Identification and characterization of beta-lactamase inhibitor protein-II (BLIP-II) interactions with beta-lactamases using phage display.

Protein-protein interactions are critical to cellular processes yet the ability to predict and rationally design interactions is limited because of incomplete knowledge of the principles governing these interactions. The beta-lactamase inhibitory protein (BLIP)/beta-lactamase interaction has become a model system to investigate protein-protein interactions and has been the focus of several structural, thermodynamic and binding specificity studies. BLIP-II also inhibits beta-lactamase but has no sequence homology with BLIP. The structure of BLIP-II in complex with TEM-1 beta-lactamase revealed that BLIP-II has a completely different structure than BLIP but it interacts with the same protruding loop-helix region of TEM-1 as does BLIP. The significance of the individual interacting residues in molecular recognition by BLIP-II is currently unknown. Therefore, a phage display vector was developed with the purpose of expressing BLIP-II onto the surface of the M13 filamentous bacteriophage. The BLIP-II displayed phage bound to TEM-1 with picomolar affinity indicating that BLIP-II is properly folded while on the surface of the phage. The phage system, as well as enzyme inhibition assays with purified proteins, revealed that BLIP-II is a more potent inhibitor than BLIP for several class A beta-lactamases with K(i) values in the low picomolar range.

Binding properties of a peptide derived from beta-lactamase inhibitory protein.

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

Identification of residues critical for metallo-beta-lactamase function by codon randomization and selection.

IMP-1 beta-lactamase is a zinc metallo-enzyme encoded by the transferable bla(IMP-1) gene, which confers resistance to virtually all beta-lactam antibiotics including carbapenems. To understand how IMP-1 recognizes and hydrolyzes beta-lactam antibiotics it is important to determine which amino acid residues are critical for catalysis and which residues control substrate specificity. We randomized 27 individual codons in the bla(IMP-1) gene to create libraries that contain all possible amino acid substitutions at residue positions in and near the active site of IMP-1. Mutants from the random libraries were selected for the ability to confer ampicillin resistance to Escherichia coli. Of the positions randomized, >50% do not tolerate amino acid substitutions, suggesting they are essential for IMP-1 function. The remaining positions tolerate amino acid substitutions and may influence the substrate specificity of the enzyme. Interestingly, kinetic studies for one of the functional mutants, Asn233Ala, indicate that an alanine substitution at this position significantly increases catalytic efficiency as compared with the wild-type enzyme.

Amino acid sequence determinants of extended spectrum cephalosporin hydrolysis by the class C P99 beta-lactamase.

Class C beta-lactamases are commonly encoded on the chromosome of Gram-negative bacterial species. Mutations leading to increased expression of these enzymes are a common cause of resistance to many cephalosporins including extended spectrum cephalosporins. Recent reports of plasmid- and integrin-encoded class C beta-lactamases are a cause for concern because these enzymes are likely to spread horizontally to susceptible strains. Because of their increasing clinical significance, it is critical to identify the determinants of catalysis and substrate specificity of these enzymes. For this purpose, the codons of a set of 21 amino acid residues that encompass the active site region of the P99 beta-lactamase were individually randomized to create libraries containing all possible amino acid substitutions. The amino acid sequence requirements for the hydrolysis of ceftazidime, an extended spectrum cephalosporin commonly used to treat serious infections, were determined by selecting resistant mutants from each of the 21 libraries. DNA sequencing identified the residue positions that are critical for ceftazidime hydrolysis. In addition, it was found that certain amino acid substitutions in the omega-loop region of the P99 enzyme result in increased ceftazidime hydrolysis suggesting the loop is an important determinant of substrate specificity.

Protein minimization by random fragmentation and selection.

Protein-protein interactions are involved 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 protein inhibitors. BLIP is a 165 amino acid protein that is a potent inhibitor of TEM-1 beta-lactamase (K(i) = 0.1 nM). To aid in the development of new inhibitors of beta-lactamase, the gene encoding BLIP was randomly fragmented and DNA segments encoding peptides that retain the ability to bind TEM-1 beta-lactamase were isolated using phage display. The selected peptides revealed a common, overlapping region that includes BLIP residues C30-D49. Synthesis and binding analysis of the C30-D49 peptide indicate that this peptide inhibits TEM-1 beta-lactamase. Therefore, a peptide derivative of BLIP that has been reduced in size by 88% compared with wild-type BLIP retains the ability to bind and inhibit beta-lactamase.

A secondary drug resistance mutation of TEM-1 beta-lactamase that suppresses misfolding and aggregation.

In Gram-negative bacteria, TEM-1 beta-lactamase provides the major mechanism of plasmid-mediated beta-lactam resistance. Natural variants of TEM-1 with increased antibiotic resistance have appeared in response to the use of extended-spectrum beta-lactam antibiotics (e.g., ceftazidime) and beta-lactamase inhibitors (e.g., clavulanic acid). Some of the variant enzymes are more efficient at catalyzing beta-lactam hydrolysis, whereas others are more resistant to inhibitors. M182T is a substitution observed in both types of variant TEM-1 beta-lactamases. This mutation is found only in combination with other amino acid substitutions, suggesting that it may correct defects introduced by other mutations that alter the specificity. An engineered core mutation, L76N, which diminishes the periplasmic beta-lactamase activity by 100-fold, was used as a model to understand the mechanism of suppression of the M182T mutation. Biochemical studies of the L76N enzyme alone and in combination with the M182T mutation indicate that the M182T substitution acts at the level of folding but does not affect the thermodynamic stability of TEM-1 beta-lactamase. Thus, the M182T substitution is an example of a naturally occurring mutation that has evolved to alter the folding pathway of a protein and confer a selective advantage during the evolution of drug resistance.

Human immune response to streptococcal inhibitor of complement, a serotype M1 group A Streptococcus extracellular protein involved in epidemics.

Streptococcal inhibitor of complement (Sic) is a highly polymorphic extracellular protein made by serotype M1 group A Streptococcus strains that contributes to bacterial persistence in the mammalian upper respiratory tract. New variants of the Sic protein arise very rapidly by positive selection in human populations during M1 epidemics. The human antibody response to Sic was analyzed. Of 636 persons living in diverse localities, 43% had anti-Sic serum antibodies, but only 16.4% had anti-M1 protein serum antibody. Anti-Sic antibody was also present in nasal wash specimens in high frequency. Linear B cell epitope mapping showed that serum antibodies recognized epitopes located in structurally variable regions of Sic and the amino terminal hypervariable region of the M1 protein. Phage display analyses confirmed that the polymorphic regions of Sic are primary targets of host antibodies. These results support the hypothesis that selection of Sic variants occurs on mucosal surfaces by a mechanism that involves acquired host antibody.

Use of the arabinose p(bad) promoter for tightly regulated display of proteins on bacteriophage.

Phage display is a widely used method to optimize the binding characteristics of protein-ligand interactions. In addition, it has been used to clone genes from genomic and cDNA libraries based on their ligand-binding characteristics. One difficulty often encountered when expressing heterologous proteins by phage display is the toxicity of the protein on the Escherichia coli host. Previous studies have shown that heterologous protein expression can be tightly controlled using plasmids with the P(BAD) promoter of the arabinose operon of E. coli, and the araC gene, which is both a positive and negative regulator of the promoter. We constructed a set of phage display vectors that utilize the P(BAD) promoter to control the expression of proteins on the surface of the M13 bacteriophage. These vectors exhibit tightly controlled expression of proteins on the surface of the phage. In addition, the amount of protein displayed on the phage is modulated by the amount of arabinose present in the growth medium during phage propagation. This may be useful for altering the stringency of binding enrichment during phage display.

Structure-function analysis of alpha-helix H4 using PSE-4 as a model enzyme representative of class A beta-lactamases.

We extracted maximum information for structure-function analysis of the PSE-4 class A beta-lactamase by random replacement mutagenesis of three contiguous codons in the H4 alpha-helix at amino acid positions Ala125, Thr126, Met127, Thr128 and Thr129. These positions were predicted to interact with suicide mechanism-based inhibitors when examining the PSE-4 three-dimensional model. Structure-function studies on positions 125-129 indicated that in PSE-4 these amino acids have a role distinct from those in TEM-1, in tolerating substitutions at Ala125 and being invariant at Met127. The importance of Met127 was suspected to be implicated in a structural role in maintaining the integrity of the H4 alpha-helix structure together, thus maintaining the important Ser130-Asp131-Asn132 motif positioned towards the active site. At the structural level, the H4 region was analyzed using energy minimization of the H4 regions of the PSE-4 YAM mutant and compared with wild-type PSE-4. The Tyr 125 of the mutant YAM formed an edge to face pi-pi interaction with Phe 124 which also interacts with the Trp 210 with the same interactions. Antibiotic susceptibilities showed that amino acid changes in the the H4 alpha-helix region of PSE-4 are particularly sensitive to mechanism based-inhibitors. However, kinetic analysis of PSE-4 showed that the two suicide inhibitors belonging to the penicillanic acid sulfone class, sulbactam and tazobactam, were less affected by changes in the H4 alpha-helix region than clavulanic acid, an inhibitor of the oxypenam class. The analysis of H4 alpha-helix in PSE-4 suggests its importance in interactions with the three clinically useful inhibitors and in general to all class A enzymes.

Design of potent beta-lactamase inhibitors by phage display of beta-lactamase inhibitory protein.

Beta-lactamase inhibitory protein (BLIP) binds tightly to several beta-lactamases including TEM-1 beta-lactamase (K(i) 0.1 nm). The TEM-1 beta-lactamase/BLIP co-crystal structure indicates that two turn regions in BLIP insert into the active site of beta-lactamase to block the binding of beta-lactam antibiotics. Residues from each turn, Asp(49) and Phe(142), mimic interactions made by penicillin G when bound in the beta-lactamase active site. Phage display was used to determine which residues within the turn regions of BLIP are critical for binding TEM-1 beta-lactamase. The sequences of a set of functional mutants from each library indicated that a few sequence types were predominant. These BLIP mutants exhibited K(i) values for beta-lactamase inhibition ranging from 0.01 to 0.2 nm. The results indicate that even though BLIP is a potent inhibitor of TEM-1 beta-lactamase, the wild-type sequence of the active site binding region is not optimal and that derivatives of BLIP that bind beta-lactamase extremely tightly can be obtained. Importantly, all of the tight binding BLIP mutants have sequences that would be predicted theoretically to form turn structures.

Susceptibility of beta-lactamase to core amino acid substitutions.

To determine which amino acids in TEM-1 beta-lactamase are important for its structure and function, random libraries were previously constructed which systematically randomized the 263 codons of the mature enzyme. A comprehensive screening of these libraries identified several TEM-1 beta-lactamase core positions, including F66 and L76, which are strictly required for wild-type levels of hydrolytic activity. An examination of positions 66 and 76 in the class A beta-lactamase gene family shows that a phenylalanine at position 66 is strongly conserved while position 76 varies considerably among other beta-lactamases. It is possible that position 76 varies in the gene family because beta-lactamase mutants with non-conservative substitutions at position 76 retain partial function. In contrast, position 66 may remain unchanged in the gene family because non-conservative substitutions at this location are detrimental for enzyme structure and function. By determining the beta-lactam resistance levels of the 38 possible mutants at positions 66 and 76 in the TEM-1 enzyme, it was confirmed that position 76 is indeed more tolerant of non-conservative substitutions. An analysis of the Protein Data Bank files for three class A beta-lactamases indicates that volume constraints at position 66 are at least partly responsible for the low tolerance of substitutions at this position.

Identification of residues in beta-lactamase critical for binding beta-lactamase inhibitory protein.

beta-Lactamase inhibitory protein (BLIP) is a potent inhibitor of several beta-lactamases including TEM-1 beta-lactamase (Ki = 0.1 nM). The co-crystal structure of TEM-1 beta-lactamase and BLIP has been solved, revealing the contact residues involved in the interface between the enzyme and inhibitor. To determine which residues in TEM-1 beta-lactamase are critical for binding BLIP, the method of monovalent phage display was employed. Random mutants of TEM-1 beta-lactamase in the 99-114 loop-helix and 235-240 B3 beta-strand regions were displayed as fusion proteins on the surface of the M13 bacteriophage. Functional mutants were selected based on the ability to bind BLIP. After three rounds of enrichment, the sequences of a collection of functional beta-lactamase mutants revealed a consensus sequence for the binding of BLIP. Seven loop-helix residues including Asp-101, Leu-102, Val-103, Ser-106, Pro-107, Thr-109, and His-112 and three B3 beta-strand residues including Ser-235, Gly-236, and Gly-238 were found to be critical for tight binding of BLIP. In addition, the selected beta-lactamase mutants A113L/T114R and E240K were found to increase binding of BLIP by over 6- and 11-fold, respectively. Combining these substitutions resulted in 550-fold tighter binding between the enzyme and BLIP with a Ki of 0.40 pM. These results reveal that the binding between TEM-1 beta-lactamase and BLIP can be improved and that there are a large number of sequences consistent with tight binding between BLIP and beta-lactamase.

Contributions of aspartate 49 and phenylalanine 142 residues of a tight binding inhibitory protein of beta-lactamases.

beta-Lactamases are bacterial enzymes that hydrolyze beta-lactam antibiotics to render them inactive. The beta-lactamase inhibitor protein (BLIP) of Streptomyces clavuligerus, is a potent inhibitor of several beta-lactamases, including the TEM-1 enzyme (Ki = 0.6 nM). Evidence from the TEM-1/BLIP co-crystal suggests that two BLIP residues, Asp-49 and Phe-142, mimic interactions made by penicillin G when bound in the active site of TEM-1. To determine the importance of these two residues, a heterologous expression system for BLIP was established in Escherichia coli. Site-directed mutagenesis was used to change Asp-49 and Phe-142 to alanine, and inhibition constants (Ki) for both mutants were determined. Each mutation increases the Ki for BLIP inhibition of TEM-1 beta-lactamase approximately 100-fold. To address how these two positions effect the specificity of beta-lactamase binding, Ki values were determined for the interaction of wild-type BLIP, as well as the D49A and F142A mutants, with two extended spectrum beta-lactamases (the G238S and the E104K TEM variants). Positions 104 and 238 are located in the BLIP/beta-lactamase interface. Interestingly, the three BLIP proteins inhibited the G238S beta-lactamase mutant to the same degree that they inhibited TEM-1. However, wild-type BLIP has a higher Ki for the E104K beta-lactamase mutant, suggesting that interactions between BLIP and beta-lactamase residue Glu-104 are important for wild-type levels of BLIP inhibition.

Mapping protein-ligand interactions using whole genome phage display libraries.

The function of many genes cannot be deduced from sequence similarity, and biochemical methods are usually required. Whole genome sequences can be thought of as not only a set of genes but also collections of functional domains. These domains can be studied by affinity methods whereby identification of the ligand can provide information on biochemical function. To take advantage of this method, one must express all functional domains in a form suitable for affinity studies. Phage display technology provides a means for accomplishing this. The pJuFo phage display system, based on the interaction between the leucine zippers Jun and Fos, has been modified and used to create a genomic phage display library from Escherichia coli MG1655. The system has been tested by using the library to map the dominant binding epitopes for an anti-RecA protein polyclonal antibody sera. This methodology provides a general biochemical approach to functional analysis of protein-ligand interactions on a genomewide basis.

Display of functional beta-lactamase inhibitory protein on the surface of M13 bacteriophage.

The display of proteins on the surface of filamentous phage has been shown to be a powerful method to select variants of a protein with altered binding properties from large combinatorial libraries of mutants. The beta-lactamase inhibitory protein (BLIP) is a 165-amino-acid protein that binds and inhibits TEM-1 beta-lactamase-catalyzed hydrolysis of the penicillin and cephalosporin antibiotics. Here we describe the construction of a new phagemid vector and the use of this vector to display BLIP on the surface of filamentous phage. It is shown that BLIP-displaying phage bind to immobilized beta-lactamase and that the binding can be competed off by the addition of soluble beta-lactamase. In addition, a two-step phage enzyme-linked immunosorbent assay procedure was used to demonstrate that the BLIP-displaying phage bind beta-lactamase with a 50% inhibitory concentration of 1 nM, which compares favorably with a previously published Ki of 0.6 nM. A system has therefore been established for protein engineering of BLIP to expand its range of binding to other beta-lactamases and penicillin-binding proteins.

Roles of amino acids 161 to 179 in the PSE-4 omega loop in substrate specificity and in resistance to ceftazidime.

The PSE-4 enzyme is a prototype carbenicillin-hydrolyzing enzyme exhibiting high activity against penicillins and early cephalosporins. To understand the mechanism that modulates substrate profiles and to verify the ability of PSE-4 to extend its substrate specificity toward expanded-spectrum cephalosporins, we used random replacement mutagenesis to generate six random libraries from amino acids 162 to 179 in the Omega loop. This region is known from studies with TEM-1 to be implicated in substrate specificity. It was found that the mechanism modulating ceftazidime hydrolysis in PSE-4 was different from that in TEM-1. The specificity of class 2c carbenicillin-hydrolyzing enzymes could not be assigned to the Omega loop of PSE-4. Analysis of the percentage of functional enzymes revealed that the hydrolysis of ampicillin was more affected than hydrolysis of carbenicillin by amino acid substitutions at positions 162 to 164 and 165 to 167.

The role of residue 238 of TEM-1 beta-lactamase in the hydrolysis of extended-spectrum antibiotics.

beta-Lactamases inactivate beta-lactam antibiotics by catalyzing the hydrolysis of the amide bond in the beta-lactam ring. The plasmid-encoded class A TEM-1 beta-lactamase is a commonly encountered beta-lactamase. It is able to inactivate penicillins and cephalosporins but not extended-spectrum antibiotics. However, TEM-1-derived natural variants containing the G238S amino acid substitution display increased hydrolysis of extended-spectrum antibiotics. Two models have been proposed to explain the role of the G238S substitution in hydrolysis of extended-spectrum antibiotics. The first proposes a direct hydrogen bond of the Ser238 side chain to the oxime group of extended-spectrum antibiotics. The second proposes that steric conflict with surrounding residues, due to increased side chain volume, leads to a more accessible active site pocket. To assess the validity of each model, TEM-1 mutants with amino acids substitutions of Ala, Ser, Cys, Thr, Asn, and Val have been constructed. Kinetic analysis of these enzymes with penicillins and cephalosporins suggests that a hydrogen bond is necessary but not sufficient to achieve the hydrolytic activity of the G238S enzyme for the extended-spectrum antibiotics cefotaxime and ceftazidime. In addition, it appears that the new hydrogen bond interaction is to a site on the enzyme rather than directly to the extended-spectrum antibiotic. The data indicate that, for the G238S substitution, a combination of an optimal side chain volume and hydrogen bonding potential results in the most versatile and advantageous antibiotic hydrolytic spectrum for bacterial resistance to extended-spectrum antibiotics.

beta-Lactamases: protein evolution in real time.

The evolution and spread of bacteria resistant to beta-lactam antibiotics has progressed at an alarming rate. Bacteria may acquire resistance to a given drug by mutation of pre-existing genes or by the acquisition of new genes from other bacteria. One ongoing example of these mechanisms is the evolution of new variants of the TEM and SHV beta-lactamases with altered substrate specificity.

Characterization of a PSE-4 mutant with different properties in relation to penicillanic acid sulfones: importance of residues 216 to 218 in class A beta-lactamases.

Class A beta-lactamases are inactivated by the suicide inactivators sulbactam, clavulanic acid, and tazobactam. An examination of multiple alignments indicated that amino acids 216 to 218 differed among class A enzymes. By random replacement mutagenesis of codons 216 to 218 in PSE-4, a complete library consisting of 40,864 mutants was created. The library of mutants with mutations at positions 216 to 218 in PSE-4 was screened on carbenicillin and ampicillin with the inactivator sulbactam; a collection of 14 mutants was selected, and their bla genes were completely sequenced. Purified wild-type and mutant PSE-4 beta-lactamases were used to measure kinetic parameters. One enzyme, V216S:T217A:G218R, was examined for its peculiar pattern of inhibition. There was an increase in the Km from 68 microM for the wild type to 271 microM for the mutant for carbenicillin and 33 to 216 microM for ampicillin. Relative to the wild-type PSE-4 enzyme, 37- and 30-fold increases in Ki values were observed for the mutant enzyme for sulbactam and tazobactam, respectively. The results that were obtained suggested that positions 216 to 218 are important for interactions with penicillanic acid sulfone inhibitors. In contrast, V216 and A217 in the TEM-1 class A beta-lactamase do not tolerate amino acid residue substitutions. However, for the PSE-4 beta-lactamase, 11 of 14 mutants from the library of mutants with mutations at positions 216 to 218 whose sequences were determined had substitutions at position 216 (G, R, A, S) and position 217 (A, S). The data showed the importance of residues 216 to 218 in their atomic interactions with inactivators in the PSE-4 beta-lactamase structure.