PMID:21238457

Citation	Yuan, J, Chow, DC, Huang, W and Palzkill, T (2011) Identification of a β-lactamase inhibitory protein variant that is a potent inhibitor of Staphylococcus PC1 β-lactamase. J. Mol. Biol. 406:730-44
Abstract	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.
Links	PubMed PMC3081586 Online version:10.1016/j.jmb.2011.01.014
Keywords	Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Calorimetry; Kinetics; Models, Molecular; Mutant Proteins/genetics; Mutant Proteins/metabolism; Peptide Library; Protein Binding; Protein Structure, Quaternary; Protein Structure, Tertiary; beta-Lactamase Inhibitors; beta-Lactamases/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
STRCL:BLIP		GO:0033252: regulation of beta-lactamase activity	ECO:0000315:		P		Figure 3 shows the interaction between K74G BLIP (a beta-lactamase inhibitory protein mutant) with S. aureus PC1 wild-type β-lactamase and its A104E (beta-lactamase) mutant. K74G BLIP has greater inhibitory effect on the wild type beta-lactamase than the mutant beta-lactamase. Table 2 includes the inhibition constant of the purified K74G BLIP variant for PC1 β-lactamase, which shows about a 10-fold increase in comparison to that of wild type BLIP.	complete CACAO 13272

References

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