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

Rudgers, Gary W.; Huang, Wanzhi; Palzkill, Timothy

doi:10.1128/aac.45.12.3279-3286.2001

Cited by 33 publications

(33 citation statements)

References 49 publications

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“…This finding is promising for inhibitor development and reinforces the results of previous efforts to develop peptide inhibitors. Peptides corresponding to residues 46 -51 of BLIP (the Asp-49 binding loop) were shown to bind TEM-1 and SHV-1 with affinities of 488 and 420 M, respectively (37). Random fragmentation and phage display of BLIP identified a peptide consisting of residues 30 -49 of BLIP that inhibits TEM-1 with 446 M affinity, and a combination of phage display and peptide arrays identified a 136 M inhibitor that has 50% sequence identity to BLIP residues 46 -51 (38,39).…”

Section: Discussionmentioning

confidence: 99%

Structural and Computational Characterization of the SHV-1 β-Lactamase-β-Lactamase Inhibitor Protein Interface

Reynolds

Thomson

Corbett

et al. 2006

Journal of Biological Chemistry

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␤-Lactamase inhibitor protein (BLIP) binds a variety of class A ␤-lactamases with affinities ranging from micromolar to picomolar. Whereas the TEM-1 and SHV-1 ␤-lactamases are almost structurally identical, BLIP binds TEM-1 ϳ1000-fold tighter than SHV-1. Determining the underlying source of this affinity difference is important for understanding the molecular basis of ␤-lactamase inhibition and mechanisms of protein-protein interface specificity and affinity. Here we present the 1.6 Å resolution crystal structure of SHV-1⅐BLIP. In addition, a point mutation was identified, SHV D104E, that increases SHV⅐BLIP binding affinity from micromolar to nanomolar. Comparison of the SHV-1⅐BLIP structure with the published TEM-1⅐BLIP structure suggests that the increased volume of Glu-104 stabilizes a key binding loop in the interface. Solution of the 1.8 Å SHV D104K⅐BLIP crystal structure identifies a novel conformation in which this binding loop is removed from the interface. Using these structural data, we evaluated the ability of EGAD, a program developed for computational protein design, to calculate changes in the stability of mutant ␤-lactamase⅐BLIP complexes. Changes in binding affinity were calculated within an error of 1.6 kcal/ mol of the experimental values for 112 mutations at the TEM-1⅐BLIP interface and within an error of 2.2 kcal/mol for 24 mutations at the SHV-1⅐BLIP interface. The reasonable success of EGAD in predicting changes in interface stability is a promising step toward understanding the stability of the ␤-lactamase⅐BLIP complexes and computationally assisted design of tight binding BLIP variants.Class A ␤-lactamases are a major cause of ␤-lactam resistance in Gram-negative bacteria. These enzymes catalyze the hydrolysis of ␤-lactam antibiotics, such as penicillins and cephalosporins, rendering them inactive. ␤-Lactamase inhibitor protein (BLIP), 6 which is secreted by the Gram-positive soil bacterium Streptomyces clavuligeris, inhibits a variety of class A ␤-lactamase enzymes with a wide spectrum of affinities. Its binding partners include Escherichia coli TEM-1, Klebsiella pneumoniae SHV-1, Serratia marcescens SME-1, Bacillus anthracis BlaI, and Proteus vulgaris K1, among others. BLIP is able to inhibit K1 with picomolar affinity and TEM-1, SME-1, and BlaI with nanomolar affinity. However, it inhibits SHV-1 with only micromolar affinity (1, 2). Whereas SHV-1 shares 67% sequence identity with TEM-1 (Fig. 1), and the crystal structures of the unbound ␤-lactamases overlay with an ␣-carbon r.m.s. deviation of 1.4 Å, BLIP exhibits a 1000-fold difference in affinity for the two (3). This poses an interesting question of binding specificity and affinity. How does BLIP bind multiple targets, and what is the source of variation in binding affinity? Recent alanine scanning mutagenesis has provided insight into the origins of BLIP affinity and specificity for an array of ␤-lactamases, including TEM-1 and SHV-1 (2, 4). However, interpretation of these data has been limited, because only the structure of the T...

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Section: Discussionmentioning

confidence: 99%

Structural and Computational Characterization of the SHV-1 β-Lactamase-β-Lactamase Inhibitor Protein Interface

Reynolds

Thomson

Corbett

et al. 2006

Journal of Biological Chemistry

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“…Nineteen different oligonucleotide primer pairs were used to replace the wildtype asparagine codon with that for the alternative amino acids. The PCR product was ligated into the pTP123-IMP-1 plasmid and electroporated into E. coli XL1-Blue cells (Stratagene) (38). The colonies were selected on LB agar supplemented with 12.5 g/ml chloramphenicol (6).…”

Section: Methodsmentioning

confidence: 99%

Analysis of the Functional Contributions of Asn233 in Metallo-β-Lactamase IMP-1

Brown

Banks

Huang

et al. 2011

Antimicrob Agents Chemother

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Metallo-␤-lactamases, such as IMP-1, are a major global health threat, as they provide for bacterial resistance to a wide range of ␤-lactam antibiotics, including carbapenems. Understanding the molecular details of the enzymatic process and the sequence requirements for function are essential aids in overcoming ␤-lactamase-mediated resistance. An asparagine residue is conserved at position 233 in approximately 67% of all metallo-␤-lactamases. Despite its conservation, the molecular basis of Asn233 function is poorly understood and remains controversial. It has previously been shown that mutations at this site exhibit context-dependent sequence requirements in that the importance of a given amino acid depends on the antibiotic being tested. To provide a more thorough examination as to the function and sequence requirements at this position, a collection of IMP-1 mutants encoding each of the 19 possible amino acid substitutions was generated. The resistance levels toward four ␤-lactam antibiotics were measured for Escherichia coli containing each of these mutants. The sequence requirements at position 233 for wild-type levels of resistance toward two cephalosporins were the most relaxed, while there were more stringent sequence requirements for resistance to ampicillin or imipenem. Enzyme kinetic analysis and determinations of steady-state protein levels indicated that the effects of the substitutions on resistance are due to changes in the kinetic parameters of the enzyme. Taken together, the results indicate that substitutions at position 233 significantly alter the kinetic parameters of the enzyme, but most substituted enzymes are able to provide for a high level of resistance to a broad range of ␤-lactams.The ␤-lactams, which include the penicillins and cephalosporins, are the most widely used class of antibiotics, and resistance to these drugs is a significant clinical problem (7,25). ␤-Lactamases are secreted into the periplasm and hydrolyze the conserved four-membered ␤-lactam ring, rendering the drug inactive. Currently, there are hundreds of ␤-lactamases reported, and they are regarded as the most common mechanism of resistance to this class of antibiotics (43).There are four classes of ␤-lactamases (A to D) based on primary amino acid sequence homology. ␤-Lactamases in classes A, C, and D hydrolyze the drugs by using a catalytic serine as the primary nucleophile (9, 15). Class B ␤-lactamases are metallo-enzymes that require either one or two zinc ions in the active site. The zinc ions in the active site decrease the pK of a coordinated water molecule, resulting in a hydroxide ion that acts as a nucleophile to attack the carbonyl group of the ␤-lactams (14). These enzymes exhibit a broad substrate profile, including carbapenems, which are often held in reserve as antibiotics of last resort (18,26). Mechanism-based inhibitors of the serine-active ␤-lactamases are available for clinical use, but these molecules do not act on class B enzymes (2). The genes encoding metallo-␤-lactamases are often found on mob...

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“…Alanine-scanning analysis has revealed that: 1) there are two hotspots on the interacting surface of BLIP, and 2) one mutation (Y50A) actually increases binding affinity for TEM-1 ␤-lactamase by 50-fold (8). Several weak binding peptide inhibitors for TEM-1 have been generated, among which two are the TEM-1-contacting fragments of BLIP (19,20). The contacts of these peptides potentially can be strengthened to create tight binding inhibitors with appropriate enhancement of binding forces.…”

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confidence: 99%

Thermodynamic Investigation of the Role of Contact Residues of β-Lactamase-inhibitory Protein for Binding to TEM-1 β-Lactamase

Wang

Zhang²,

Palzkill

et al. 2007

Journal of Biological Chemistry

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We have determined the thermodynamics of binding for the interaction between TEM-1 ␤-lactamase and a set of alanine substituted contact residue mutants of ␤-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°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-1 ␤-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 ␤-lactamase, explaining a large loss of entropy-driven binding energy of the K74A mutant and the resulting anti-canonical behavior. The anticanonical 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 .Protein-protein interactions are critical for any living organism. Their roles are vital in most biological processes, including metabolism, immune responses, signal transduction, gene regulation, and enzymatic regulation. Most noncovalent protein-protein interactions involve many contacts and interactions, which in turn combine into a macromolecular binding interaction with the needed specificity and affinity. Understanding the principles behind the affinities and specificities of protein-protein interactions will provide leads in developing new methods and reagents for protein-protein interactions. Recent advances in the structural biology of protein-protein interactions have revealed the complexity of structure-function relationships (1).In general, a protein-protein binding interaction involves a relatively planar interface with a large contact interface area (typically Ͼ1000...

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

Cited by 33 publications

References 49 publications

Structural and Computational Characterization of the SHV-1 β-Lactamase-β-Lactamase Inhibitor Protein Interface

Structural and Computational Characterization of the SHV-1 β-Lactamase-β-Lactamase Inhibitor Protein Interface

Analysis of the Functional Contributions of Asn233 in Metallo-β-Lactamase IMP-1

Thermodynamic Investigation of the Role of Contact Residues of β-Lactamase-inhibitory Protein for Binding to TEM-1 β-Lactamase

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