Kinetic and Structural Analysis of Tight Complexes of TEM‐1 Beta‐Lactamase and BLIP mutants

Abstract

The S. clavuligerus ß‐Lactamase Inhibitor Protein (BLIP) is an effective inhibitor of class A ß‐lactamases, including the TEM‐1 β‐Lactamase (TEM1). A previous report from our groups shows that Y143A mutant of BLIP binds to TEM1 with similar affinity as the wild type, but with a larger entropy contribution to binding. The Y50A mutant binds to TEM1 50 fold stronger than wild type, which is the result of increased enthalpy driving forces (Wang J., et al. JBC. 2007; 282:17676–84). In addition, binding of BLIP Y50A with TEM1 exhibits a less negative ΔCp than that of the wild type complex. These findings suggest that binding between BLIP Y50A and TEM1 is enhanced by hydrophilic interactions. Interestingly, our stopped‐flow kinetic measurements show that the association rate of BLIP Y50A with TEM1 is similar to that of the wild type, demonstrating the increased affinity is mainly due to the slower dissociation. To understand the changes in binding thermodynamics and kinetics, we initiated structural studies of these complexes. After extensive crystallization screens, some high quality crystals were obtained for the BLIP Y50A/TEM1 complex and the BLIP Y143A/TEM1 complex crystallized in micro‐needle form. We are currently optimizing conditions to obtain high‐quality crystals. Future X‐ray diffraction analysis will determine if the crystals are suitable for structure determination.Support from AHA & UH to D.C. and NIH to T.P.