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

Abstract

The β-lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A β-lactamases. Widespread resistance to β-lactam antibiotics currently limits treatment strategies for Staphylococcus infections. The goal of this study was to determine the binding affinity of BLIP for S. aureus PC1 β-lactamase and to identify mutants that alter binding affinity. The BLIP inhibition constant (Ki) for the PC1 β-lactamase was measured at 350 nM and isothermal titration calorimetry (ITC) experiments indicated a binding constant (Kd) of 380 nM. A total of 23 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 Ki of 42 nM while calorimetry indicated a Kd of 26 nM. Molecular modeling studies suggested 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 BLIP residue Lys74 that is important for the stability of the complex. This hypothesis was confirmed by showing that the A104E PC1 enzyme binds BLIP with 15-fold greater affinity than wild type PC1 β-lactamase. Kinetic measurements indicated similar association rates for all complexes with the variation in affinity due to altered dissociation rate constants suggesting changes in short-range interactions are responsible for the altered binding properties of the mutants.

Identification of the β-Lactamase Inhibitor Protein-II (BLIP-II) Interface Residues Essential for Binding Affinity and Specificity for Class A β-Lactamases

“…This finding is similar to observations made with other protein-protein interactions (23,31,50,51). In the BLIP-␤-lactamase system, the BLIP Glu-73-Lys-74 motif has been shown to be a specificity determinant for interactions with several different ␤-lactamases (8,32,35). Another example is the cross-reactivity between bovine and human growth hormone and the human growth hormone receptor that occurs when the human growth hormone Arg-43 and receptor Asp-171 salt bridge is eliminated (50).…”

Analysis of the Binding Forces Driving the Tight Interactions between β-Lactamase Inhibitory Protein-II (BLIP-II) and Class A β-Lactamases

“…In this study we show that BLIP-II has a higher affinity for class A ␤-lactamases than BLIP and that the association rate constants are consistent with diffusion controlled rates (46). However, it is the exceptionally slow off-rates of BLIP-II that cause its overall affinity to be several orders of magnitude tighter than that observed for BLIP (34,77).…”

“…f The dissociation constant (K d ) was calculated from the dissociation rate constant and averaged association rate constant at ambient temperature (23°C). g The BLIP affinities to these ␤-lactamases were determined previously (34,40).…”

Engineering Specificity from Broad to Narrow: Design of a β-Lactamase Inhibitory Protein (BLIP) Variant That Exclusively Binds and Detects KPC β-Lactamase

“…Randomization of only five positions in BLIP (Y50, E73, K74, W112, and R160) identified four single position substitutions that maintained binding to KPC-2 while losing affinity for TEM-1 β-lactamase. The high success rate for finding mutants that changed binding specificity is due to the choice of BLIP positions to interrogate, which was based on knowledge from previous studies that substitutions at these positions can change the binding specificity of BLIP 9, 11, 17, 28 . The strategy for engineering further changes in specificity was to make double mutant combinations of a subset of the single mutants with a focus on double mutants containing the E73K and K74T substitutions, which had the most significant changes in binding specificity as single substitutions.…”