title: Engineering Specificity from Broad to Narrow: Design of a β-Lactamase Inhibitory Protein (BLIP) Variant That Exclusively Binds and Detects KPC β-Lactamase.
abstract: The β-lactamase inhibitory protein (BLIP) binds and inhibits a wide range of class A β-lactamases including the TEM-1 β-lactamase (Ki= 0.5 nM), which is widely present in Gram-negative bacteria, and the KPC-2 β-lactamase (Ki= 1.2 nM), which hydrolyzes virtually all clinically useful β-lactam antibiotics. The extent to which the specificity of a protein that binds a broad range of targets can be modified to display narrow specificity was explored in this study by engineering BLIP to bind selectively to KPC-2 β-lactamase. A genetic screen for BLIP function in Escherichia coli was used to narrow the binding specificity of BLIP by identifying amino acid substitutions that retain affinity for KPC-2 while losing affinity for TEM-1 β-lactamase. The combination of single substitutions yielded the K74T:W112D BLIP variant, which was shown by inhibition assays to retain high affinity for KPC-2 with a Kiof 0.4 nM, while drastically losing affinity for TEM-1 with a Ki> 10 μM. The K74T:W112D mutant therefore binds KPC-2 β-lactamase 3 times more tightly while binding TEM-1 > 20000-fold more weakly than wild-type BLIP. The K74T:W112D BLIP variant also exhibited low affinity (Ki> 10 μM) for other class A β-lactamases. The high affinity and narrow specificity of BLIP K74T:W112D for KPC-2 β-lactamase suggest it could be a useful sensor for the presence of this enzyme in multidrug-resistant bacteria. This was demonstrated with an assay employing BLIP K74T:W112D conjugated to a bead to specifically pull-down and detect KPC-2 β-lactamase in lysates from clinical bacterial isolates containing multiple β-lactamases.
authors.affiliation: Departments of Pharmacology, ‡Medicine, and §Molecular Virology and Microbiology, Baylor College of Medicine , One Baylor Plaza, Houston, Texas 77030, United States.
authors.forename: Dar-Chone
authors.initials: DC
authors.lastname: Chow
authors.affiliation: Departments of Pharmacology, ‡Medicine, and §Molecular Virology and Microbiology, Baylor College of Medicine , One Baylor Plaza, Houston, Texas 77030, United States.
authors.forename: Kacie
authors.initials: K
authors.lastname: Rice
authors.affiliation: Departments of Pharmacology, ‡Medicine, and §Molecular Virology and Microbiology, Baylor College of Medicine , One Baylor Plaza, Houston, Texas 77030, United States.
authors.forename: Wanzhi
authors.initials: W
authors.lastname: Huang
authors.affiliation: Departments of Pharmacology, ‡Medicine, and §Molecular Virology and Microbiology, Baylor College of Medicine , One Baylor Plaza, Houston, Texas 77030, United States.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Atmar
authors.affiliation: Departments of Pharmacology, ‡Medicine, and §Molecular Virology and Microbiology, Baylor College of Medicine , One Baylor Plaza, Houston, Texas 77030, United States.
authors.forename: Timothy
authors.initials: T
authors.lastname: Palzkill
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D015202:Protein Engineering; D065093:beta-Lactamase Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D065093:beta-Lactamase Inhibitors; C491478:beta-lactamase KPC-2; D001618:beta-Lactamases
keywords: antibiotic resistance; diagnostics; enzyme inhibition; protein engineering; protein−protein interactions; β-lactamase