title: Structural and kinetic characterization of a beta-lactamase-inhibitor protein.
abstract: The past decade has seen an alarming worldwide increase in resistance to beta-lactam antibiotics among many pathogenic bacteria, which is due mainly to plasmid- or chromosomally encoded beta-lactamases that specifically cleave penicillin and cephalosporins, rendering them inactive. There is therefore a need to develop new strategies in the design of effective inhibitors of beta-lactamase. All the small-molecule inhibitors in clinical use are not very effective and are rapidly degraded. Furthermore, newly characterized mutants of the plasmid-mediated beta-lactamase TEM-1 are highly resistant to these small-molecule inhibitors, including clavulanic acid and tazobactam. It has been shown that Streptomyces clavuligerus produces an exocellular beta-lactamase inhibitory protein (BLIP; M(r) 17.5 K). Here we present data defining BLIP as the most effective known inhibitor of a variety of beta-lactamases, with Ki values in the subnanomolar to picomolar range. To identify those features in BLIP that make it such a potent inhibitor, we have determined its molecular structure at 2.1 A resolution. BLIP is a relatively flat molecule with a unique fold, comprising a tandem repeat of a 76-amino-acid domain. Each domain consists of a helix-loop-helix motif that packs against a four-stranded antiparallel beta-sheet (Fig. 1a). To our knowledge, BLIP is the first example of a protein inhibitor having two similarly folded domains that interact with and inhibit a single target enzyme.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Canada.
authors.forename: N C
authors.initials: NC
authors.lastname: Strynadka
authors.forename: S E
authors.initials: SE
authors.lastname: Jensen
authors.forename: K
authors.initials: K
authors.lastname: Johns
authors.forename: H
authors.initials: H
authors.lastname: Blanchard
authors.forename: M
authors.initials: M
authors.lastname: Page
authors.forename: A
authors.initials: A
authors.lastname: Matagne
authors.forename: J M
authors.initials: JM
authors.lastname: Frère
authors.forename: M N
authors.initials: MN
authors.lastname: James
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors; D006602:Hexosyltransferases; D008958:Models, Molecular; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000037:antagonists & inhibitors; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000737:chemistry*; D065093:beta-Lactamase Inhibitors
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D065093:beta-Lactamase Inhibitors; C065596:beta-lactamase-inhibitor protein, Streptomyces; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase