrogen gas to the DNA sample at 5 min intervals for a total of 30 min. After each interval, the nebulizer was centrifuged at 150 g for 1 min at 4°C to collect the DNA sample in the bottom of the nebulizer. A 40% glycerol, 10 mM Tris, pH 7.5 solution was added to the nebulized sample after each centrifugation to bring the final volume of the sample back to 2 ml. The nebulized DNA was concentrated and cleaned using a QIAquick PCR purification kit (Qiagen) and resuspended in 50 µl of sterile, distilled, deionized water, pH 7.0.
The sheared BLIP DNA fragments were treated with T4-DNA polymerase according to the manufacturer (New England Biolabs) and incubated at 11°C for 1 h. The resulting DNA fragments were gel purified from a 0.8% agarose gel using a QIAquick (Qiagen) column to remove DNA fragments outside the desired range of 100500 bp.
The BLIP fragment library gVIII phage display vector, pGR48, was constructed by replacing the 780 bp SalIXbaI fragment of pTP167, which contains the blaTEM-1 gene, with fragmented bli DNA. A 3 µg amount of restriction enzyme-treated pTP167 vector was gel purified to remove the blaTEM-1 DNA fragments and treated with calf-intestinal phosphatase followed by Klenow as described by the manufacturer (New England Biolabs). A 25 µl volume of the treated fragmented bli DNA was ligated into the digested pTP167 vector. Plasmid DNA was electroporated into E.coli XL1-Blue according to the manufacturer (Stratagene) and transformed bacteria were selected on LB agar containing 12.5 µg/ml chloramphenicol. A library size of ~40 000 inserts was obtained as determined by colony polymerase chain reaction (PCR) using the primer Blasig (5'-CAACATTTCCGTGTCGCCC-3') and G8bamHI (5'GGGCGGATCCTATCAGCTTTCGAGGTGAATTTCTT-3') (Hanke and Wink, 1994). The resulting colonies were pooled using 1 ml of LB medium. A 1/50 volume of the pooled colonies was used to prepare bacteriophage library stocks.
Preparation, enrichment and titering of phage particles
The naive phagmid library was packaged and titered as described previously (Huang et al., 2000). Panning was performed in 96-well microtiter plates coated with 0.2 ml of a 10 µg/ml solution of purified TEM-1 ß-lactamase or BSA as described (Huang et al., 2000
). Phage amplification and phage titer determinations were performed as described (Huang et al., 2000
).
Expression and purification of ß-lactamase
ß-Lactamase was purified to >90% homogeneity using a zinc chelating Sepharose (fast flow) column (Pharmacia) and Sephadex G-75 gel filtration chromatography as described previously (Cantu et al., 1997). His-BLIP was expressed using vector pGR32 and purified to >90% homogeneity as described previously (Petrosino et al., 1999
).
Phage ELISA
Phage ELISA was performed in 96-well microtiter wells as described (Huang et al., 2000). A total of 5x1011 phage displaying one of the eight selected BLIP peptides in 0.2 ml of blocking buffer [2% milk powder in 1x Tris-buffered saline, pH 7.5 (Sambrook et al., 1989
)] were added in duplicate to the blocked BSA, ß-lactamase or anti-His6-tagged antibody (New England Biolabs) wells. Bound phage were detected using anti-M13 phage antibody conjugated to horseradish peroxidase (Pharmacia).
Peptide synthesis and inhibition assays
Peptides K8D49 and C30D49 were synthesized by Research Genetics (Huntsville, AL). The K8D49 peptide was N-terminal biotinylated, cyclized and purified by HPLC to >90% purity. The C30D49 peptide was cyclized and purified by HPLC to >90% purity. No modification of the C-terminus was performed for either peptide. Disulfide bonds were removed in the K8D49 and C30D49 peptides by the addition of 10 mM DTT to each peptide stock prior to performing inhibition assays. Inhibition assays were performed as described previously by incubating various concentrations of the cyclized or reduced K8D49 peptide, C30D49 peptide or a control peptide (protein kinase C substrate [PLSRTLSVAAKK]) (Sigma) with TEM-1 ß-lactamase for 2 h at 25°C to establish equilibrium. Enzyme assays were performed in 0.05 M phosphate buffer (pH 7.0) containing 1 mg/ml BSA. Following incubation, 70 µM cephaloridine was added and hydrolysis of the substrate was monitored at 260 nm. The final volume of the reaction was 0.05 ml and the extinction coefficient used for cephaloridine was = 10 200 m-1 cm-1 (Petrosino et al., 1999
). DTT concentrations were adjusted such that all samples contained equal concentrations of the reducing agent. No significant loss of wild-type TEM-1 ß-lactamase activity was detected in assays containing DTT alone.
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Results |
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The minimum sequence and structural requirements of BLIP to inhibit TEM-1 ß-lactamase are unknown and difficult to predict. To identify the minimum peptide sequence of BLIP that is sufficient to inhibit TEM-1 ß-lactamase, random peptide fragments of BLIP ranging in size from ~33 to 166 amino acid residues were fused to the gene VIII protein (gVIIIp) of M13 bacteriophage. To generate the fragmented BLIP peptides for phage display, the PCR-amplified BLIP gene, bli, was randomly fragmented into 100500 bp segments (see Methods). The sheared DNA fragments were ligated into the phage display vector, pTP167, which is designed to express the peptides as fusion proteins to a His6-tag at their N-terminus and to the M13 gVIIIp at their C-terminus. A library size of ~40 000 independent inserts was generated. Analysis of fragments within the library revealed that a range of DNA inserts from 100 to 500 bp were present with no evidence of bias towards a particular insert size (data not shown). Sequence analysis of 13 randomly chosen inserts revealed that all the inserts between the His6-tag and M13 gVIII consisted of bli DNA, none of which contained in-frame BLIP sequences with both the His6-tag and gVIIIp.
Although there was no preference for the size of DNA inserts in the phage display library, there was a bias for the insertion of fragments containing the extreme 3' terminus of the bli gene. Eleven of the 13 inserts sequenced contained the same 3' terminus but different 5' starting termini. The bias may be due to insufficient treatment of the sheared PCR fragments by T4-DNA polymerase. This would favor DNA inserts that required only one terminus to be filled by the DNA polymerase, such as fragments harboring the original, blunt-end 5' or 3' terminus of the PCR product. However, it is not known why the 3' end of the PCR fragment was so strongly favored over the 5' terminus.
Enrichment of BLIP peptides that bind ß-lactamase
To identify BLIP peptides that bind to ß-lactamase, the phage display library was enriched for phage particles that display peptides that bind to immobilized TEM-1 ß-lactamase. After extensive washing to remove unbound phage, the bound phage were eluted from the immobilized ß-lactamase and used to infect E.coli. The phage were amplified in the infected bacteria and used for another round of binding enrichment. After each round of binding enrichment, representative clones were randomly selected to determine if the library was converging on a particular BLIP insert. Colony PCR allowed the rapid determination of the DNA insert size by using primers that flanked the BLIP inserts. After one round of panning, the enriched library remained highly diverse with inserts ranging from 100 to 500 bp, similar to the starting library. However, by round two the library began to converge on a 120 bp insert and by round three 15 of the 16 selected clones examined contained a single 120 bp insert (data not shown). The panning experiment was repeated with the naive phage display library and the library again began to converge on a 120 bp insert during the third round of binding enrichment. By round four the library had completely converged on a 120 bp DNA insert.
DNA inserts from clones isolated from the final two rounds of each binding enrichment experiment were sequenced to determine the identity of the enriched fragments. All inserts were BLIP sequences in-frame with both the His6-tag and the M13 gVIIIp. Out of 55 clones sequenced, inserts encoding a total of eight different BLIP peptides were identified, with 43 of the inserts encoding a BLIP peptide consisting of residues K8D49 and also containing the mutation F14L (Figure 2). The convergence of the library on the K8D49 sequence in two independent panning experiments suggests that the K8D49 peptide binds to ß-lactamase. Six peptides were derived from domain 1 of BLIP and had in common the BLIP C30D49 residues (Figure 2
). The fact that all of the clones isolated contained in-frame BLIP fragments with the His6-tag and gVIII in the proper orientation whereas the starting library did not suggest that functional peptides were selected from the naive library. The finding of peptides derived from domain 1 after phage display is also striking considering the bias in the naive library towards clones containing domain 2.
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Inhibition of ß-lactamase by minimized BLIP peptides
Although the K8D49 peptide was selected based on the ability to bind TEM-1 ß-lactamase, it is possible that the peptide could bind without inhibiting function. To establish if the K8D49 peptide inhibits TEM-1 ß-lactamase, the full-length peptide was synthesized and used for in vitro inhibition studies. Since the crystal structure of BLIP reveals a disulfide bond between residues C30 and C42, it was hypothesized that this bond formation may be important in the inhibition of ß-lactamase by the K8D49 peptide and therefore the peptide was oxidized to form the disulfide bond. The inhibition assay involved incubating various concentrations of the K8D49 peptide in the presence of TEM-1 ß-lactamase until equilibrium was achieved. After the incubation phase, the ß-lactamase substrate, cephaloridine, was added and the hydrolysis of the substrate was monitored to determine the concentration of peptide required to inhibit TEM-1 ß-lactamase. The Ki for the BLIP peptide was determined as 352 µM, indicating that the selected peptide retained the ability both to bind and to inhibit TEM-1 ß-lactamase.
To determine the importance of the disulfide bond in the inhibition of ß-lactamase by the K8D49 peptide, the disulfide bridge between the C30 and C42 residues was reduced. The inhibition assay using the reduced peptide and TEM-1 ß-lactamase revealed a small increase in binding with Ki = 294 µM, indicating that the disulfide bond is not critical for the inhibition of ß-lactamase by the K8D49 peptide (Figure 3). Inhibition of ß-lactamase also appears specific to the K8D49 peptide in that no inhibition of TEM-1 ß-lactamase was observed when using the 12 amino acid protein kinase C substrate peptide (Figure 4
).
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Discussion |
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Of the BLIP peptides that were identified, the K8D49 peptide represented 78% of the insert population. It is of interest that both the K8D49 and the G2D49 peptides identified by phage display contained the F14L substitution (Figure 2). This mutation was most likely introduced during PCR amplification of the BLIP gene during the construction of the phage-display library. The occurrence of the substitution in two independent peptide sequences suggests that it may act to stabilize the peptides from proteolysis of bacterial enzymes or contribute to binding to TEM-1 ß-lactamase.
It has been demonstrated that peptides fused to M13 gVIIIp vary widely in efficiency of display on the surface of the bacteriophage (Malik et al., 1996). For example, the addition of just nine residues to a well-displayed hexapeptide greatly decreased the efficiency of display (Malik et al., 1996
). The K8D49 peptide described here may be displayed more efficiently on the surface of the M13 phage compared with other peptides or wild-type BLIP. This may explain why the G2D49 and K8V165 peptides (Figure 2
), despite containing all of the amino acids found in the K8D49 peptide, are not substantially enriched in the phage display experiments. The additional residues on these peptides may decrease the amount of peptide displayed on the phage, giving a slight advantage to the K8D49 peptide that could quickly result in the K8D49 peptide overtaking the phage population and providing for the observed enrichment.
In virtually all the peptides selected by phage display, the 20-residue sequence, C30D49, was identified, suggesting that this region contains several contact residues with TEM-1 ß-lactamase. If contact residues in the wild-type BLIP-ß-lactamase complex are retained in a peptide ß-lactamase complex, it is possible that peptides containing the highest density of contact residues would be selected in the phage display experiment. To test this possibility, a window encompassing 20 contiguous amino acids was scanned through the BLIP sequence and scored for the number of residues that contact TEM-1 ß-lactamase based on the contact residues reported for the X-ray structure of the BLIP-ß-lactamase complex (Strynadka et al., 1996) (Figure 5
). It is evident that domain 1 of BLIP contains the windows with the highest density of contact residues. Note that the C30D49 window resides within a cluster of windows that contain 1011 contact residues (Figure 5
). This suggests that peptides containing domain 1 sequences including positions 3049 were the most prevalent in the phage-display enrichment because they contain the highest density of contact residues. This result also implies that the peptides selected by phage display use the same set of contact residues as those found in the wild-type BLIP-ß-lactamase crystal structure. In contrast, the residues located in domain 2 of BLIP that make direct contact with TEM-1 ß-lactamase are widely dispersed in small clusters of two to four residues (Figure 5
). Therefore, phage-displaying peptides derived from domain 2 may not have been enriched because domain 2 does not contain a high density of contact residues.
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Proteinprotein interactions are important events in most biological processes and are important targets for drug design. Over the past decade, there has been increased interest in the design of small molecules that mimic functional epitopes of proteins (Leatherbarrow and Salacinski, 1991; Dennis et al., 1995
; Li et al., 1995
; Huang et al., 2000
). However, interfaces between two binding partners may consist of as many as 40 contact residues, making it difficult to construct small peptide inhibitors of such large and often discontinuous regions. Recent studies indicate that only a small subset of the contact residues in the interface contribute substantially to binding, making the design of small molecular inhibitors potentially less daunting (Jin et al., 1992
; Nuss et al., 1993
; Clackson and Wells, 1995
). In addition, several potent inhibitors of biologically important proteins have been identified in which the critical residues involved in binding the target molecule cluster together in a single domain of the inhibitor (Leatherbarrow and Salacinski, 1991
; Buckle et al., 1994
; Dennis and Lazarus, 1994
; Li et al., 1995
). However, identification of these domains has relied on crystal structure analysis and extensive mutagenic studies. Here we used phage display to identify a small domain of BLIP that contains a cluster of residues that are sufficient for binding and inhibition of TEM-1 ß-lactamase. This method can easily be applied to other proteinprotein complexes to identify critical binding residues in either protein partner. Although a co-crystal structure of the inhibitorenzyme complex has been previously solved and used in the method described here (Strynadka et al., 1996
), this information is not required for these experiments.
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Notes |
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Acknowledgments |
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References |
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Received January 15, 2001; revised April 3, 2001; accepted May 5, 2001.