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ß-Lactamase inhibitory protein (BLIP) consists of a tandem repeat of αß domains conjugated by an interdomain loop and can effectively bind and inactivate class A ß-lactamases, which are responsible for resistance of bacteria to ß-lactam antibiotics. The varied ability of BLIP to bind different ß-lactamases and the structural determinants for significant enhancement of BLIP variants with a point mutation are poorly understood. Here, we investigated the conformational dynamics of BLIP upon binding to three clinically prevalent class A ß-lactamases (TEM1, SHV1, and PC1) with dissociation constants between subnanomolar and micromolar. Hydrogen deuterium exchange mass spectrometry revealed that the flexibility of the interdomain region was significantly suppressed upon strong binding to TEM1, but was not significantly changed upon weak binding to SHV1 or PC1. E73M and K74G mutations in the interdomain region improved binding affinity toward SHV1 and PC1, respectively, showing significantly increased flexibility of the interdomain region compared to the wild-type and favorable conformational changes upon binding. In contrast, more rigidity of the interfacial loop 135-145 was observed in these BLIP mutants in both free and bound states. Consistently, molecular dynamics simulations of BLIP exhibited drastic changes in the flexibility of the loop 135-145 in all complexes. Our results indicated for the first time that higher flexibility of the interdomain linker, as well as more rigidity of the interfacial loop 135-145, could be desirable determinants for enhancing inhibition of BLIP to class A ß-lactamases. Together, these findings provide unique insights into the design of enhanced inhibitors.

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Protein-protein interactions govern many cellular processes, and identifying binding interaction sites on proteins can facilitate the discovery of inhibitors to block such interactions. Here we identify peptides from a randomly fragmented plasmid encoding the ß-lactamase inhibitory protein (BLIP) and the Lac repressor (LacI) that represent regions of protein-protein interactions. We utilized a Jun-Fos-assisted phage display system that has previously been used to screen cDNA and genomic libraries to identify antibody antigens. Affinity selection with polyclonal antibodies against LacI or BLIP resulted in the rapid enrichment of in-frame peptides from various regions of the proteins. Further, affinity selection with ß-lactamase enriched peptides that encompass regions of BLIP previously shown to contribute strongly to the binding energy of the BLIP/ß-lactamase interaction, i.e., hotspot residues. Further, one of the regions enriched by affinity selection encompassed a disulfide-constrained region of BLIP that forms part of the BLIP interaction surface in the native complex that we show also binds to ß-lactamase as a disulfide-constrained macrocycle peptide with a KD of ∼1 µM. Fragmented open reading frame (ORF) libraries may efficiently identify such naturally constrained peptides at protein-protein interaction interfaces. With sufficiently deep coverage of ORFs by peptide-coding inserts, phage display and deep sequencing can provide detailed information on the domains or peptides that contribute to an interaction. Such information should enable the design of potentially therapeutic macrocycles or peptidomimetics that block the interaction.

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BACKGROUND: Mostly BLIPs are identified in soil bacteria Streptomyces and originally isolated from Streptomyces clavuligerus and can be utilized as a model system for biophysical, structural, mutagenic and computational studies. BLIP possess homology with two proteins viz., BLIP-I (Streptomyces exofoliatus) and BLP (beta-lactamase inhibitory protein like protein from S. clavuligerus). BLIP consists of 165 amino acid, possessing two homologues domains comprising helix-loop-helix motif packed against four stranded beta-sheet resulting into solvent exposed concave surface with extended four stranded beta-sheet. BLIP-I is a 157 amino acid long protein obtained from S. exofoliatus having 37% sequence identity to BLIP and inhibits beta-lactamase. METHODS: This review is intended to briefly illustrate the beta-lactamase inhibitory activity of BLIP via proteinprotein interaction and aims to open up a new avenue to combat antimicrobial resistance using peptide based inhibition. RESULTS: D49A mutation in BLIP-I results in a decrease in affinity for TEM-1 from 0.5 nM to 10 nM (Ki). It is capable of inhibiting TEM-1 and bactopenemase and differs from BLIP only in modulating cell wall synthesis enzyme. Whereas, BLP is a 154 amino acid long protein isolated from S. clavuligerus via DNA sequencing analysis of Cephamycin-Clavulanate gene bunch. It shares 32% sequence similarity with BLIP and 42% with BLIP-I. Its biological function is unclear and lacks beta-lactamase inhibitory activity. CONCLUSION: Protein-protein interactions mediate a significant role in regulation and modulation of cellular developments and processes. Specific biological markers and geometric characteristics are manifested by active site binding clefts of protein surfaces which determines the specificity and affinity for their targets. TEM1.BLIP is a classical model to study protein-protein interaction. ß-Lactamase inhibitory proteins (BLIPs) interacts and inhibits various ß-lactamases with extensive range of affinities.

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ß-Lactamase inhibitory protein (BLIP), a low molecular weight protein from Streptomyces clavuligerus, has a wide range of potential applications in the fields of biotechnology and pharmaceutical industry because of its tight interaction with and potent inhibition on clinically important class A ß-lactamases. To meet the demands for considerable amount of highly pure BLIP, this study aimed at developing an efficient expression system in eukaryotic Pichia pastoris (a methylotrophic yeast) for production of BLIP. With methanol induction, recombinant BLIP was overexpressed in P. pastoris X-33 and secreted into the culture medium. A high yield of ~ 300 mg/L culture secretory BLIP recovered from the culture supernatant without purification was found to be > 90% purity. The recombinant BLIP was fully active and showed an inhibition constant (Ki) for TEM-1 ß-lactamase (0.55 ± 0.07 nM) comparable to that of the native S. clavuligerus-expressed BLIP (0.5 nM). Yeast-produced BLIP in combination with ampicillin effectively inhibited the growth of ß-lactamase-producing Gram-positive Bacillus. Our approach of expressing secretory BLIP in P. pastoris gave 71- to 1200-fold more BLIP with high purity than the other conventional methods, allowing efficient production of large amount of highly pure BLIP, which merits fundamental science studies, drug development and biotechnological applications.