Dataset Information

Identification of a ?-lactamase inhibitory protein variant that is a potent inhibitor of Staphylococcus PC1 ?-lactamase.

ABSTRACT: ?-Lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A ?-lactamases. Widespread resistance to ?-lactam antibiotics currently limits the treatment strategies for Staphylococcus infections. The goals of this study were to determine the binding affinity of BLIP for Staphylococcus aureus PC1 ?-lactamase and to identify mutants that alter binding affinity. The BLIP inhibition constant (K(i)) for PC1 ?-lactamase was measured at 350 nM, and isothermal titration calorimetry experiments indicated a binding constant (K(d)) of 380 nM. Twenty-three residue positions in BLIP that contact ?-lactamase were randomized, and phage display was used to sort the libraries for tight binders to immobilized PC1 ?-lactamase. The BLIP(K74G) mutant was the dominant clone selected, and it was found to inhibit the PC1 ?-lactamase with a K(i) of 42 nM, while calorimetry indicated a K(d) of 26 nM. Molecular modeling studies suggested that BLIP binds weakly to the PC1 ?-lactamase due to the presence of alanine at position 104 of PC1. This position is occupied by glutamate in the TEM-1 enzyme, where it forms a salt bridge with the BLIP residue Lys74 that is important for the stability of the complex. This hypothesis was confirmed by showing that the PC1(A104E) enzyme binds BLIP with 15-fold greater affinity than wild-type PC1 ?-lactamase. Kinetic measurements indicated similar association rates for all complexes with variation in affinity due to altered dissociation rate constants, suggesting that changes in short-range interactions are responsible for the altered binding properties of the mutants.

SUBMITTER: Yuan J

PROVIDER: S-EPMC3081586 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Identification of a β-lactamase inhibitory protein variant that is a potent inhibitor of Staphylococcus PC1 β-lactamase.

Yuan Ji J Chow Dar-Chone DC Huang Wanzhi W Palzkill Timothy T

Journal of molecular biology 20110113 5

β-Lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A β-lactamases. Widespread resistance to β-lactam antibiotics currently limits the treatment strategies for Staphylococcus infections. The goals of this study were to determine the binding affinity of BLIP for Staphylococcus aureus PC1 β-lactamase and to identify mutants that alter binding affinity. The BLIP inhibition constant (K(i)) for PC1 β-lactamase was measured at 350 nM, and isothermal titration calorim ...[more]

PMID: 21238457

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Project description:BackgroundThe Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity.ResultsIn this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31-121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding.ConclusionConformational epitopes recognized by human antibodies have been mapped on the CHIPS surface and amino acid residues involved in both antibody and C5aR interaction could be defined. This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG.

Project description:Beta-lactamases are enzymes that catalyze the hydrolysis of beta-lactam antibiotics. beta-lactamase/beta-lactamase inhibitor protein (BLIP) complexes are emerging as a well characterized experimental model system for studying protein-protein interactions. BLIP is a 165 amino acid protein that inhibits several class A beta-lactamases with a wide range of affinities: picomolar affinity for K1; nanomolar affinity for TEM-1, SME-1, and BlaI; but only micromolar affinity for SHV-1 beta-lactamase. The large differences in affinity coupled with the availability of extensive mutagenesis data and high-resolution crystal structures for the TEM-1/BLIP and SHV-1/BLIP complexes make them attractive systems for the further development of computational design methodology. We used EGAD, a physics-based computational design program, to redesign BLIP in an attempt to increase affinity for SHV-1. Characterization of several of designs and point mutants revealed that in all cases, the mutations stabilize the interface by 10- to 1000-fold relative to wild type BLIP. The calculated changes in binding affinity for the mutants were within a mean absolute error of 0.87 kcal/mol from the experimental values, and comparison of the calculated and experimental values for a set of 30 SHV-1/BLIP complexes yielded a correlation coefficient of 0.77. Structures of the two complexes with the highest affinity, SHV-1/BLIP (E73M) and SHV-1/BLIP (E73M, S130K, S146M), are presented at 1.7 A resolution. While the predicted structures have much in common with the experimentally determined structures, they do not coincide perfectly; in particular a salt bridge between SHV-1 D104 and BLIP K74 is observed in the experimental structures, but not in the predicted design conformations. This discrepancy highlights the difficulty of modeling salt bridge interactions with a protein design algorithm that approximates side chains as discrete rotamers. Nevertheless, while local structural features of the interface were sometimes miscalculated, EGAD is globally successful in designing complexes with increased affinity.

Dataset Information

Identification of a ?-lactamase inhibitory protein variant that is a potent inhibitor of Staphylococcus PC1 ?-lactamase.

Publications

Identification of a β-lactamase inhibitory protein variant that is a potent inhibitor of Staphylococcus PC1 β-lactamase.

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