Identification of a β-Lactamase Inhibitory Protein Variant That Is a Potent Inhibitor of Staphylococcus PC1 β-Lactamase

• Published in: Journal of molecular biology 2011-03, Vol.406 (5), p.730-744
• Main Authors: Yuan, Ji, Chow, Dar-Chone, Huang, Wanzhi, Palzkill, Timothy
• Format: Article
• Language: English
• Subjects: alanine; antibiotic resistance; antibiotics; Bacterial Proteins; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; bacteriophages; beta-lactamase; beta-Lactamase Inhibitors; beta-Lactamases; beta-Lactamases - metabolism; binding capacity; Calorimetry; dissociation; enzyme inhibitors; genetics; glutamic acid; Kinetics; metabolism; Models, Molecular; Mutant Proteins; Mutant Proteins - genetics; Mutant Proteins - metabolism; mutants; Peptide Library; phage display; Protein Binding; Protein Structure, Quaternary; Protein Structure, Tertiary; protein–protein interactions; salt bridge; Staphylococcus aureus; titration
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2011.01.014

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 calorimetry experiments indicated a binding constant ( K d) of 380 nM. Twenty-three residue positions in BLIP that contact β-lactamase were randomized, and phage display was used to sort the libraries for tight binders to immobilized PC1 β-lactamase. The BLIP K74G mutant was the dominant clone selected, and it was found to inhibit the PC1 β-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 β-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 β-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. ▪ ► Phage display screening of a combinatorial library of BLIP mutants was used to identify a potent inhibitor of Staphylococcus β-lactamase. ► Charge complementarity at the protein–protein interface is the mechanism for the tight binding of PC1 β-lactamase by the inhibiting BLIP variant. ► Charge complementarity can be achieved by neutralizing the net charge in the interface, either by introducing an oppositely charged residue or by eliminating charged residues.