Identification of a β-lactamase inhibitory protein variant that is a potent inhibitor of Staphylococcus PC1 β-lactamase.
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.
Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Ji
J
Yuan
Dar-Chone
DC
Chow
Wanzhi
W
Huang
Timothy
T
Palzkill
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002151:Calorimetry; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000378:metabolism
D001426:Bacterial Proteins; D050505:Mutant Proteins; D019151:Peptide Library; D065093:beta-Lactamase Inhibitors; C065596:beta-lactamase-inhibitor protein, Streptomyces; C054232:beta-lactamase PC1; D001618:beta-Lactamases
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