Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0276640 (TEM)
 20,729 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The past decade has seen an alarming worldwide increase in resistance to beta-lactam antibiotics among many pathogenic bacteria, which is due mainly to plasmid- or chromosomally encoded beta-lactamases that specifically cleave penicillin and cephalosporins, rendering them inactive. There is therefore a need to develop new strategies in the design of effective inhibitors of beta-lactamase. All the small-molecule inhibitors in clinical use are not very effective and are rapidly degraded. Furthermore, newly characterized mutants of the plasmid-mediated beta-lactamase TEM-1 are highly resistant to these small-molecule inhibitors, including clavulanic acid and tazobactam. It has been shown that Streptomyces clavuligerus produces an exocellular beta-lactamase inhibitory protein (BLIP; M(r) 17.5 K). Here we present data defining BLIP as the most effective known inhibitor of a variety of beta-lactamases, with Ki values in the subnanomolar to picomolar range. To identify those features in BLIP that make it such a potent inhibitor, we have determined its molecular structure at 2.1 A resolution. BLIP is a relatively flat molecule with a unique fold, comprising a tandem repeat of a 76-amino-acid domain. Each domain consists of a helix-loop-helix motif that packs against a four-stranded antiparallel beta-sheet (Fig. 1a). To our knowledge, BLIP is the first example of a protein inhibitor having two similarly folded domains that interact with and inhibit a single target enzyme.
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PMID:Structural and kinetic characterization of a beta-lactamase-inhibitor protein. 814 54

Crystallization of the 1:1 molecular complex between the beta-lactamase TEM-1 and the beta-lactamase inhibitory protein BLIP has provided an opportunity to put a stringent test on current protein-docking algorithms. Prior to the successful determination of the structure of the complex, nine laboratory groups were given the refined atomic coordinates of each of the native molecules. Other than the fact that BLIP is an effective inhibitor of a number of beta-lactamase enzymes (KI for TEM-1 approximately 100 pM) no other biochemical or structural data were available to assist the practitioners in their molecular docking. In addition, it was not known whether the molecules underwent conformational changes upon association or whether the inhibition was competitive or non-competitive. All six of the groups that accepted the challenge correctly predicted the general mode of association of BLIP and TEM-1.
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PMID:Molecular docking programs successfully predict the binding of a beta-lactamase inhibitory protein to TEM-1 beta-lactamase. 860 17

The structure of TEM-1 beta-lactamase complex with the inhibitor BLIP has been determined at 1.7 angstrom resolution. The two tandemly repeated domains of BLIP form a polar, concave surface that docks onto a predominantly polar, convex protrusion on the enzyme. The ability of BLIP to adapt to a variety of class A beta-lactamases is most likely due to an observed flexibility between the two domains of the inhibitor and to an extensive layer of water molecules entrapped between the enzyme and inhibitor. A beta-hairpin loop from domain 1 of BLIP is inserted into the active site of the beta-lactamase. The carboxylate of Asp 49 forms hydrogen bonds to four conserved, catalytic residues in the beta-lactamase, thereby mimicking the position of the penicillin G carboxylate observed in the acyl-enzyme complex of TEM-1 with substrate. This beta-hairpin may serve as a template with which to create a new family of peptide-analogue beta-lactamase inhibitors.
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PMID:A potent new mode of beta-lactamase inhibition revealed by the 1.7 A X-ray crystallographic structure of the TEM-1-BLIP complex. 860 17

BLIP is a secreted protein from Streptomyces clavuligerus that inhibits a wide range of beta-lactamases. Here we investigate the tight interaction of BLIP, expressed heterologousely in E. coli, with TEM-1. Kinetic and thermodynamic constants were determined using methods with the proteins either in a homogeneous or in a heterogeneous phase. While values of Delta DeltaG(mut-wt) are similar whether measured by fluorescence quench, enzyme inhibition, or surface plasmon resonance, absolute values of DeltaG and kinetic constants vary. Association and dissociation rate constants of 10(5) M-1 s-1 and 10(-)4 s-1, respectively, and a nanomolar affinity were determined for the wild-type proteins. The highest affinity is measured at pH 7.5, with a decreasing association rate constant at higher pH values, and an increasing dissociation rate constant at lower pH values. The marginal effect of salt on the kinetics of binding, as well as the calculated surface potentials, suggests a limited role for electrostatic forces in guiding this reaction. Still, mutations of interfacial residues affect the rate of association significantly, so that an increase in the net negative charge on either protein reduces the association rate constant. We show that simple electrostatic rules can explain this behavior. BLIP inhibits the catalytic activity of TEM-1 by binding its active site. Yet, mutations of active site residues on TEM-1 only have a moderate though cooperative effect on the binding energy. This can be explained in light of the peripheral location of the active site in the interface between the two proteins.
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PMID:Biophysical characterization of the interaction of the beta-lactamase TEM-1 with its protein inhibitor BLIP. 989 Aug 78

The X-ray crystallographic structure of the SHV-1 beta-lactamase has been established. The enzyme crystallizes from poly(ethylene glycol) at pH 7 in space group P212121 with cell dimensions a = 49.6 A, b = 55.6 A, and c = 87.0 A. The structure was solved by the molecular replacement method, and the model has been refined to an R-factor of 0.18 for all data in the range 8.0-1.98 A resolution. Deviations of model bonds and angles from ideal values are 0.018 A and 1.8 degrees, respectively. Overlay of all 263 alpha-carbon atoms in the SHV-1 and TEM-1 beta-lactamases results in an rms deviation of 1.4 A. Largest deviations occur in the H10 helix (residues 218-224) and in the loops between strands in the beta-sheet. All atoms in residues 70, 73, 130, 132, 166, and 234 in the catalytic site of SHV-1 deviate only 0.23 A (rms) from atoms in TEM-1. However, the width of the substrate binding cavity in SHV-1, as measured from the 104-105 and 130-132 loops on one side to the 235-238 beta-strand on the other side, is 0.7-1.2 A wider than in TEM-1. A structural analysis of the highly different affinity of SHV-1 and TEM-1 for the beta-lactamase inhibitory protein BLIP focuses on interactions involving Asp/Glu104.
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PMID:Structure of the SHV-1 beta-lactamase. 1023 22

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.
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PMID:New beta -lactamase inhibitory protein (BLIP-I) from Streptomyces exfoliatus SMF19 and its roles on the morphological differentiation. 1074 83

An experimental approach to evaluate the net binding free energy of buried hydrogen bonds and salt bridges is presented. The approach, which involves a modified multiple-mutant cycle protocol, was applied to selected interactions between TEM-1-beta-lactamase and its protein inhibitor, BLIP. The selected interactions (two salt bridges and two hydrogen bonds) all involving BLIP-D49, define a distinct binding unit. The penta mutant, where all side-chains constructing the binding unit were mutated to Ala, was used as a reference state to which combinations of side-chains were introduced. At first, pairs of interacting residues were added allowing the determination of interaction energies in the absence of neighbors, using double mutant cycles. Addition of neighboring residues allowed the evaluation of their cooperative effects on the interaction. The two isolated salt bridges were either neutral or repulsive whereas the two hydrogen bonds contribute 0.3 kcal mol(-1 )each. Conversely, a double mutant cycle analysis of these interactions in their native environment showed that they all stabilize the complex by 1-1.5 kcal mol(-1). Examination of the effects of neighboring residues on each of the interactions revealed that the formation of a salt bridge triad, which involves two connected salt bridges, had a strong cooperative effect on stabilizing the complex independent of the presence or absence of additional neighbors. These results demonstrate the importance of forming net-works of buried salt bridges. We present theoretical electrostatic calculations which predict the observed mode of cooperativity, and suggest that the cooperative networking effect results from the favorable contribution of the protein to the interaction. Furthermore, a good correlation between calculated and experimentally determined interaction energies for the two salt bridges, and to a lesser extent for the two hydrogen bonds, is shown. The data analysis was performed on values of DeltaDeltaG(double dagger)K(d) which reflect the strength of short range interactions, while DeltaDeltaG(o)K(D) values which include the effects of long range electrostatic forces that alter specifically DeltaDeltaG(double dagger)k(a) were treated separately.
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PMID:Evaluation of direct and cooperative contributions towards the strength of buried hydrogen bonds and salt bridges. 1077 66

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.
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PMID:Protein minimization by random fragmentation and selection. 1152 22

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.
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PMID:Crystal structure and kinetic analysis of beta-lactamase inhibitor protein-II in complex with TEM-1 beta-lactamase. 1157 88

We have determined the thermodynamics of binding for the interaction between TEM-1 beta-lactamase and a set of alanine substituted contact residue mutants ofbeta-lactamase-inhibitory protein (BLIP) using isothermal titration calorimetry. The binding enthalpies for these interactions are highly temperature dependent, with negative binding heat capacity changes ranging from -800 to -271 cal mol(-1) K(-1). The isoenthalpic temperatures (at which the binding enthalpy is zero) of these interactions range from 5 to 38 degrees C. The changes in isoenthalpic temperature were used as an indicator of the changes in enthalpy and entropy driving forces, which in turn are related to hydrophobic and hydrophilic interactions. A contact residue of BLIP is categorized as a canonical residue if its alanine substitution mutant exhibits a change of isoenthalpic temperature matching the change of hydrophobicity because of the mutation. A contact position exhibiting a change in isoenthalpic temperature that does not match the change in hydrophobicity is categorized as an anti-canonical residue. Our experimental results reveal that the majority of residues where alanine substitution results in a loss of affinity are canonical (7 of 10), and about half of the residues where alanine substitutions have a minor effect are canonical. The interactions between TEM-1beta-lactamase and BLIP canonical contact residues contribute directly to binding free energy, suggesting potential anchoring sites for binding partners. The anti-canonical behavior of certain residues may be the result of mutation-induced modifications such as structural rearrangements affecting contact residue configurations. Structural inspection of BLIP suggests that the Lys(74) side chain electrostatically holds BLIP loop 2 in position to bind to TEM-1 beta-lactamase, explaining a large loss of entropy-driven binding energy of the K74A mutant and the resulting anti-canonical behavior. The anti-canonical behavior of the W150A mutant may also be due to structural rearrangements. Finally, the affinity enhancing effect of the contact residue mutant Y50A may be due to energetic coupling interactions between Asp(49) and His(41).
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PMID:Thermodynamic investigation of the role of contact residues of beta-lactamase-inhibitory protein for binding to TEM-1 beta-lactamase. 1743 Aug 99


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