Design of potent beta-lactamase inhibitors by phage display of beta-lactamase inhibitory protein.
Beta-lactamase inhibitory protein (BLIP) binds tightly to several beta-lactamases including TEM-1 beta-lactamase (K(i) 0.1 nm). The TEM-1 beta-lactamase/BLIP co-crystal structure indicates that two turn regions in BLIP insert into the active site of beta-lactamase to block the binding of beta-lactam antibiotics. Residues from each turn, Asp(49) and Phe(142), mimic interactions made by penicillin G when bound in the beta-lactamase active site. Phage display was used to determine which residues within the turn regions of BLIP are critical for binding TEM-1 beta-lactamase. The sequences of a set of functional mutants from each library indicated that a few sequence types were predominant. These BLIP mutants exhibited K(i) values for beta-lactamase inhibition ranging from 0.01 to 0.2 nm. The results indicate that even though BLIP is a potent inhibitor of TEM-1 beta-lactamase, the wild-type sequence of the active site binding region is not optimal and that derivatives of BLIP that bind beta-lactamase extremely tightly can be obtained. Importantly, all of the tight binding BLIP mutants have sequences that would be predicted theoretically to form turn structures.
Huang
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W
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.
Zhang
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Z
Palzkill
T
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000494:pharmacology*; D001665:Binding Sites; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D004926:Escherichia coli; D007700:Kinetics; D008958:Models, Molecular; D010400:Penicillin G / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D065093:beta-Lactamase Inhibitors
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D019151:Peptide Library; D010455:Peptides; D065093:beta-Lactamase Inhibitors; C065596:beta-lactamase-inhibitor protein, Streptomyces; D010400:Penicillin G