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

beta-Lactamases hydrolyze beta-lactam antibiotics to provide drug resistance to bacteria. beta-Lactamase inhibitory protein-II (BLIP-II) is a potent proteinaceous inhibitor that exhibits low picomolar affinity for class A beta-lactamases. This study examines the driving forces for binding between BLIP-II and beta-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 beta-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 beta-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 beta-lactamases.