Identification and characterization of beta-lactamase inhibitor protein-II (BLIP-II) interactions with beta-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 beta-lactamase inhibitory protein (BLIP)/beta-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 beta-lactamase but has no sequence homology with BLIP. The structure of BLIP-II in complex with TEM-1 beta-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 beta-lactamases with K(i) values in the low picomolar range.

Fig. 1

Schematic representation of the plasmids constructed for this study. (A) Construction of pNK621 phage display plasmid. (B) Construction of pET-BLIP-II expression plasmid. Restriction sites utilized for the construction of the plasmids are shown. (C) The amino acid sequence of BLIP-II-g3p present in pNK621. The signal sequence and the signal peptidase cleavage site are illustrated followed by an amino acid linker segment used to introduce the SalI restriction site. The first three amino acids of the BLIP-II protein is shown as ‘start BLIP-II’. The amber codon position is marked at the C-terminus of BLIP-II as a glutamine which is followed in the plasmid by the full-length g3p with a trypsin cleavage site between the BLIP-II and g3p.

Fig. 2

Bacteriophage displaying BLIP-II interacts with TEM-1 β-lactamase with binding affinity in the picomolar concentration range. (A) Phage ELISA results with a dose-response curve. The x-axis indicates the amount of BLIP-II displaying phage added to immobilized β-lactamase, and the y-axis shows the absorbance after addition of HRP reagent that reacts with the anti-M13 HRP antibody. (B) Phage ELISA with competition from soluble TEM-1 β-lactamase. A subsaturating level of BLIP-II phage (1.7 × 10⁻⁹ M) was used for binding with increasing concentrations of TEM-1 β-lactamase competitor (x-axis). The y-axis indicates the fractional amount of BLIP-II phage bound in the well. This figure reveals that the BLIP-II phage has an IC₅₀ value of 320 pM. The IC₅₀ value is the concentration of soluble TEM-1 competitor that results in half maximal binding of the BLIP-II phage with immobilized β-lactamase.

Fig. 3

Bacteriophage displaying BLIP-II interacts with class A β-lactamases. This diagram shows the results of phage ELISA experiments with BLIP-II phage tested for binding to immobilized β-lactamases (TEM-1, PC1, Bla1—grey bars). P99 and Oxa-10 are representative class C and D β-lactamases, respectively. The results of phage ELISA measurement of KM13 helper phage binding to the immobilized targets are shown with black bars.

Fig. 4

Illustration of the structures of the (A) BLIP–TEM-1 β-lactamase (1JTG) and (B) BLIP-II–TEM-1 (1JTD) β-lactamase complexes (Strynadka et al., 1996; Lim et al., 2001). The protruding loop-helix region of β-lactamase, in red, is adjacent to the active site of TEM-1 β-lactamase, which is shown in green. BLIP and BLIP-II are shown in blue. (C) Sequence alignment of the β-lactamase loop-helix region (residues 99–114). The name of each β-lactamase used is at the left of the sequence. The residue 99–114 numbering refers to the canonical numbering scheme for class A β-lactamases.