Beta-lactamase Inhibitor Protein Research Articles

(Invited) Predicting the Response of Carbon Nanotube Field-Effect Biosensors Using Simulations

Objectives Carbon nanotube field-effect biosensors (bioCNTFETs) are a promising platform to detect biomolecular analytes because they are highly sensitive, specific, low-cost, portable, and integrable into arrays for multiplexed sensing [1]. Their sensitivity originates from carbon nanotubes having electrical properties that are strongly dependent on nearby biomolecules. As a result, the biosensor detects an electrical current through the network of nanotubes that depends on the concentration of analyte. To achieve specificity, carbon nanotubes need to be functionalized with a probe molecule that binds to a specific target analyte. However, it has been demonstrated that the response of bioCNTFETs strongly depends on the attachment site on the probe molecule to the carbon nanotube [2], and this variability is impairing the development of bioCNTFETs.To address this challenge, we are developing an integrated approach combining simulation and experiment: simulations are used to predict which attachment sites on the binding protein are expected to generate the greatest response, then devices are fabricated using phenyl azide photochemistry to precisely attach the binding protein via these sites on the nanotubes. To validate our approach, we characterized a bioCNTFET covalently functionalized with the beta-lactamase inhibitor protein II (BLIP-II) to detect beta-lactamases such as TEM-1 and KPC-2 that are associated with antimicrobial resistance. Results Our simulation protocol is based on molecular dynamics to sample the biomolecule-nanotube interactions and then on Poisson-Boltzmann calculations to estimate the electrostatic potential generated on the nanotube’s surface by the conformations of the biomolecule [3]. Since electrostatic potential changes on the surface of the nanotubes in the BLIP-II biosensor are expected to cause the electrical signal changes detected, we specifically quantify that as TEM-1 or KPC-2 binds to the covalently attached BLIP-II to the nanotube. We looked at two different attachment sites on BLIP-II – A41 and T213 – that are expected to produce a different response. For the A41 site, we show that the electrostatic potential doesn’t change significantly as TEM-1 or KPC-2 binds to BLIP-II because they are too far from the nanotube. However, the T213 site allows significant electrostatic potential changes: TEM-1 shifts the potential to more negative values, while KPC-2 shifts the potential to less negative values.The predictions from our simulations agree with measurements done on devices. For the A41 site, the measured conductance doesn’t change significantly upon binding of TEM-1 or KPC-2, indicating that the electrostatic potential on the nanotube doesn’t change much, as predicted. For the T213 site, the measured conductance increases significantly upon binding of TEM-1, while it decreases significantly for KPC-2. This suggests more p-type carriers in the nanotube (due to a more negative potential on the nanotube) as TEM-1 binds, but less p-type carriers (due to a less negative potential) as KPC-2 binds, as predicted. Conclusions We have developed a simulation protocol that predicts, in agreement with experimental measurements, the impact of the probe’s attachment site on the conductance modulation detected by carbon-nanotube field-effect biosensors. This supports the development of a truly integrated approach combining simulation and experiment to consistently design and fabricate these biosensors to achieve optimized sensitivity.

Rheostat positions: A new classification of protein positions relevant to pharmacogenomics.

To achieve the full potential of pharmacogenomics, one must accurately predict the functional outcomes that arise from amino acid substitutions in proteins. Classically, researchers have focused on understanding the consequences of individual substitutions. However, literature surveys have shown that most substitutions were created at evolutionarily conserved positions. Awareness of this bias leads to a shift in perspective, from considering the outcomes of individual substitutions to understanding the roles of individual protein positions. Conserved positions tend to act as “toggle” switches, with most substitutions abolishing function. However, nonconserved positions have been found equally capable of affecting protein function. Indeed, many nonconserved positions act like functional dimmer switches (“rheostat” positions): this is revealed when multiple substitutions are made at a single position. Each substitution has a different functional outcome; the set of substitutions spans a range of outcomes. Finally, some nonconserved positions appear neutral, capable of accommodating all amino acid types without modifying function. This paper reviews the currently-known properties of rheostat positions, with examples shown for pyruvate kinase, organic anion transporting polypeptide 1B1, the beta-lactamase inhibitory protein, and angiotensin-converting enzyme 2. Outcomes observed for rheostat positions have implications for the rational design of drug analogs and allosteric drugs. Furthermore, this new framework—comprising three types of protein positions—provides a new approach to interpreting disease and population-based databases of amino acid changes. In conclusion, although a full understanding of substitution outcomes at rheostat positions poses a challenge, utilization of this new frame of reference will further advance the application of pharmacogenomics.

Insight into Structure-Function Relationships of β-Lactamase and BLIPs Interface Plasticity using Protein-Protein Interactions.

Mostly BLIPs are identified in soil bacteria Streptomyces and originally isolated from Streptomyces clavuligerus and can be utilized as a model system for biophysical, structural, mutagenic and computational studies. BLIP possess homology with two proteins viz., BLIP-I (Streptomyces exofoliatus) and BLP (beta-lactamase inhibitory protein like protein from S. clavuligerus). BLIP consists of 165 amino acid, possessing two homologues domains comprising helix-loop-helix motif packed against four stranded beta-sheet resulting into solvent exposed concave surface with extended four stranded beta-sheet. BLIP-I is a 157 amino acid long protein obtained from S. exofoliatus having 37% sequence identity to BLIP and inhibits beta-lactamase. This review is intended to briefly illustrate the beta-lactamase inhibitory activity of BLIP via proteinprotein interaction and aims to open up a new avenue to combat antimicrobial resistance using peptide based inhibition. D49A mutation in BLIP-I results in a decrease in affinity for TEM-1 from 0.5 nM to 10 nM (Ki). It is capable of inhibiting TEM-1 and bactopenemase and differs from BLIP only in modulating cell wall synthesis enzyme. Whereas, BLP is a 154 amino acid long protein isolated from S. clavuligerus via DNA sequencing analysis of Cephamycin-Clavulanate gene bunch. It shares 32% sequence similarity with BLIP and 42% with BLIP-I. Its biological function is unclear and lacks beta-lactamase inhibitory activity. Protein-protein interactions mediate a significant role in regulation and modulation of cellular developments and processes. Specific biological markers and geometric characteristics are manifested by active site binding clefts of protein surfaces which determines the specificity and affinity for their targets. TEM1.BLIP is a classical model to study protein-protein interaction. β-Lactamase inhibitory proteins (BLIPs) interacts and inhibits various β-lactamases with extensive range of affinities.

A novel chimeric peptide with antimicrobial activity.

Beta-lactamase-mediated bacterial drug resistance exacerbates the prognosis of infectious diseases, which are sometimes treated with co-administration of beta-lactam type antibiotics and beta-lactamase inhibitors. Antimicrobial peptides are promising broad-spectrum alternatives to conventional antibiotics in this era of evolving bacterial resistance. Peptides based on the Ala46-Tyr51 beta-hairpin loop of beta-lactamase inhibitory protein (BLIP) have been previously shown to inhibit beta-lactamase. Here, our goal was to modify this peptide for improved beta-lactamase inhibition and cellular uptake. Motivated by the cell-penetrating pVEC sequence, which includes a hydrophobic stretch at its N-terminus, our approach involved the addition of LLIIL residues to the inhibitory peptide N-terminus to facilitate uptake. Activity measurements of the peptide based on the 45-53 loop of BLIP for enhanced inhibition verified that the peptide was a competitive beta-lactamase inhibitor with a K(i) value of 58 μM. Incubation of beta-lactam-resistant cells with peptide decreased the number of viable cells, while it had no effect on beta-lactamase-free cells, indicating that this peptide had antimicrobial activity via beta-lactamase inhibition. To elucidate the molecular mechanism by which this peptide moves across the membrane, steered molecular dynamics simulations were carried out. We propose that addition of hydrophobic residues to the N-terminus of the peptide affords a promising strategy in the design of novel antimicrobial peptides not only against beta-lactamase but also for other intracellular targets.

Communication between the active site and the allosteric site in class A beta-lactamases

Bacterial production of beta-lactamases, which hydrolyze beta-lactam type antibiotics, is a common antibiotic resistance mechanism. Antibiotic resistance is a high priority intervention area and one strategy to overcome resistance is to administer antibiotics with beta-lactamase inhibitors in the treatment of infectious diseases. Unfortunately, beta-lactamases are evolving at a rapid pace with new inhibitor resistant mutants emerging every day, driving the design and development of novel beta-lactamase inhibitors. Here, we examined the inhibitor recognition mechanism of two common beta-lactamases using molecular dynamics simulations. Binding of beta-lactamase inhibitor protein (BLIP) caused changes in the flexibility of regions away from the binding site. One of these regions was the H10 helix, which was previously identified to form a lid over an allosteric inhibitor binding site. Closer examination of the H10 helix using sequence and structure comparisons with other beta-lactamases revealed the presence of a highly conserved Trp229 residue, which forms a stacking interaction with two conserved proline residues. Molecular dynamics simulations on the Trp229Ala mutants of TEM-1 and SHV-1 resulted in decreased stability in the apo form, possibly due to loss of the stacking interaction as a result of the mutation. The mutant TEM-1 beta-lactamase had higher H10 fluctuations in the presence of BLIP, higher affinity to BLIP and higher cross-correlations with BLIP. Our results suggest that the H10 helix and specifically W229 are important modulators of the allosteric communication between the active site and the allosteric site.

Communication Between an Allosteric Inhibitor Binding Site and the Active Site in Beta-Lactamase

Beta-lactamases hydrolyze the beta-lactam ring of antibiotics, rendering them ineffective. Understanding the inhibitor recognition mechanism of beta-lactamases will give important information toward the fight against beta-lactamase mediated antibiotic resistance. Most inhibitors of beta-lactamase bind to the active site and thus inhibit beta-lactamase in a competitive manner. An allosteric inhibitor binding site, near the H10 helix (residues 218-230), has also been discovered in TEM-1 and KPC-2 beta-lactamases. Multiple sequence alignment of beta-lactamases shows that Trp 229, which resides on the H10 helix, is a highly conserved residue within the beta-lactamase family. In this study, computational mutagenesis of Trp 229 on two clinically relevant beta-lactamases, TEM-1 and SHV-1, was performed. Beta-lactamase inhibitor protein (BLIP) is known to bind TEM-1 with 1000 fold higher affinity than it binds to SHV-1. Molecular dynamics simulations were carried out on the unbound and BLIP bound forms of the wild type and W229A mutant beta-lactamases. The simulation trajectories were analyzed to obtain information about the changes in mobility. The binding free energies of the beta-lactamase - BLIP complexes were obtained and the individual energy terms contributing to the difference in affinity in the different mutants were determined. The results were compared for TEM-1 and SHV-1 to elucidate the difference in affinity toward BLIP. The change in binding dynamics upon mutation of Trp 229 was examined.

Identification and characterization of β-lactamase inhibitor protein-II (BLIP-II) interactions with β-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.

Structural and biochemical characterization of the interaction between KPC-2 beta-lactamase and beta-lactamase inhibitor protein.

KPC beta-lactamases hydrolyze the "last resort" beta-lactam antibiotics (carbapenems) used to treat multidrug resistant infections and are compromising efforts to combat life-threatening Gram-negative bacterial infections in hospitals worldwide. Consequently, the development of novel inhibitors is essential for restoring the effectiveness of existing antibiotics. The beta-lactamase inhibitor protein (BLIP) is a competitive inhibitor of a number of class A beta-lactamases. In this study, we characterize the previously unreported interaction between KPC-2 beta-lactamase and BLIP. Biochemical results show that BLIP is an extremely potent inhibitor of KPC enzymes, binding KPC-2 and KPC-3 with subnanomolar affinity. To understand the basis of affinity and specificity in the beta-lactamase-BLIP system, the crystallographic structure of the KPC-2-BLIP complex was determined to 1.9 A resolution. Computational alanine scanning was also conducted to identify putative hot spots in the KPC-2-BLIP interface. Interestingly, the two complexes making up the KPC-2-BLIP asymmetric unit are distinct, and in one structure, the BLIP F142 loop is absent, in contrast to homologous structures in which it occupies the active site. This finding and other sources of structural plasticity appear to contribute to BLIP's promiscuity, enabling it to respond to mutations at the beta-lactamase interface. Given the continuing emergence of antibiotic resistance, the high-resolution KPC-2-BLIP structure will facilitate its use as a template for the rational design of new inhibitors of this problematic enzyme.

Fine Mapping of the Sequence Requirements for Binding of β-Lactamase Inhibitory Protein (BLIP) to TEM-1 β-Lactamase Using a Genetic Screen for BLIP Function

Beta-lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A beta-lactamases with a wide range of affinities. Alanine-scanning mutagenesis was previously performed to identify the amino acid sequence requirements of BLIP for binding the TEM-1, SME-1, SHV-1, and Bla1 beta-lactamases. Twenty-three BLIP residues that contact TEM-1 beta-lactamase in the structure of the complex were mutated to alanine and assayed for inhibition (K(i)) of beta-lactamase to identify two hotspots of binding energy. These studies have been extended by the development of a genetic screen for BLIP function in Escherichia coli. The bla(TEM-1) gene encoding TEM-1 beta-lactamase was inserted into the E. coli pyrF chromosomal locus. Expression of wild-type BLIP from a plasmid in this strain resulted in a large decrease in ampicillin resistance, while introduction of the same plasmid lacking BLIP had no effect on ampicillin resistance. In addition, it was found that when the BLIP alanine-scanning mutants were tested in the strain, the level of ampicillin resistance was proportional to the K(i) of the BLIP mutant. These results indicate that BLIP function can be monitored by the level of ampicillin resistance of the genetic test strain. Each of the 23 BLIP positions examined by alanine scanning was randomized to create libraries containing all possible substitutions at each position. The genetic screen for BLIP function was used to sort the libraries for active mutants, and DNA sequence analysis of functional BLIP mutants identified the sequences required for binding TEM-1 beta-lactamase. The results indicate the BLIP surface is tolerant of substitutions in that many contact positions can be substituted with other amino acid types and retain wild-type levels of function.

Dynamic Analysis of Beta-lactamase Ligand Recognition

A serious public health threat today is the emergence of pathogens that are resistant to commonly used antibiotics. One of the mechanisms of acquired drug resistance is the bacterial production of beta-lactamases, which break down these antibiotics. Currently used beta-lactamase inhibitors are not effective at targeting the 700 types and new mutants of beta-lactamases. Beta lactamase is therefore an important drug target in combating antibiotic resistance. Beta lactamase inhibitory protein (BLIP) is an effective inhibitor of TEM-1 and SHV-1, but binds and inhibits the two variants with different affinities. We hypothesize that elucidating the mechanism whereby the differential binding results will guide the design of new peptide inhibitors based on the BLIP structure. Molecular dynamics simulations are performed to examine the binding properties of BLIP and BLIP based peptides to TEM-1 and SHV-1 beta lactamase. These simulations on the complex will guide the design of new peptides.

Modeling of Protein Adsorption on a Metal Surface: Brownian Dynamics Simulations

The ability of proteins to bind selectively to different kinds of solid surfaces is widely used in advanced technologies in medicine, pharmacy, nanodevices and bioengineering. However, experimental data on the interfacial behavior of proteins is limited and our knowledge of the driving forces for protein-solid surface binding is still very poor. The present study is aimed at building a computational model for understanding and predicting the chemical and physical properties of protein-solid surface systems.As a test example, adsorption of the BLIP (beta-lactamase inhibitor protein) protein fused with different homotripeptides to the gold surface is explored. The computational algorithm is based on Brownian dynamics simulations of a protein in the presence of a metal surface with interactions described by electrostatic, Lennard-Jones (LJ), and desolvation energy terms. The interatomic LJ potential describes both the van der Waals and the chemical interaction between amino acids and the gold surface with parameters derived from ab initio calculations and experimental data. The desolvation term includes protein desolvation as well as surface water desorption effects. The results of the computer simulations are compared with the experimentally observed binding characteristics of the systems under consideration.We thank our partners in the Prosurf project, in particular G. Schreiber and D. Reichmann for sharing experimental data, K. Gottschak and M. Hofling for caring out MD simulations, and S. Corni and F. Iori for providing force field parameters for peptides on the gold surface.This project is carried out with financial support from KTS Klaus Tschira Foundation gGmbH and the European Community.

The solution structure of the outer membrane lipoprotein OmlA from Xanthomonas axonopodis pv. citri reveals a protein fold implicated in protein–protein interaction

The outer membrane lipoprotein A (OmlA) belongs to a family of bacterial small lipoproteins widely distributed across the beta and gamma proteobacteria. Although the role of numerous bacterial lipoproteins is known, the biological function of OmlA remains elusive. We found that in the citrus canker pathogen, Xanthomonas axonopodis pv. citri (X. citri), OmlA is coregulated with the ferric uptake regulator (Fur) and their expression is enhanced when X. citri is grown on citrus leaves, suggesting that these proteins are involved in plant-pathogen interaction. To gain insights into the function of OmlA, its conformational and dynamic features were determined by nuclear magnetic resonance. The protein has highly flexible N- and C- termini and a structurally well defined core composed of three beta-strands and two small alpha-helices, which pack against each other forming a two-layer alpha/beta scaffold. This protein fold resembles the domains of the beta-lactamase inhibitory protein BLIP, involved in protein-protein binding. In conclusion, the structure of OmlA does suggest that this protein may be implicated in protein-protein interactions required during X. citri infection.

Action‐at‐a‐distance interactions enhance protein binding affinity

The identification of protein mutations that enhance binding affinity may be achieved by computational or experimental means, or by a combination of the two. Sources of affinity enhancement may include improvements to the net balance of binding interactions of residues forming intermolecular contacts at the binding interface, such as packing and hydrogen-bonding interactions. Here we identify noncontacting residues that make substantial contributions to binding affinity and that also provide opportunities for mutations that increase binding affinity of the TEM1 beta-lactamase (TEM1) to the beta-lactamase inhibitor protein (BLIP). A region of BLIP not on the direct TEM1-binding surface was identified for which changes in net charge result in particularly large increases in computed binding affinity. Some mutations to the region have previously been characterized, and our results are in good correspondence with this results of that study. In addition, we propose novel mutations to BLIP that were computed to improve binding significantly without contacting TEM1 directly. This class of noncontacting electrostatic interactions could have general utility in the design and tuning of binding interactions.

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

beta-Lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A beta-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 beta-lactamase and SME-1 beta-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 beta-lactamase, which is a poor binding substrate (Ki= 1.1 microm), and the Bacillus anthracis Bla1 enzyme (Ki= 2.5 nm). The two hotspots previously identified as important for binding TEM-1 and SME-1 beta-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 beta-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 beta-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 beta-lactamase.

Determinants of Binding Affinity and Specificity for the Interaction of TEM-1 and SME-1 β-Lactamase with β-Lactamase Inhibitory Protein

The hydrolysis of beta-lactam antibiotics by class A beta-lactamases is a common cause of bacterial resistance to these agents. The beta-lactamase inhibitory protein (BLIP) is able to bind and inhibit several class A beta-lactamases, including TEM-1 beta-lactamase and SME-1 beta-lactamase. Although the TEM-1 and SME-1 enzymes share 33% amino acid sequence identity and a similar fold, they differ substantially in surface electrostatic properties and the conformation of a loop-helix region that BLIP binds. Alanine-scanning mutagenesis was performed to identify the residues on BLIP that contribute to its binding affinity for each of these enzymes. The results indicate that the sequence requirements for binding are similar for both enzymes with most of the binding free energy provided by two patches of aromatic residues on the surface of BLIP. Polar residues such as several serines in the interface do not make significant contributions to affinity for either enzyme. In addition, the specificity of binding is significantly altered by mutation of two charged residues, Glu73 and Lys74, that are buried in the structure of the TEM-1.BLIP complex as well as by residues located on two loops that insert into the active site pocket. Based on the results, a E73A/Y50A double mutant was constructed that exhibited a 220,000-fold change in binding specificity for the TEM-1 versus SME-1 enzymes.

Beta-turn formation by a six-residue linear peptide in solution.

A model peptide AAGDYY-NH2 (B1), which is found to adopt a beta-turn conformation in the TEM-1 beta-lactamase inhibitor protein (BLIP) in the TEM-1/BLIP co-crystal, was synthesized to elucidate the mechanism of its beta-turn formation and stability. Its structural preferences in solution were comprehensively characterized using CD, FT-IR and 1H NMR spectroscopy, respectively. The set of observed diagnostic NOEs, the restrained molecular dynamics simulation, CD and FT-IR spectroscopy confirmed the formation of a beta-turn in solution by the model peptide. The dihedral angles [(phi3, phi3) (phi4, phi4)] of [(-52 degrees, -32 degrees ) (-38 degrees, -44 degrees )] of Gly-Asp fragment in the model peptide are consistent with those of a type III beta-turn. In a conclusion, the conformational preference of the linear hexapeptide B1 in solution was determined, and it would provide a simple template to study the mechanism of beta-turn formation and stability.

In vitro selection for catalytic activity with ribosome display.

We report what is, to our knowledge, the first in vitro selection for catalytic activity based on catalytic turnover by using ribosome display, a method which does not involve living cells at any step. RTEM-beta-lactamase was functionally displayed on ribosomes as a complex with its encoding mRNA. We designed and synthesized a mechanism-based inhibitor of beta-lactamase, biotinylated ampicillin sulfone, appropriate for selection of catalytic activity of the ribosome-displayed beta-lactamase. This derivative of ampicillin inactivated beta-lactamase in a specific and irreversible manner. Under appropriate selection conditions, active RTEM-beta-lactamase was enriched relative to an inactive point mutant over 100-fold per ribosome display selection cycle. Selection for binding, carried out with beta-lactamase inhibitory protein (BLIP), gave results similar to selection with the suicide inhibitor, indicating that ribosome display is similarly efficient in catalytic activity and affinity selections. In the future, the capacity to select directly for enzymatic activity using an entirely in vitro process may allow for a significant increase in the explorable sequence space relative to existing strategies.

Crystal structure and kinetic analysis of beta-lactamase inhibitor protein-II in complex with TEM-1 beta-lactamase.

The structure of the 28 kDa beta-lactamase inhibitor protein-II (BLIP-II) in complex with the TEM-1 beta-lactamase has been determined to 2.3 A resolution. BLIP-II is a secreted protein produced by the soil bacterium Streptomyces exfoliatus SMF19 and is able to bind and inhibit TEM-1 with subnanomolar affinity. BLIP-II is a seven-bladed beta-propeller with a unique blade motif consisting of only three antiparallel beta-strands. The overall fold is highly similar to the core structure of the human regulator of chromosome condensation (RCC1). Although BLIP-II does not share the same fold with BLIP, the first beta-lactamase inhibitor protein for which structural data was available, a comparison of the two complexes reveals a number of similarities and provides further insights into key components of the TEM-1-BLIP and TEM-1-BLIP-II interfaces. Our preliminary results from gene knock-out studies and scanning electron microscopy also reveal a critical role of BLIP-II in sporulation.

New β-Lactamase Inhibitory Protein (BLIP-I) from Streptomyces exfoliatus SMF19 and Its Roles on the Morphological Differentiation

A new beta-lactamase inhibitory protein (BLIP-I) from Streptomyces exfoliatus SMF19 was purified and characterized. The molecular mass of BLIP-I was estimated to be 17.5 kDa by gel filtration fast protein liquid chromatography. The N-terminal sequence was NH(2)-Asn-Ser-Gly-Phe-Ser-Ala-Glu-Lys-Tyr-Glu-Gln-Ile-Gln-Phe-Gly. BLIP-I inhibited Bacto(R) Penase (Difco), and plasmid encoded TEM-1 beta-lactamase, whereas it did not inhibit Enterobacter cloacae beta-lactamases. The K(i) value of BLIP-I against TEM-1 beta-lactamase was determined to be 0.047 nm. The gene (bliA) encoding BLIP-I protein was identified by screening a genomic library using an oligonucleotide probe with a sequence based on the N-terminal sequence of BLIP-I. Analysis of the nucleotide sequence revealed that the gene was 558 base pairs in length and encoded a mature protein of 157 amino acid residues preceded by a 29-amino acid signal sequence. Pairwise comparison of the deduced amino acid sequence showed 38% identity with BLIP of Streptomyces clavuligerus. Furthermore, the 49th amino acid residue of BLIP-I was identical to Asp-49 of BLIP that was characterized to be an important residue for the inhibitory activity of BLIP. A modified BLIP-I in which Asp-49 was replaced by alanine (D49A) was obtained by site-directed mutagenesis. The inhibitory activities of recombinant (r) BLIP-I and its D49A mutant derivative, expressed in Escherichia coli, were compared. The K(i) value of rBLIP-I against TEM-1 beta-lactamase was similar to that of wild-type BLIP-I, but the D49A mutation increased the K(i) of rBLIP-I inhibition approximately 200-fold. A disruption mutant of the bliA gene in S. exfoliatus SMF19 was obtained by replacing the wild-type bliA gene with a copy inactivated by inserting a hygromycin resistance gene. The disruption mutant showed a bald phenotype, indicating that the bliA gene plays a role in morphological differentiation.

Design of Potent β-Lactamase Inhibitors by Phage Display of β-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.