Identification and characterization of β-lactamase inhibitor protein-II (BLIP-II) interactions with β-lactamases using phage display

Abstract

Protein-protein interactions are critical to cellular processes yet the ability to predict and rationally design interactions is limited because of incomplete knowledge of the principles governing these interactions. The β-lactamase inhibitory protein (BLIP)/β-lactamase interaction has become a model system to investigate protein-protein interactions and has been the focus of several structural, thermodynamic and binding specificity studies. BLIP-II also inhibits β-lactamase but has no sequence homology with BLIP. The structure of BLIP-II in complex with TEM-1 β-lactamase revealed that BLIP-II has a completely different structure than BLIP but it interacts with the same protruding loop-helix region of TEM-1 as does BLIP. The significance of the individual interacting residues in molecular recognition by BLIP-II is currently unknown. Therefore, a phage display vector was developed with the purpose of expressing BLIP-II onto the surface of the M13 filamentous bacteriophage. The BLIP-II displayed phage bound to TEM-1 with picomolar affinity indicating that BLIP-II is properly folded while on the surface of the phage. The phage system, as well as enzyme inhibition assays with purified proteins, revealed that BLIP-II is a more potent inhibitor than BLIP for several class A β-lactamases with Ki values in the low picomolar range. © 2010 The Author.