STRUCTURAL AND KINETIC CHARACTERIZATION OF A BETA-LACTAMASE-INHIBITORPROTEIN

THE past decade has seen an alarming worldwide increase in resistance to beta-lactam antibiotics among many pathogenic bacteria1, which is d ue mainly to plasmid- or chromosomally encoded beta-lactamases that sp ecifically cleave penicillin and cephalosporins, rendering them inacti ve. There is therefore a need to develop new strategies in the design of effective inhibitors of beta-lactamase. All the small-molecule inhi bitors in clinical use are not very effective and are rapidly degraded 2,3. Furthermore, newly characterized mutants of the plasmid-mediated beta-lactamase TEM-1 are highly resistant to these small-molecule inhi bitors, including clavulanic acid and tazobactam4. It has been shown t hat Streptomyces clavuligerus produces an exocellular beta-lactamase i nhibitory protein (BLIP; M(r) 17.5 K)5. Here we present data defining BLIP as the most effective known inhibitor of a variety of beta-lactam ases, with K(i) values in the subnanomolar to picomolar range. To iden tify those features in BLIP that make it such a potent inhibitor, we h ave determined its molecular structure at 2.1 angstrom resolution. BLI P is a relatively flat molecule with a unique fold, comprising a tande m repeat of a 76-amino-acid domain. Each domain consists of a helix-lo op-helix motif that packs against a four-stranded antiparallel beta-sh eet (Fig. 1a). To our knowledge, BLIP is the first example of a protei n inhibitor having two similarly folded domains that interact with and inhibit a single target enzyme.