Analysis of the binding forces driving the tight interactions between beta-lactamase inhibitory protein-II (BLIP-II) and class A beta-lactamases.

β-Lactamases hydrolyze β-lactam antibiotics to provide drug resistance to bacteria. β-Lactamase inhibitory protein-II (BLIP-II) is a potent proteinaceous inhibitor that exhibits low picomolar affinity for class A β-lactamases. This study examines the driving forces for binding between BLIP-II and β-lactamases using a combination of presteady state kinetics, isothermal titration calorimetry, and x-ray crystallography. The measured dissociation rate constants for BLIP-II and various β-lactamases ranged from 10(-4) to 10(-7) s(-1) and are comparable with those found in some of the tightest known protein-protein interactions. The crystal structures of BLIP-II alone and in complex with Bacillus anthracis Bla1 β-lactamase revealed no significant side-chain movement in BLIP-II in the complex versus the monomer. The structural rigidity of BLIP-II minimizes the loss of the entropy upon complex formation and, as indicated by thermodynamics experiments, may be a key determinant of the observed potent inhibition of β-lactamases.