Structural and kinetic characterization of a β-lactamase-inhibitor protein

Abstract

THE past decade has seen an alarming worldwide increase in resistance to β-lactam antibiotics among many pathogenic bacteria¹, which is due mainly to plasmid- or chromosomally encoded p-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 β-lactamase. All the small-molecule inhibitors in clinical use are not very effective and are rapidly degraded^2,3. Furthermore, newly characterized mutants of the plasmid-mediated β-lactamase TEM-1 are highly resistant to these small-molecule inhibitors, including clavulanic acid and tazobactam⁴. It has been shown that Streptomyces clavuligerus produces an β-lactamase inhibitory protein (BLIP; M_T 17.5 K)⁵. Here we present data defining BLIP as the most effective known inhibitor of a variety of β-lactamase, 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 Å 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 β-sheet (Fig. la). 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.