Identification of Residues in β-Lactamase Critical for Binding β-Lactamase Inhibitory Protein

Rudgers, Gary W.; Palzkill, Timothy

doi:10.1074/jbc.274.11.6963

Cited by 31 publications

(38 citation statements)

References 35 publications

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“…Pro-107 is conserved through all class A ␤-lactamases that are inhibited by BLIP, and most of these enzymes have a valine or isoleucine at position 108. Both residues 107 and 108 in TEM-1 ␤-lactamase were previously shown to be important for BLIP binding (28). The fact that these hydrophobic interactions are important for binding between class A ␤-lactamases and BLIP is consistent with previous systematic analyses of protein-protein interfaces (14).…”

Section: Determination Of the Functional Epitope For Blip Binding Tosupporting

confidence: 76%

“…The interaction area between BLIP and ␤-lactamase (2636 Å 2 ) is one of the largest among known protein-protein interactions (23). Several previous studies have focused on the TEM-1⅐BLIP complex (1,(23)(24)(25)(26)(27)(28)(29). We previously employed alanine-scanning mutagenesis to determine the functional epitopes of BLIP for binding the TEM-1 and SME-1 ␤-lactamases (1).…”

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confidence: 99%

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Dissecting the Protein-Protein Interface between β-Lactamase Inhibitory Protein and Class A β-Lactamases

Zhang

Palzkill

2004

Journal of Biological Chemistry

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124

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␤-Lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A ␤-lactamases at a wide range of affinities. Alanine-scanning mutagenesis was previously performed to identify the amino acid sequence requirements of BLIP for inhibiting TEM-1 ␤-lactamase and SME-1 ␤-lactamase. Two hotspots of binding energy, one from each domain of BLIP, were identified (Zhang, Z., and Palzkill, T. (2003) J. Biol. Chem. 278, 45706 -45712). This study has been extended to examine the amino acid sequence requirements of BLIP for binding to the SHV-1 ␤-lactamase, which is a poor binding substrate (K i ‫؍‬ 1.1 M), and the Bacillus anthracis Bla1 enzyme (K i ‫؍‬ 2.5 nM). The two hotspots previously identified as important for binding TEM-1 and SME-1 ␤-lactamase were also found to be important for binding Bla1. The hotspot from the second domain of BLIP, however, does not make substantial contributions to SHV-1 binding. This may explain why BLIP binds to SHV-1 ␤-lactamase with much weaker affinity than to the other three enzymes. Three regions, including two loops that insert into the active pocket of TEM-1 ␤-lactamase and the Glu-73-Lys-74 buried charge motif, exhibit strikingly different effects on the binding affinity of BLIP toward the various enzymes when mutated and, therefore, act as specificity determinants. Analysis of double mutants of BLIP that combine specificity-determining residues suggests that these residues contribute to the poor affinity between the second domain of BLIP and SHV-1 ␤-lactamase.

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Section: Determination Of the Functional Epitope For Blip Binding Tosupporting

confidence: 76%

mentioning

confidence: 99%

Dissecting the Protein-Protein Interface between β-Lactamase Inhibitory Protein and Class A β-Lactamases

Zhang

Palzkill

2004

Journal of Biological Chemistry

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124

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“…Previous studies have shown that the binding between the TEM-1 ␤-lactamase and BLIP is not optimal and can be improved (38,40). Phage display has been shown to be an effective means of optimizing protein-protein interactions and therefore could be used to improve the binding between the BP46-51 peptide and various ␤-lactamases.…”

Section: Discussionmentioning

confidence: 99%

“…S. clavuligerus also produces ␤-lactam antibiotics such as cephamycins as well as a ␤-lactamase inhibitor, clavulanic acid (23). BLIP has been shown to bind to and inhibit the TEM-1 ␤-lactamase with a K i of 0.1 to 0.6 nM (38,40,45). In addition, BLIP binds to and inhibits the class A ␤-lactamases from Staphylococcus aureus, Bacillus cereus, and Bacillus licheniformis with K i values of 1 to 3 M. BLIP does not efficiently bind to class B, C, or D ␤-lactamases (45).…”

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confidence: 99%

Binding Properties of a Peptide Derived from β-Lactamase Inhibitory Protein

Rudgers¹,

Huang²,

Palzkill

2001

Antimicrob Agents Chemother

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To overcome the antibiotic resistance mechanism mediated by ␤-lactamases, small-molecule ␤-lactamase inhibitors, such as clavulanic acid, have been used. This approach, however, has applied selective pressure for mutations that result in ␤-lactamases no longer sensitive to ␤-lactamase inhibitors. On the basis of the structure of ␤-lactamase inhibitor protein (BLIP), novel peptide inhibitors of ␤-lactamase have been constructed. BLIP is a 165-amino-acid protein that is a potent inhibitor of TEM-1 ␤-lactamase (K i ‫؍‬ 0.3 nM). The cocrystal structure of TEM-1 ␤-lactamase and BLIP indicates that residues 46 to 51 of BLIP make critical interactions with the active site of TEM-1 ␤-lactamase. A peptide containing this six-residue region of BLIP was found to retain sufficient binding energy to interact with TEM-1 ␤-lactamase. Inhibition assays with the BLIP peptide reveal that, in addition to inhibiting TEM-1 ␤-lactamase, the peptide also inhibits a class A ␤-lactamase and a class C ␤-lactamase that are not inhibited by BLIP. The crystal structures of class A and C ␤-lactamases and two penicillin-binding proteins (PBPs) reveal that the enzymes have similar threedimensional structures in the vicinity of the active site. This similarity suggests that the BLIP peptide inhibitor may have a broad range of activity that can be used to develop novel small-molecule inhibitors of various classes of ␤-lactamases and PBPs.

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“…Crystallographic analyses of BLIP and TEM alone and in complex gave insights into the nature of the inhibitory interaction and defined a peptide loop exposed on the surface of BLIP which interacts with the active site of the ␤-lactamase (20). While the proteinaceous nature of BLIP limits its value as a therapeutic agent, the fact that BLIP is structurally unrelated to existing small-molecule ␤-lactamase inhibitors, such as clavulanic acid and tazobactam, suggests that new classes of ␤-lactamase inhibitors based on smallmolecule derivatives of BLIP might hold clinical potential (15)(16)(17). However, this theory is predicated upon the assumption that clavulanic acid-resistant forms of ␤-lactamases will not show a parallel resistance to inhibition by BLIP.…”

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confidence: 99%

Resistance to β-Lactamase Inhibitor Protein Does Not Parallel Resistance to Clavulanic Acid in TEM β-Lactamase Mutants

Schroeder

Locke

Jensen

2002

Antimicrob Agents Chemother

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In order to compare patterns of resistance to inhibition by clavulanic acid with patterns of resistance to inhibition by a ␤-lactamase inhibitor protein (BLIP), R164S, R244S, and R164S/R244S mutant forms of TEM ␤-lactamase were prepared by site-directed mutagenesis. When kinetic parameters were determined for these mutant and wild-type forms of TEM, the single mutants showed properties that were similar to those in the literature but the double mutant showed properties that were very different. The R164S/R244S double mutant form of TEM retained its resistance to inhibition by clavulanic acid (characteristic of the R244S mutation) but lost all its ability to hydrolyze ceftazidime (characteristic of the R164S mutation). While these characteristics are contrary to those previously reported for an R164S/R244S double mutant, this discrepancy resulted from the use of a defective mutant in the earlier study. Both the single and double mutant forms of TEM remained highly sensitive when tested for inhibition by BLIP, showing only slightly increased resistance compared to that of the wild type; this pattern of resistance is quite different from the pattern of clavulanic acid resistance. The slight increases in resistance to inhibition by BLIP seen in the mutants may have been related to the fact that all of the mutations effected changes in the net charge on the TEM protein that could impede interactions with BLIP.Bacterial resistance to antibiotics is an important and growing concern in the treatment of infectious diseases. Although ␤-lactam compounds still compose more than 50% of all prescribed antibiotics, the development of resistant strains represents a serious threat to the continued usefulness of these agents. In particular, the emergence of mutant forms of the ␤-lactamase TEM, the single most prevalent ␤-lactamase found in gram-negative bacteria, provides a striking example of the evolution of antibiotic resistance. In response to the appearance of microbial resistance mediated by TEM and other ␤-lactamases, aminothiazole oxime-containing, ␤-lactamaseresistant antibiotics such as ceftazidime, cefotaxime, and aztreonam were developed. As an alternative approach, ␤-lactamase inhibitors used in combination with conventional penicillins and cephalosporins were also introduced. However, the advent of each new control strategy was met with the emergence of mutant forms of TEM that are resistant to the new agent. The number of naturally occurring mutant forms of TEM now exceeds 100 (http://www.lahey.org/studies/temtable .htm). Although there are many examples of TEM mutants showing activity against extended-spectrum cephalosporins, and others that are insensitive to inhibition by clavulanic acid (3-6, 10, 12, 14; D. Sirot, C. Chanal, R. Bonnet, C. DeChamps, and J. Sirot, Letter, Antimicrob. Agents Chemother. 45:2179-2180, 2001), the coexistence of mutations associated with these resistance characteristics within a single enzyme is less common and typically results in the loss of one phenotype or the other (9, 13, 18). T...

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Identification of Residues in β-Lactamase Critical for Binding β-Lactamase Inhibitory Protein

Cited by 31 publications

References 35 publications

Dissecting the Protein-Protein Interface between β-Lactamase Inhibitory Protein and Class A β-Lactamases

Dissecting the Protein-Protein Interface between β-Lactamase Inhibitory Protein and Class A β-Lactamases

Binding Properties of a Peptide Derived from β-Lactamase Inhibitory Protein

Resistance to β-Lactamase Inhibitor Protein Does Not Parallel Resistance to Clavulanic Acid in TEM β-Lactamase Mutants

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